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Sullenger, Bruce Alan

Overview:

The main focus of my translational research laboratory is to develop RNA based therapeutic agents for the potential treatment of a range of diseases. To this end, we have and will continue to take advantage of the fact that RNA is not just a passive carrier of genetic instructions inside of cells during the conversion of information from DNA to RNA to protein. Rather, RNA is an extremely versatile biological macromolecule. Certian RNAs can bind to specific protiens with high affinities, while others can for catalytic centers and perform enzymatic reactions. These facets of RNA coupled with the ease with which RNA can be manipulated in vitro make it a very powerful and unique therapeutic agent whose potential is largely untapped. Durring our endeavors, we plan to work closely with the members of the Molecular Therapeutics program as well as other faculty at the Duke University Medical Center to expedite the development and testing of these therapeutics.

The specific aims of my laboratory are:

1. To isolate and characterize RNA and DNA aptamers which block therapeutically relavent proteins such as those involved in cardiovascular diseases and immune modulation.

2. To develop RNA-based tumor targeting strategies for delivering siRNAs and miRNAs to tumor cells.

3. To reprogram cells using mRNA delivery.

4. To explore novel methods to control inflammation.

Positions:

Joseph W. and Dorothy W. Beard Professor of Experimental Surgery, in the School of Medicine

Surgery, Surgical Sciences
School of Medicine

Professor of Surgery

Surgery, Surgical Sciences
School of Medicine

Director of the Duke Center for Translational Research

Surgery
School of Medicine

Associate Professor in Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1990

Ph.D. — Cornell University

News:

Grants:

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2022

Regional Oncolytic Poliovirus Immunotherapy for Breast Cancer

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Investigator
Start Date
August 01, 2016
End Date
July 31, 2021

Genetics Training Grant

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 1979
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Targeting DAMP-induced inflammation to prevent metastasis

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Partnering PI
Start Date
September 30, 2016
End Date
September 29, 2019

Targeting DAMP-induced inflammation to prevent metastasis

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Principal Investigator
Start Date
September 30, 2016
End Date
September 29, 2019

Viral Oncology Training Grant

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1980
End Date
June 30, 2019

Antithrombotic Aptamers and Antidotes

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2014
End Date
March 31, 2019

DTMI-Center for Thrombotic and Hemostatic Disorders

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 15, 2012
End Date
April 30, 2018

Duke-UNC Clinical Hematology and Transfusion Research Career Development Program

Administered By
Medicine, Hematology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 28, 2006
End Date
April 30, 2018

Studying Anticoagulant Synergy of Factor Xa Aptamer and Catalytic Site Inhibitors

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2015
End Date
March 31, 2018

Neutralizing Antibody & AAV VIX Gene Therapy

Administered By
Surgery, Surgical Sciences
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
February 01, 2017
End Date
January 31, 2018

TRC-THD Administrative Coordinating Center (ACC)

Administered By
Duke Clinical Research Institute
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
May 15, 2012
End Date
April 30, 2017

Aptamer Targeted Drug and Toxin Delivery to Prostate Cancer

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Mentor
Start Date
September 30, 2014
End Date
November 29, 2016

Targeting DNA Neutrophil Extracellular Traps in Sickle Cell Disease

Administered By
Surgery, Surgical Sciences
AwardedBy
University of North Carolina - Chapel Hill
Role
Principal Investigator
Start Date
September 01, 2013
End Date
August 31, 2016

Research Training In Neuro-Oncology

Administered By
Neurosurgery, Neuro-Oncology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1998
End Date
August 31, 2016

Using Aptamer Coated Nanoparticles Encapsulating Prostate Tumor Antigen Encoding mRNA to Target Dendritic Cells In Vivo

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Partnering PI
Start Date
September 01, 2012
End Date
August 31, 2015

RNA Therapeutics for Pancreatic Cancer

Administered By
Surgery, Advanced Oncologic and Gastrointestinal Surgery
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
August 17, 2011
End Date
July 31, 2015

Anticoagulant Synergy of Factor Xa Aptamer and Catalytic Site Inhibitors: Mechanistic and Translational Studies

Administered By
Surgery, Surgical Sciences
AwardedBy
PhRMA Foundation
Role
Principal Investigator
Start Date
January 01, 2015
End Date
March 31, 2015

In Vivo Selection of Tumor-Specific RNA Binding Motifs

Administered By
Surgery, Advanced Oncologic and Gastrointestinal Surgery
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 01, 2011
End Date
February 28, 2015

Regulation of inflammatory responses by nucleic-acid binding polymers

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2012
End Date
August 31, 2014

RNA aptamers as cell surface receptor agonists and siRNA delivery agents

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 24, 2009
End Date
April 30, 2014

Nucleic Acid Binding Polymers as Anti-Inflammatory Agents

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2011
End Date
August 31, 2013

CTSA UL

Administered By
Institutes and Centers
AwardedBy
National Institutes of Health
Role
Director
Start Date
September 30, 2006
End Date
September 01, 2012

In Vivo Selection of Aptamers Targeting Pancreatic Cancer

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2011
End Date
June 30, 2012

Research Training In Neuro-Oncology

Administered By
Neurosurgery, Neuro-Oncology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 15, 2005
End Date
August 31, 2010

Development and Analysis of Aptamer-Antidote Pairs

Administered By
Surgery
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2000
End Date
April 30, 2010

Enhancement of Thrombolysis through Inhibition of PAI-1 and Antiplasmin

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2007
End Date
June 24, 2008

E2F-Mediated Control of Vascular Growth and Remodeling

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2003
End Date
March 31, 2008

The Angiopoietins in Vascular Maintenance and Remodeling

Administered By
Medicine, Cardiology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
April 01, 2002
End Date
March 31, 2007

Analysis of Group I and II Introns in Mammalian Cells

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1995
End Date
March 31, 2007

Development of Oligonucleotide-based CTLA-4 Inhibitors.

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
April 01, 2004
End Date
August 31, 2006

Disrupting Viral Gene with Group II Introns

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2001
End Date
January 31, 2006

Rna Ligands: E2f Function and Cell Proliferation

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
January 14, 1999
End Date
December 31, 2003

Developing RNA-Mimotopes Of Hiv Antigens

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1998
End Date
September 29, 2001

Ribozyme Mediated Repair of Sickle B-Globin Transcripts

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1996
End Date
August 31, 2001

Trans-splicing by a Group I Ribozyme in Mammalial Cells.

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1998
End Date
March 31, 2001

Probing expressed genome organization with ribozymes

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1997
End Date
July 31, 2000

Ribozyme-Mediated Repair Of Sickle B-Globin Transcripts

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1996
End Date
August 31, 1999

Trans Splicing By A Group I Ribozyme In Mammalian Cells

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1996
End Date
March 31, 1999

Trans-Splicing By A Group I Ribozyme In Mammalian Cells

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 1995
End Date
March 31, 1999
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Publications:

A kallikrein-targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release.

Essentials Kallikrein amplifies contact activation and is a potential target for preventing thrombosis. We developed and characterized a kallikrein aptamer using convergent evolution and kinetic assays. Kall1-T4 prolongs intrinsic clotting time by inhibiting factor XIIa-mediated prekallikrein activation. Kall1-T4 decreases high-molecular-weight kininogen cleavage and bradykinin release.Background Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non-enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose-dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein activation. Additionally, Kall1-T4 significantly reduces kallikrein-mediated HK cleavage and subsequent BK release. Conclusions We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation.

Authors
Steen Burrell, K-A; Layzer, J; Sullenger, BA
MLA Citation
Steen Burrell, K-A, Layzer, J, and Sullenger, BA. "A kallikrein-targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release." Journal of thrombosis and haemostasis : JTH 15.9 (September 2017): 1807-1817.
PMID
28632925
Source
epmc
Published In
Journal of Thrombosis and Haemostasis
Volume
15
Issue
9
Publish Date
2017
Start Page
1807
End Page
1817
DOI
10.1111/jth.13760

Generation and characterization of aptamers targeting factor XIa.

The plasma protease factor XIa (FXIa) has become a target of interest for therapeutics designed to prevent or treat thrombotic disorders.We used a solution-based, directed evolution approach called systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that target the FXIa catalytic domain.Two aptamers, designated 11.16 and 12.7, were identified that bound to previously identified anion binding and serpin bindings sites on the FXIa catalytic domain. The aptamers were non-competitive inhibitors of FXIa cleavage of a tripeptide chromogenic substrate and of FXIa activation of factor IX. In normal human plasma, aptamer 12.7 significantly prolonged the aPTT clotting time.The results show that novel inhibitors of FXIa can be prepared using SELEX techniques. RNA aptamers can bind to distinct sites on the FXIa catalytic domain and noncompetitively inhibit FXIa activity toward its primary macromolecular substrate factor IX with different levels of potency. Such compounds can be developed for use as therapeutic inhibitors.

Authors
Woodruff, RS; Ivanov, I; Verhamme, IM; Sun, M-F; Gailani, D; Sullenger, BA
MLA Citation
Woodruff, RS, Ivanov, I, Verhamme, IM, Sun, M-F, Gailani, D, and Sullenger, BA. "Generation and characterization of aptamers targeting factor XIa." Thrombosis research 156 (August 2017): 134-141.
PMID
28644959
Source
epmc
Published In
Thrombosis Research
Volume
156
Publish Date
2017
Start Page
134
End Page
141
DOI
10.1016/j.thromres.2017.06.015

Differential Induction of Immunogenic Cell Death and Interferon Expression in Cancer Cells by Structured ssRNAs.

Activation of the RNA-sensing pattern recognition receptor (PRR) in cancer cells leads to cell death and cytokine expression. This cancer cell death releases tumor antigens and damage-associated molecular patterns (DAMPs) that induce anti-tumor immunity. However, these cytokines and DAMPs also cause adverse inflammatory and thrombotic complications that can limit the overall therapeutic benefits of PRR-targeting anti-cancer therapies. To overcome this problem, we generated and evaluated two novel and distinct ssRNA molecules (immunogenic cell-killing RNA [ICR]2 and ICR4). ICR2 and ICR4 differentially stimulated cell death and PRR signaling pathways and induced different patterns of cytokine expression in cancer and innate immune cells. Interestingly, DAMPs released from ICR2- and ICR4-treated cancer cells had distinct patterns of stimulation of innate immune receptors and coagulation. Finally, ICR2 and ICR4 inhibited in vivo tumor growth as effectively as poly(I:C). ICR2 and ICR4 are potential therapeutic agents that differentially induce cell death, immune stimulation, and coagulation when introduced into tumors.

Authors
Lee, J; Lee, Y; Xu, L; White, R; Sullenger, BA
MLA Citation
Lee, J, Lee, Y, Xu, L, White, R, and Sullenger, BA. "Differential Induction of Immunogenic Cell Death and Interferon Expression in Cancer Cells by Structured ssRNAs." Molecular Therapy 25.6 (June 2017): 1295-1305.
PMID
28372998
Source
epmc
Published In
Molecular Therapy
Volume
25
Issue
6
Publish Date
2017
Start Page
1295
End Page
1305
DOI
10.1016/j.ymthe.2017.03.014

Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis.

Trauma patients produce a host of danger signals and high levels of damage-associated molecular patterns (DAMPs) after cellular injury and tissue damage. These DAMPs are directly and indirectly involved in the pathogenesis of various inflammatory and thrombotic complications in patients with severe injuries. No effective therapeutic agents for the removal of DAMPs from blood or tissue fluid have been developed. Herein, we demonstrated that nucleic acid binding polymers, e.g., polyethylenimine (PEI) and polyamidoamine dendrimers, immobilized onto electrospun microfiber mesh can effectively capture various DAMPs, such as extracellular DNAs and high mobility group box 1 (HMGB1). Furthermore, treatment with PEI-immobilized microfiber mesh abrogated the ability of DAMPs, released from dead and dying cells in culture or found in patients following traumatic injury, to activate innate immune responses and coagulation in vitro and in vivo. Nucleic acid scavenging microfiber meshes represent an effective strategy to combat inflammation and thrombosis in trauma.

Authors
Lee, J; Jackman, JG; Kwun, J; Manook, M; Moreno, A; Elster, EA; Kirk, AD; Leong, KW; Sullenger, BA
MLA Citation
Lee, J, Jackman, JG, Kwun, J, Manook, M, Moreno, A, Elster, EA, Kirk, AD, Leong, KW, and Sullenger, BA. "Nucleic acid scavenging microfiber mesh inhibits trauma-induced inflammation and thrombosis." Biomaterials 120 (March 2017): 94-102.
PMID
28049065
Source
epmc
Published In
Biomaterials
Volume
120
Publish Date
2017
Start Page
94
End Page
102
DOI
10.1016/j.biomaterials.2016.12.024

Aptamers in Bordeaux, 24–25 June 2016

Authors
Toulmé, J-J; Giangrande, P; Mayer, G; Suess, B; Ducongé, F; Sullenger, B; de Franciscis, V; Darfeuille, F; Peyrin, E
MLA Citation
Toulmé, J-J, Giangrande, P, Mayer, G, Suess, B, Ducongé, F, Sullenger, B, de Franciscis, V, Darfeuille, F, and Peyrin, E. "Aptamers in Bordeaux, 24–25 June 2016." Pharmaceuticals 10.1 (March 2017): 14-14.
Source
crossref
Published In
Pharmaceuticals
Volume
10
Issue
1
Publish Date
2017
Start Page
14
End Page
14
DOI
10.3390/ph10010014

Aptamers as Therapeutics.

Aptamers are single-stranded nucleic acid molecules that bind to and inhibit proteins and are commonly produced by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers undergo extensive pharmacological revision, which alters affinity, specificity, and therapeutic half-life, tailoring each drug for a specific clinical need. The first therapeutic aptamer was described 25 years ago. Thus far, one aptamer has been approved for clinical use, and numerous others are in preclinical or clinical development. This review presents a short history of aptamers and SELEX, describes their pharmacological development and optimization, and reviews potential treatment of diseases including visual disorders, thrombosis, and cancer.

Authors
Nimjee, SM; White, RR; Becker, RC; Sullenger, BA
MLA Citation
Nimjee, SM, White, RR, Becker, RC, and Sullenger, BA. "Aptamers as Therapeutics." Annual review of pharmacology and toxicology 57 (January 2017): 61-79.
PMID
28061688
Source
epmc
Published In
Annual Review of Pharmacology and Toxicology
Volume
57
Publish Date
2017
Start Page
61
End Page
79
DOI
10.1146/annurev-pharmtox-010716-104558

RNA Aptamer Against FXa Synergizes with FXa Catalytic Site Inhibitors to Effectively and Reversibly Anticoagulate Blood in an Ex Vivo Oxygenator Circuit

Authors
Gunaratne, R; Frederiksen, J; Thalji, NK; Ho, MD; Arepally, GM; Camire, RM; Sullenger, BA
MLA Citation
Gunaratne, R, Frederiksen, J, Thalji, NK, Ho, MD, Arepally, GM, Camire, RM, and Sullenger, BA. "RNA Aptamer Against FXa Synergizes with FXa Catalytic Site Inhibitors to Effectively and Reversibly Anticoagulate Blood in an Ex Vivo Oxygenator Circuit." December 2, 2016.
Source
wos-lite
Published In
Blood
Volume
128
Issue
22
Publish Date
2016

Cell-Free DNA Is Elevated after Acute Arterial Injury in Infants

Authors
Kim, J; Naqvi, I; Nag, UP; Leraas, HJ; Otto, JC; Borst, AJ; Hill, KD; Fleming, GA; Sullenger, BA; Tracy, ET
MLA Citation
Kim, J, Naqvi, I, Nag, UP, Leraas, HJ, Otto, JC, Borst, AJ, Hill, KD, Fleming, GA, Sullenger, BA, and Tracy, ET. "Cell-Free DNA Is Elevated after Acute Arterial Injury in Infants." December 2, 2016.
Source
wos-lite
Published In
Blood
Volume
128
Issue
22
Publish Date
2016

Aptamer Mediated Inhibition of Protein S

Authors
Plautz, WE; Layzer, J; Sullenger, BA; Majumder, R
MLA Citation
Plautz, WE, Layzer, J, Sullenger, BA, and Majumder, R. "Aptamer Mediated Inhibition of Protein S." December 2, 2016.
Source
wos-lite
Published In
Blood
Volume
128
Issue
22
Publish Date
2016

Polycationic Nanofibers for Nucleic Acid Scavenging.

Dying cells release nucleic acids (NA) and NA-containing complexes that activate inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation frequently encountered in autoimmune and inflammatory diseases. In this study, grafting of cationic polymers onto a nanofibrous mesh enabled local scavenging of negatively charged pro-inflammatory molecules in the extracellular space. Nucleic acid scavenging nanofibers (NASFs) formed from poly(styrene-alt-maleic anhydride) conjugated with 1.8 kDa bPEI resulted in nanofibers of diameters 486 ± 9 nm. NASFs inhibited the NF-κB response stimulated by the negatively charged agonists, CpG and poly(I:C), in Ramos-blue cells but not Pam3CSK4, a nonanionic agonist. Moreover, NASFs significantly impeded NF-κB activation in cells stimulated with damage-associated molecular pattern molecules (DAMPs) released from doxorubicin killed cancer cells. In vivo application of NASFs to open wounds demonstrated nucleic acid scavenging in wounds of diabetic mice infected with Pseudomonas aeruginosa, suggesting the in vivo efficacy of NASFs. This simple technique of generating NASF results in effective localized anti-inflammation in vitro and local nucleic acid scavenging in vivo.

Authors
Jackman, JG; Juwarker, H; Poveromo, LP; Levinson, H; Leong, KW; Sullenger, BA
MLA Citation
Jackman, JG, Juwarker, H, Poveromo, LP, Levinson, H, Leong, KW, and Sullenger, BA. "Polycationic Nanofibers for Nucleic Acid Scavenging." Biomacromolecules 17.11 (November 2016): 3706-3713.
PMID
27741396
Source
epmc
Published In
Biomacromolecules
Volume
17
Issue
11
Publish Date
2016
Start Page
3706
End Page
3713
DOI
10.1021/acs.biomac.6b01236

Toll-Like Receptor Signaling as a Prognostic Tool in Trauma Patients

Authors
Darrabie, MD; Lee, J; Cheeseman, J; Limkakeng, AT; Vaslef, SN; Sullenger, BA; Kirk, AD
MLA Citation
Darrabie, MD, Lee, J, Cheeseman, J, Limkakeng, AT, Vaslef, SN, Sullenger, BA, and Kirk, AD. "Toll-Like Receptor Signaling as a Prognostic Tool in Trauma Patients." October 2016.
Source
wos-lite
Published In
Journal of The American College of Surgeons
Volume
223
Issue
4
Publish Date
2016
Start Page
S159
End Page
S160

Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

Authors
Kahsai, AW; Wisler, JW; Lee, J; Ahn, S; Cahill Iii, TJ; Dennison, SM; Staus, DP; Thomsen, ARB; Anasti, KM; Pani, B; Wingler, LM; Desai, H; Bompiani, KM; Strachan, RT; Qin, X; Alam, SM; Sullenger, BA; Lefkowitz, RJ
MLA Citation
Kahsai, AW, Wisler, JW, Lee, J, Ahn, S, Cahill Iii, TJ, Dennison, SM, Staus, DP, Thomsen, ARB, Anasti, KM, Pani, B, Wingler, LM, Desai, H, Bompiani, KM, Strachan, RT, Qin, X, Alam, SM, Sullenger, BA, and Lefkowitz, RJ. "Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor." Nature chemical biology 12.9 (September 2016): 709-716.
PMID
27398998
Source
epmc
Published In
Nature Chemical Biology
Volume
12
Issue
9
Publish Date
2016
Start Page
709
End Page
716
DOI
10.1038/nchembio.2126

Differential effects of toll-like receptor stimulation on mRNA-driven myogenic conversion of human and mouse fibroblasts.

Transfection with in vitro transcribed mRNAs is a safe and effective tool to convert somatic cells to any cell type of interest. One caveat of mRNA transfection is that mRNAs are recognized by multiple RNA-sensing toll like receptors (TLRs). These TLRs can both promote and inhibit cellular reprogramming. We demonstrated that mRNA transfection stimulated TLR3 and TLR7 and induced cytotoxicity and IFN-β expression in human and mouse fibroblasts. Furthermore, mRNA transfection induced paracrine inhibition of repeated mRNA transfection through type I IFNs. Modified mRNAs (mmRNAs) containing pseudouridine and 5-methycytosine reduced TLR stimulation, cytotoxicity and IFN-β expression in fibroblasts. Repeated liposomal transfection with MyoD mmRNAs significantly enhanced myogenic conversion of human and mouse fibroblasts compared with repeated transfection with MyoD mRNAs. Interestingly, electroporation of mRNAs and mmRNAs completely abrogated cytotoxicity and IFN-β expression and also abolished myogenic conversion of fibroblasts. At a low concentration, TLR7/8 agonist R848 enhanced MyoD mmRNA-driven conversion of human fibroblasts into skeletal muscle cells, whereas high concentrations of R848 inhibited myogenic conversion of fibroblasts. Our study suggests that deliberate control of TLR signaling is a key factor in the success of mRNA-driven cellular reprogramming.

Authors
Lee, J; Xu, L; Gibson, TM; Gersbach, CA; Sullenger, BA
MLA Citation
Lee, J, Xu, L, Gibson, TM, Gersbach, CA, and Sullenger, BA. "Differential effects of toll-like receptor stimulation on mRNA-driven myogenic conversion of human and mouse fibroblasts." Biochemical and biophysical research communications 478.3 (September 2016): 1484-1490.
PMID
27586271
Source
epmc
Published In
Biochemical and Biophysical Research Communications
Volume
478
Issue
3
Publish Date
2016
Start Page
1484
End Page
1490
DOI
10.1016/j.bbrc.2016.08.159

Scavenging nucleic acid debris to combat autoimmunity and infectious disease.

Nucleic acid-containing debris released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Inappropriate activation of the innate immune response can engender pathological inflammation and autoimmune disease. To combat such diseases, major efforts have been made to therapeutically target the pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or the downstream effector molecules they engender, to limit inflammation. Unfortunately, such strategies can limit the ability of the immune system to combat infection. Previously, we demonstrated that nucleic acid-binding polymers can act as molecular scavengers and limit the ability of artificial nucleic acid ligands to activate PRRs. Herein, we demonstrate that nucleic acid scavengers (NASs) can limit pathological inflammation and nucleic acid-associated autoimmunity in lupus-prone mice. Moreover, we observe that such NASs do not limit an animal's ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. These results indicate that molecules that scavenge extracellular nucleic acid debris represent potentially safer agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

Authors
Holl, EK; Shumansky, KL; Borst, LB; Burnette, AD; Sample, CJ; Ramsburg, EA; Sullenger, BA
MLA Citation
Holl, EK, Shumansky, KL, Borst, LB, Burnette, AD, Sample, CJ, Ramsburg, EA, and Sullenger, BA. "Scavenging nucleic acid debris to combat autoimmunity and infectious disease." Proceedings of the National Academy of Sciences of the United States of America 113.35 (August 15, 2016): 9728-9733.
PMID
27528673
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
35
Publish Date
2016
Start Page
9728
End Page
9733
DOI
10.1073/pnas.1607011113

2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors.

Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-β expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.

Authors
Lee, Y; Urban, JH; Xu, L; Sullenger, BA; Lee, J
MLA Citation
Lee, Y, Urban, JH, Xu, L, Sullenger, BA, and Lee, J. "2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors." Nucleic acid therapeutics 26.3 (June 2016): 173-182.
PMID
26789413
Source
epmc
Published In
Nucleic Acid Therapeutics
Volume
26
Issue
3
Publish Date
2016
Start Page
173
End Page
182
DOI
10.1089/nat.2015.0575

Translation and Clinical Development of Antithrombotic Aptamers.

Thrombosis is a necessary physiological process to protect the body from uncontrolled bleeding. Pathological thrombus formation can lead to devastating clinical events including heart attack, stroke, deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. Numerous drugs have been developed to inhibit thrombosis. These have been targeted to coagulation factors along with proteins and receptors that activate platelets. While these drugs are effective at preventing blood clotting, their major side effect is inadvertent hemorrhage that can result in significant morbidity and mortality. There exists a need for anticoagulants that are not only effective at preventing thrombosis but can also be readily reversed. Aptamers offer a potential solution, representing a new class of drug agents that can be isolated to any protein and where antidote oligonucleotides can be designed based on the sequence of the aptamer. We present a summary of the anticoagulant and antithrombotic aptamers that have been identified and their stage of development and comment on the future of aptamer-based drug development to treat thrombosis.

Authors
Nimjee, SM; Povsic, TJ; Sullenger, BA; Becker, RC
MLA Citation
Nimjee, SM, Povsic, TJ, Sullenger, BA, and Becker, RC. "Translation and Clinical Development of Antithrombotic Aptamers." Nucleic acid therapeutics 26.3 (June 2016): 147-155.
PMID
26882082
Source
epmc
Published In
Nucleic Acid Therapeutics
Volume
26
Issue
3
Publish Date
2016
Start Page
147
End Page
155
DOI
10.1089/nat.2015.0581

Aptamers Coming of Age at Twenty-Five.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Aptamers Coming of Age at Twenty-Five." Nucleic acid therapeutics 26.3 (June 2016): 119-.
PMID
27258064
Source
epmc
Published In
Nucleic Acid Therapeutics
Volume
26
Issue
3
Publish Date
2016
Start Page
119
DOI
10.1089/nat.2016.29001.sul

From the RNA world to the clinic.

The study of RNA has continually emphasized the structural and functional versatility of RNA molecules. This versatility has inspired translational and clinical researchers to explore the utility of RNA-based therapeutic agents for a wide variety of medical applications. Several RNA therapeutics, with diverse modes of action, are being evaluated in large late-stage clinical trials, and many more are in early clinical development. Hundreds of patients are enrolled in large trials testing messenger RNAs to combat cancer, small interfering RNAs to treat renal and hepatic disorders, and aptamers to combat ocular and cardiovascular disease. Results from these studies are generating considerable interest among the biomedical community and the public and will be important for the future development of this emerging class of therapeutic agents.

Authors
Sullenger, BA; Nair, S
MLA Citation
Sullenger, BA, and Nair, S. "From the RNA world to the clinic." Science (New York, N.Y.) 352.6292 (June 2016): 1417-1420. (Review)
PMID
27313039
Source
epmc
Published In
Science
Volume
352
Issue
6292
Publish Date
2016
Start Page
1417
End Page
1420
DOI
10.1126/science.aad8709

Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer.

Authors
Ganson, NJ; Povsic, TJ; Sullenger, BA; Alexander, JH; Zelenkofske, SL; Sailstad, JM; Rusconi, CP; Hershfield, MS
MLA Citation
Ganson, NJ, Povsic, TJ, Sullenger, BA, Alexander, JH, Zelenkofske, SL, Sailstad, JM, Rusconi, CP, and Hershfield, MS. "Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer." The Journal of allergy and clinical immunology 137.5 (May 2016): 1610-1613.e7.
PMID
26688515
Source
epmc
Published In
Journal of Allergy and Clinical Immunology
Volume
137
Issue
5
Publish Date
2016
Start Page
1610
End Page
1613.e7
DOI
10.1016/j.jaci.2015.10.034

In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9.

The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs) that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery.

Authors
Mi, J; Ray, P; Liu, J; Kuan, C-T; Xu, J; Hsu, D; Sullenger, BA; White, RR; Clary, BM
MLA Citation
Mi, J, Ray, P, Liu, J, Kuan, C-T, Xu, J, Hsu, D, Sullenger, BA, White, RR, and Clary, BM. "In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9." Molecular Therapy. Nucleic Acids 5 (April 26, 2016): e315-.
PMID
27115840
Source
epmc
Published In
Molecular Therapy - Nucleic Acids
Volume
5
Publish Date
2016
Start Page
e315
DOI
10.1038/mtna.2016.27

Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent

Authors
Freedman, JA; Robinson, TJ; LaCroix, B; Patierno, BM; George, DJ; Sullenger, BA; Patierno, SR
MLA Citation
Freedman, JA, Robinson, TJ, LaCroix, B, Patierno, BM, George, DJ, Sullenger, BA, and Patierno, SR. "Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent." March 2016.
Source
crossref
Published In
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology
Volume
25
Issue
3 Supplement
Publish Date
2016
Start Page
B18
End Page
B18
DOI
10.1158/1538-7755.DISP15-B18

Targeting Two Coagulation Cascade Proteases with a Bivalent Aptamer Yields a Potent and Antidote-Controllable Anticoagulant.

Potent and rapid-onset anticoagulation is required for several clinical settings, including cardiopulmonary bypass surgery. In addition, because anticoagulation is associated with increased bleeding following surgery, the ability to rapidly reverse such robust anticoagulation is also important. Previously, we observed that no single aptamer was as potent as heparin for anticoagulating blood. However, we discovered that combinations of two aptamers were as potent as heparin. Herein, we sought to combine two individual anticoagulant aptamers into a single bivalent RNA molecule in an effort to generate a single molecule that retained the potent anticoagulant activity of the combination of individual aptamers. We created four bivalent aptamers that can inhibit Factor X/Xa and prothrombin/thrombin and anticoagulate plasma, as well as the combination of individual aptamers. Detailed characterization of the shortest bivalent aptamer indicates that each aptamer retains full binding and functional activity when presented in the bivalent context. Finally, reversal of this bivalent aptamer with a single antidote was explored, and anticoagulant activity could be rapidly turned off in a dose-dependent manner. These studies demonstrate that bivalent anticoagulant aptamers represent a novel and potent approach to actively and reversibly control coagulation.

Authors
Soule, EE; Bompiani, KM; Woodruff, RS; Sullenger, BA
MLA Citation
Soule, EE, Bompiani, KM, Woodruff, RS, and Sullenger, BA. "Targeting Two Coagulation Cascade Proteases with a Bivalent Aptamer Yields a Potent and Antidote-Controllable Anticoagulant." Nucleic acid therapeutics 26.1 (February 2016): 1-9.
PMID
26584417
Source
epmc
Published In
Nucleic Acid Therapeutics
Volume
26
Issue
1
Publish Date
2016
Start Page
1
End Page
9
DOI
10.1089/nat.2015.0565

RNA-Mediated Reprogramming of Primary Adult Human Dermal Fibroblasts into c-kit(+) Cardiac Progenitor Cells.

Cardiovascular disease is the leading cause of death in the United States. Heart failure is a common, costly, and potentially fatal condition that is inadequately managed by pharmaceuticals. Cardiac repair therapies are promising alternative options. A potential cardiac repair therapy involves reprogramming human fibroblasts toward an induced cardiac progenitor-like state. We developed a clinically useful and safer reprogramming method by nonintegrative delivery of a cocktail of cardiac transcription factor-encoding mRNAs into autologous human dermal fibroblasts obtained from skin biopsies. Using this method, adult and neonatal dermal fibroblasts were reprogrammed into cardiac progenitor cells (CPCs) that expressed c-kit, Isl-1, and Nkx2.5. Furthermore, these reprogrammed CPCs differentiated into cardiomyocytes (CMs) in vitro as judged by increased expression of cardiac troponin T, α-sarcomeric actinin, RyR2, and SERCA2 and displayed enhanced caffeine-sensitive calcium release. The ability to reprogram patient-derived dermal fibroblasts into c-kit(+) CPCs and differentiate them into functional CMs provides clinicians with a potential new source of CPCs for cardiac repair from a renewable source and an alternative therapy in the treatment of heart failure.

Authors
Pratico, ED; Feger, BJ; Watson, MJ; Sullenger, BA; Bowles, DE; Milano, CA; Nair, SK
MLA Citation
Pratico, ED, Feger, BJ, Watson, MJ, Sullenger, BA, Bowles, DE, Milano, CA, and Nair, SK. "RNA-Mediated Reprogramming of Primary Adult Human Dermal Fibroblasts into c-kit(+) Cardiac Progenitor Cells." Stem cells and development 24.22 (November 2015): 2622-2633.
PMID
26176491
Source
epmc
Published In
Stem Cells and Development
Volume
24
Issue
22
Publish Date
2015
Start Page
2622
End Page
2633
DOI
10.1089/scd.2015.0073

Modulation of the Coagulation Cascade Using Aptamers.

As a novel class of therapeutics, aptamers, or nucleic acid ligands, have garnered clinical interest because of the ease of isolating a highly specific aptamer against a wide range of targets, their chemical flexibility and synthesis, and their inherent ability to have their function reversed. The following review details the development and molecular mechanisms of aptamers targeting specific proteases in the coagulation cascade. The ability of these anticoagulant aptamers to bind to and inhibit exosite function rather than binding within the active site highlights the importance of exosites in blocking protein function. As both exosite inhibitors and reversible agents, the use of aptamers is a promising strategy for future therapeutics.

Authors
Woodruff, RS; Sullenger, BA
MLA Citation
Woodruff, RS, and Sullenger, BA. "Modulation of the Coagulation Cascade Using Aptamers." Arteriosclerosis, thrombosis, and vascular biology 35.10 (October 2015): 2083-2091. (Review)
PMID
26315404
Source
epmc
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
35
Issue
10
Publish Date
2015
Start Page
2083
End Page
2091
DOI
10.1161/atvbaha.115.300131

A novel aptamer targeting agent for prostate cancer

Authors
Gray, BP; Kelly, L; Levy, M; Sullenger, BA
MLA Citation
Gray, BP, Kelly, L, Levy, M, and Sullenger, BA. "A novel aptamer targeting agent for prostate cancer." August 1, 2015.
Source
wos-lite
Published In
Cancer Research
Volume
75
Publish Date
2015
DOI
10.1158/1538-7445.AM2015-5523

Development and characterization of an RNA aptamer targeting FV/FVA

Authors
Soule, E; Krishnaswamy, S; Sullenger, BA
MLA Citation
Soule, E, Krishnaswamy, S, and Sullenger, BA. "Development and characterization of an RNA aptamer targeting FV/FVA." June 2015.
Source
wos-lite
Published In
Journal of Thrombosis and Haemostasis
Volume
13
Publish Date
2015
Start Page
54
End Page
55

Identification and Characterization of a B Cell Aptamer That Targets Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Myelogenous Leukemia (CML)

Authors
Pratico, ED; Nair, SK; Sullenger, BA
MLA Citation
Pratico, ED, Nair, SK, and Sullenger, BA. "Identification and Characterization of a B Cell Aptamer That Targets Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Myelogenous Leukemia (CML)." May 2015.
Source
wos-lite
Published In
Molecular Therapy
Volume
23
Publish Date
2015
Start Page
S29
End Page
S29

A Novel Aptamer Targeting Agent for Prostate Cancer

Authors
Gray, BP; Kelly, L; Levy, M; Sullenger, BA
MLA Citation
Gray, BP, Kelly, L, Levy, M, and Sullenger, BA. "A Novel Aptamer Targeting Agent for Prostate Cancer." May 2015.
Source
wos-lite
Published In
Molecular Therapy
Volume
23
Publish Date
2015
Start Page
S28
End Page
S29

Laboratory Assessment of Anti-Coagulant Properties of a Von Willebrand Factor Targeted Aptamer

Authors
Lewis, DA; Nimjee, SM; Pitoc, GA; Khan, S; Becker, RC; Sullenger, BA; Ortel, TL
MLA Citation
Lewis, DA, Nimjee, SM, Pitoc, GA, Khan, S, Becker, RC, Sullenger, BA, and Ortel, TL. "Laboratory Assessment of Anti-Coagulant Properties of a Von Willebrand Factor Targeted Aptamer." December 6, 2014.
Source
wos-lite
Published In
Blood
Volume
124
Issue
21
Publish Date
2014

X-Ray Structure of an Anticoagulant RNA Aptamer Bound to Factor Xa. Structural Basis for Its Ability to Disrupt Interactions Between Xa and Va within Prothrombinase

Authors
Kumar, S; Sullenger, BA; Stayrook, S; Krishnaswamy, S
MLA Citation
Kumar, S, Sullenger, BA, Stayrook, S, and Krishnaswamy, S. "X-Ray Structure of an Anticoagulant RNA Aptamer Bound to Factor Xa. Structural Basis for Its Ability to Disrupt Interactions Between Xa and Va within Prothrombinase." December 6, 2014.
Source
wos-lite
Published In
Blood
Volume
124
Issue
21
Publish Date
2014

Development of immunocytotoxic RNA therapeutics for human melanoma

Authors
Lee, J; Lee, Y; Sullenger, BA
MLA Citation
Lee, J, Lee, Y, and Sullenger, BA. "Development of immunocytotoxic RNA therapeutics for human melanoma." October 1, 2014.
Source
wos-lite
Published In
Cancer Research
Volume
74
Issue
19
Publish Date
2014
DOI
10.1158/1538-7445.AM2014-2639

The nucleic acid scavenger polyamidoamine third-generation dendrimer inhibits fibroblast activation and granulation tissue contraction.

Pathologic cutaneous scarring affects over 40 million people worldwide and costs billions of dollars annually. Understanding mechanisms of fibroblast activation and granulation tissue contraction is the first step toward preventing pathologic scarring. The authors hypothesize that nucleic acids increase fibroblast activation and cause granulation tissue contraction and that sequestration of nucleic acids by application of a nucleic acid scavenger dendrimer, polyamidoamine third-generation dendrimer, will decrease pathologic scarring.In vitro experiments were performed to assess the effect of nucleic acids on pathologic scar-associated fibroblast activity. The effect of nucleic acids on cytokine production and migration on mouse fibroblasts was evaluated. Immunofluorescence microscopy was used to determine the effect of nucleic acids on the differentiation of human primary fibroblasts into myofibroblasts. Using a murine model, the effect of polyamidoamine third-generation dendrimer on granulation tissue contraction was evaluated by gross and histologic parameters.Mouse fibroblasts stimulated with nucleic acids had increased cytokine production (i.e., transforming growth factor-β, monocyte chemotactic protein 1, interleukin-10, tumor necrosis factor-α, and interferon-γ), migration, and differentiation into myofibroblasts. Polyamidoamine third-generation dendrimer blocked cytokine production, migration, and differentiation into myofibroblasts. Using a murine model of granulation tissue contraction, polyamidoamine third-generation dendrimer decreased wound contraction and angiogenesis. Collagen deposition in polyamidoamine third-generation dendrimer-treated tissues was aligned more randomly and whorl-like compared with control tissue.The data demonstrate that nucleic acid-stimulated fibroblast activation and granulation tissue contraction are blocked by polyamidoamine third-generation dendrimer. Sequestration of pathogen-associated molecular patterns may be an approach for preventing pathologic scarring.

Authors
Holl, EK; Bond, JE; Selim, MA; Ehanire, T; Sullenger, B; Levinson, H
MLA Citation
Holl, EK, Bond, JE, Selim, MA, Ehanire, T, Sullenger, B, and Levinson, H. "The nucleic acid scavenger polyamidoamine third-generation dendrimer inhibits fibroblast activation and granulation tissue contraction." Plastic and reconstructive surgery 134.3 (September 2014): 420e-433e.
PMID
25158719
Source
epmc
Published In
Plastic and Reconstructive Surgery
Volume
134
Issue
3
Publish Date
2014
Start Page
420e
End Page
433e
DOI
10.1097/prs.0000000000000471

Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation.

Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends.

Authors
Bompiani, KM; Lohrmann, JL; Pitoc, GA; Frederiksen, JW; Mackensen, GB; Sullenger, BA
MLA Citation
Bompiani, KM, Lohrmann, JL, Pitoc, GA, Frederiksen, JW, Mackensen, GB, and Sullenger, BA. "Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation." Chemistry & biology 21.8 (August 2014): 935-944.
PMID
25065530
Source
epmc
Published In
Chemistry & Biology
Volume
21
Issue
8
Publish Date
2014
Start Page
935
End Page
944
DOI
10.1016/j.chembiol.2014.05.016

Nucleic-Acid Binding Polymers as Anti-Inflammatory Agents

Authors
Juwarker, HA; Holl, EK; Lao, Y-H; Leong, KW; Sullenger, BA
MLA Citation
Juwarker, HA, Holl, EK, Lao, Y-H, Leong, KW, and Sullenger, BA. "Nucleic-Acid Binding Polymers as Anti-Inflammatory Agents." May 2014.
Source
wos-lite
Published In
Molecular Therapy
Volume
22
Publish Date
2014
Start Page
S4
End Page
S5

Targeted disruption of β-arrestin 2-mediated signaling pathways by aptamer chimeras leads to inhibition of leukemic cell growth.

β-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis in several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of β-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the β-arrestin 2-targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple β-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as β-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy.An RNA aptamer inhibits β-arrestin 2 activity.Inhibiting β-arrestin 2 impedes multiple tumorigenic pathways simultaneously.The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer.Targeting β-arrestin 2 inhibits tumor progression in CML models and patient samples.

Authors
Kotula, JW; Sun, J; Li, M; Pratico, ED; Fereshteh, MP; Ahrens, DP; Sullenger, BA; Kovacs, JJ
MLA Citation
Kotula, JW, Sun, J, Li, M, Pratico, ED, Fereshteh, MP, Ahrens, DP, Sullenger, BA, and Kovacs, JJ. "Targeted disruption of β-arrestin 2-mediated signaling pathways by aptamer chimeras leads to inhibition of leukemic cell growth." PloS one 9.4 (January 2014): e93441-.
PMID
24736311
Source
epmc
Published In
PloS one
Volume
9
Issue
4
Publish Date
2014
Start Page
e93441
DOI
10.1371/journal.pone.0093441

Further characterization of the target of a potential aptamer biomarker for pancreatic cancer: cyclophilin B and its posttranslational modifications.

Posttranslational modifications on proteins can serve as useful biomarkers for disease. However, their discovery and detection in biological fluids is challenging. Aptamers are oligonucleotide ligands that demonstrate high affinity toward their target proteins and can discriminate closely related proteins with superb specificity. Previously, we generated a cyclophilin B aptamer (M9-5) that could discriminate sera from pancreatic cancer patients and healthy volunteers with high specificity and sensitivity. In our present work we further characterize the aptamer and the target protein, cyclophilin B, and demonstrate that the aptamer could discriminate between cyclophilin B expressed in human cells versus bacteria. Using mass-spectrometric analysis, we discovered post-translational modifications on cyclophilin B that might be responsible for the M9-5 selectivity. The ability to distinguish between forms of the same protein with differing post-translational modifications is an important advantage of aptamers as tools for identification and detection of biomarkers.

Authors
Ray, P; Sullenger, BA; White, RR
MLA Citation
Ray, P, Sullenger, BA, and White, RR. "Further characterization of the target of a potential aptamer biomarker for pancreatic cancer: cyclophilin B and its posttranslational modifications." Nucleic Acid Ther 23.6 (December 2013): 435-442.
PMID
24152208
Source
pubmed
Published In
Nucleic Acid Therapeutics
Volume
23
Issue
6
Publish Date
2013
Start Page
435
End Page
442
DOI
10.1089/nat.2013.0439

Aptamer inhibition of an exosite of Factor (F)Xa or thrombin synergizes with non-aptamer inhibition of the catalytic site of FXa or thrombin, respectively

Authors
Frederiksen, JW; Lohrmann, J; Bompiani, KM; Smock-Woodruff, RBS; Gunaratne, RBA; Sullenger, BA
MLA Citation
Frederiksen, JW, Lohrmann, J, Bompiani, KM, Smock-Woodruff, RBS, Gunaratne, RBA, and Sullenger, BA. "Aptamer inhibition of an exosite of Factor (F)Xa or thrombin synergizes with non-aptamer inhibition of the catalytic site of FXa or thrombin, respectively." July 2013.
Source
wos-lite
Published In
Journal of Thrombosis and Haemostasis
Volume
11
Publish Date
2013
Start Page
123
End Page
124

Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer.

BACKGROUND: Exposure of the plasma protein factor XII (FXII) to an anionic surface generates activated FXII that not only triggers the intrinsic pathway of blood coagulation through the activation of FXI but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of FXII are not associated with excessive bleeding, thrombosis models in factor-deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, FXII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage. OBJECTIVES: Using an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease-resistant RNA aptamer that binds specifically to FXII and directly inhibits FXII coagulant function. METHODS AND RESULTS: We describe the isolation and characterization of a high-affinity RNA aptamer targeting FXII/activated FXII (FXIIa) that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of FXII, as well as inhibiting intrinsic pathway activation (FXI activation). However, the aptamer does not affect the FXIIa-mediated activation of the proinflammatory kallikrein-kinin system (plasma kallikrein activation). CONCLUSIONS: We have generated a specific and potent FXII/FXIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation.

Authors
Woodruff, RS; Xu, Y; Layzer, J; Wu, W; Ogletree, ML; Sullenger, BA
MLA Citation
Woodruff, RS, Xu, Y, Layzer, J, Wu, W, Ogletree, ML, and Sullenger, BA. "Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer." J Thromb Haemost 11.7 (July 2013): 1364-1373.
PMID
23692437
Source
pubmed
Published In
Journal of Thrombosis and Haemostasis
Volume
11
Issue
7
Publish Date
2013
Start Page
1364
End Page
1373
DOI
10.1111/jth.12302

Identification and characterization of an agonistic aptamer against the T cell costimulatory receptor, OX40.

Induction of an effective immune response that can target and eliminate malignant cells or virus-infected cells requires the stimulation of antigen-specific effector T cells. A productive and long-lasting memory response requires 2 signals: a specific signal provided by antigen recognition through engagement of the T cell receptor and a secondary signal via engagement of costimulatory molecules (such as OX40) on these newly activated T cells. The OX40-OX40-ligand interaction is critical for the generation of productive effector and memory T cell functions. Thus agonistic antibodies that stimulate OX40 on activated T cells have been used as adjuvants to augment immune responses. We previously demonstrated that an aptamer modified to stimulate murine OX40 enhanced vaccine-mediated immune responses in a murine melanoma model. In this study, we describe the development of an agonistic aptamer that targets human OX40 (hOX40). This hOX40 aptamer was isolated using systematic evolution of ligands by exponential enrichment and binds the target purified protein with high affinity [dissociation constants (K(d))<10 nM]. Moreover, the hOX40 aptamer-streptavidin complex has an apparent binding affinity of ~50 nM for hOX40 on activated T cells as determined by flow cytometry and specifically binds activated human T cells. A multivalent version of the aptamer, but not a mutant version of the aptamer, was able to stimulate OX40 on T cells and enhance cell proliferation and interferon-gamma production. Future studies will assess the therapeutic potential of hOX40 aptamers for ex vivo stimulation of antigen specific T cells in conjunction with dendritic cell-based vaccines for adoptive cellular therapy.

Authors
Pratico, ED; Sullenger, BA; Nair, SK
MLA Citation
Pratico, ED, Sullenger, BA, and Nair, SK. "Identification and characterization of an agonistic aptamer against the T cell costimulatory receptor, OX40." Nucleic Acid Ther 23.1 (February 2013): 35-43.
PMID
23113766
Source
pubmed
Published In
Nucleic Acid Therapeutics
Volume
23
Issue
1
Publish Date
2013
Start Page
35
End Page
43
DOI
10.1089/nat.2012.0388

The effect of surface contact activation and temperature on plasma coagulation with an RNA aptamer directed against factor IXa.

The anticoagulant properties of a novel RNA aptamer that binds FIXa depend collectively on the intensity of surface contact activation of human blood plasma, aptamer concentration, and its binding affinity for FIXa. Accordingly, anticoagulation efficiency of plasma containing any particular aptamer concentration is low when coagulation is strongly activated by hydrophilic surfaces compared to the anticoagulation efficiency in plasma that is weakly activated by hydrophobic surfaces. Anticoagulation efficiency is lower at hypothermic temperatures possibly because aptamer-FIXa binding decreases with decreasing temperatures. Experimental results demonstrating these trends are qualitatively interpreted in the context of a previously established model of anticoagulation efficiency of thrombin-binding DNA aptamers that exhibit anticoagulation properties similar to the FIXa aptamer. In principle, FIXa aptamer anticoagulants should be more efficient and therefore more clinically useful than thrombin-binding aptamers because aptamer binding to FIXa competes only with FX that is at much lower blood concentration than fibrinogen (FI) that competes with thrombin-binding aptamers. Our findings may have translatable relevance in the application of aptamer anticoagulants for clinical conditions in which blood is in direct contact with non-biological surfaces such as those encountered in cardiopulmonary bypass circuits.

Authors
Krishnan, A; Vogler, EA; Sullenger, BA; Becker, RC
MLA Citation
Krishnan, A, Vogler, EA, Sullenger, BA, and Becker, RC. "The effect of surface contact activation and temperature on plasma coagulation with an RNA aptamer directed against factor IXa." J Thromb Thrombolysis 35.1 (January 2013): 48-56.
PMID
23054460
Source
pubmed
Published In
Journal of Thrombosis and Thrombolysis
Volume
35
Issue
1
Publish Date
2013
Start Page
48
End Page
56
DOI
10.1007/s11239-012-0778-7

Nucleic acid scavenging polymers inhibit extracellular DNA-mediated innate immune activation without inhibiting anti-viral responses.

Toll-like receptor (TLR) family members, 3, 7 and 9 are key components in initiation and progression of autoimmune disorders such as systemic lupus erythematosus (SLE). These TLRs are often referred to as nucleic acid-sensing TLRs based on their ability to recognize DNAs or RNAs produced by pathogens or damaged cells. During autoimmune disease progression these receptors recognize self nucleic acids as well as self nucleic acid-containing complexes and contribute to inflammatory cytokine production and subsequent enhancement of serum autoantibody levels. We have recently discovered that nucleic-acid scavenging polymers (NASPs) can neutralize the proinflammatory effects of nucleic acids. Here, we begin to explore what effects such NASPs have on normal immune function. We show that such NASPs can inhibit TLR activation without affecting nucleic acid-independent T cell activation. Moreover, we observe that stimulation of immune cells by encapsulated nucleic acids, such as those found in viral particles, is unaffected by NASPs. Thus NASPs only limit the activation of the immune system by accessible extra-cellular nucleic acid and do not engender non-specific immune suppression. These important findings suggest that NASPs represent a new approach toward anti-inflammatory drug development as these agents can potentially be utilized to block overt autoimmune disorders and inflammation while allowing normal immune responses to occur.

Authors
Holl, EK; Shumansky, KL; Pitoc, G; Ramsburg, E; Sullenger, BA
MLA Citation
Holl, EK, Shumansky, KL, Pitoc, G, Ramsburg, E, and Sullenger, BA. "Nucleic acid scavenging polymers inhibit extracellular DNA-mediated innate immune activation without inhibiting anti-viral responses." PloS one 8.7 (January 2013): e69413-.
PMID
23936008
Source
epmc
Published In
PloS one
Volume
8
Issue
7
Publish Date
2013
Start Page
e69413
DOI
10.1371/journal.pone.0069413

Aptamer-mediated delivery of chemotherapy to pancreatic cancer cells.

Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type-specific therapy can be adapted to other targets and to other types of therapeutic cargo.

Authors
Ray, P; Cheek, MA; Sharaf, ML; Li, N; Ellington, AD; Sullenger, BA; Shaw, BR; White, RR
MLA Citation
Ray, P, Cheek, MA, Sharaf, ML, Li, N, Ellington, AD, Sullenger, BA, Shaw, BR, and White, RR. "Aptamer-mediated delivery of chemotherapy to pancreatic cancer cells." Nucleic Acid Ther 22.5 (October 2012): 295-305.
PMID
23030589
Source
pubmed
Published In
Nucleic Acid Therapeutics
Volume
22
Issue
5
Publish Date
2012
Start Page
295
End Page
305
DOI
10.1089/nat.2012.0353

Nucleic acid scavengers inhibit thrombosis without increasing bleeding.

Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH(2)(CH(2))(2)NH(2)](G = 3);dendri PAMAM(NH(2))(32) (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.

Authors
Jain, S; Pitoc, GA; Holl, EK; Zhang, Y; Borst, L; Leong, KW; Lee, J; Sullenger, BA
MLA Citation
Jain, S, Pitoc, GA, Holl, EK, Zhang, Y, Borst, L, Leong, KW, Lee, J, and Sullenger, BA. "Nucleic acid scavengers inhibit thrombosis without increasing bleeding." Proceedings of the National Academy of Sciences of the United States of America 109.32 (August 2012): 12938-12943.
PMID
22837404
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
32
Publish Date
2012
Start Page
12938
End Page
12943
DOI
10.1073/pnas.1204928109

Antidote control of aptamer therapeutics: the road to a safer class of drug agents.

Aptamers, or nucleic acid ligands, have gained clinical interest over the past 20 years due to their unique characteristics, which are a combination of the best facets of small molecules and antibodies. The high binding affinity and specificity of aptamers allows for isolation of an artificial ligand for theoretically any therapeutic target of interest. Chemical manipulations of aptamers also allow for fine-tuning of their bioavailability, and antidote control greatly expands their clinical use. Here we review the various methods of antidote control of aptamer therapeutics--matched oligonucleotide antidotes and universal antidotes. We also describe the development, recent progress, and potential future therapeutic applications of these types of aptamer-antidote pairs.

Authors
Bompiani, KM; Woodruff, RS; Becker, RC; Nimjee, SM; Sullenger, BA
MLA Citation
Bompiani, KM, Woodruff, RS, Becker, RC, Nimjee, SM, and Sullenger, BA. "Antidote control of aptamer therapeutics: the road to a safer class of drug agents." Curr Pharm Biotechnol 13.10 (August 2012): 1924-1934. (Review)
PMID
22352726
Source
pubmed
Published In
Current Pharmaceutical Biotechnology
Volume
13
Issue
10
Publish Date
2012
Start Page
1924
End Page
1934

Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures.

UNLABELLED: Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation. FROM THE CLINICAL EDITOR: Thrombin inhibition via DNA aptamers has recently become a possibility. In this study, thrombin-binding DNA aptamers were further optimized by nanoscale organization on DNA nanostructures. The authors have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than that of free aptamer. Reversal of thrombin inhibition was also achieved by single-stranded DNA antidotes, enabling significant control over the coagulation pathway.

Authors
Rangnekar, A; Zhang, AM; Li, SS; Bompiani, KM; Hansen, MN; Gothelf, KV; Sullenger, BA; LaBean, TH
MLA Citation
Rangnekar, A, Zhang, AM, Li, SS, Bompiani, KM, Hansen, MN, Gothelf, KV, Sullenger, BA, and LaBean, TH. "Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures." Nanomedicine 8.5 (July 2012): 673-681.
PMID
21889476
Source
pubmed
Published In
Nanomedicine: Nanotechnology, Biology and Medicine
Volume
8
Issue
5
Publish Date
2012
Start Page
673
End Page
681
DOI
10.1016/j.nano.2011.08.011

Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells.

To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.

Authors
Kotula, JW; Pratico, ED; Ming, X; Nakagawa, O; Juliano, RL; Sullenger, BA
MLA Citation
Kotula, JW, Pratico, ED, Ming, X, Nakagawa, O, Juliano, RL, and Sullenger, BA. "Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells." Nucleic Acid Ther 22.3 (June 2012): 187-195.
PMID
22703281
Source
pubmed
Published In
Nucleic Acid Therapeutics
Volume
22
Issue
3
Publish Date
2012
Start Page
187
End Page
195
DOI
10.1089/nat.2012.0347

A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity.

BACKGROUND: The conversion of prothrombin to thrombin is one of two non-duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti-thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D-14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. OBJECTIVES: We sought to explore the structure of R9D-14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNA(R9D-14T)), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer's anticoagulant activity. METHODS AND RESULTS: RNA(R9D-14T) binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I-mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNA(R9D-14T) significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC-183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNA(R9D-14T) anticoagulation in vitro. CONCLUSIONS: Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA(R9D-14T) may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.

Authors
Bompiani, KM; Monroe, DM; Church, FC; Sullenger, BA
MLA Citation
Bompiani, KM, Monroe, DM, Church, FC, and Sullenger, BA. "A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity." J Thromb Haemost 10.5 (May 2012): 870-880.
PMID
22385910
Source
pubmed
Published In
Journal of Thrombosis and Haemostasis
Volume
10
Issue
5
Publish Date
2012
Start Page
870
End Page
880
DOI
10.1111/j.1538-7836.2012.04679.x

Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker.

Most cases of pancreatic cancer are not diagnosed until they are no longer curable with surgery. Therefore, it is critical to develop a sensitive, preferably noninvasive, method for detecting the disease at an earlier stage. In order to identify biomarkers for pancreatic cancer, we devised an in vitro positive/negative selection strategy to identify RNA ligands (aptamers) that could detect structural differences between the secretomes of pancreatic cancer and non-cancerous cells. Using this molecular recognition approach, we identified an aptamer (M9-5) that differentially bound conditioned media from cancerous and non-cancerous human pancreatic cell lines. This aptamer further discriminated between the sera of pancreatic cancer patients and healthy volunteers with high sensitivity and specificity. We utilized biochemical purification methods and mass-spectrometric analysis to identify the M9-5 target as cyclophilin B (CypB). This molecular recognition-based strategy simultaneously identified CypB as a serum biomarker and generated a new reagent to recognize it in body fluids. Moreover, this approach should be generalizable to other diseases and complementary to traditional approaches that focus on differences in expression level between samples. Finally, we suggest that the aptamer we identified has the potential to serve as a tool for the early detection of pancreatic cancer.

Authors
Ray, P; Rialon-Guevara, KL; Veras, E; Sullenger, BA; White, RR
MLA Citation
Ray, P, Rialon-Guevara, KL, Veras, E, Sullenger, BA, and White, RR. "Comparing human pancreatic cell secretomes by in vitro aptamer selection identifies cyclophilin B as a candidate pancreatic cancer biomarker." J Clin Invest 122.5 (May 2012): 1734-1741.
PMID
22484812
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
122
Issue
5
Publish Date
2012
Start Page
1734
End Page
1741
DOI
10.1172/JCI62385

Rapidly regulating platelet activity in vivo with an antidote controlled platelet inhibitor.

Millions of individuals are prescribed platelet inhibitors, such as aspirin and clopidogrel, to reduce their risk of thrombosis-related clinical events. Unfortunately many platelet inhibitors are contraindicated in surgical settings because of their inherent bleeding risk complicating the treatment of patients who require surgery. We describe the development of a potent antiplatelet agent, an RNA aptamer-termed Ch-9.14-T10 that binds von Willebrand factor (VWF) with high affinity and inhibits thrombosis in a murine carotid artery damage model. As expected, when this potent antiplatelet agent is administered, it greatly increases bleeding from animals that are surgically challenged. To improve this antiplatelet agent's safety profile, we describe the generation of antidotes that can rapidly reverse the activity of Ch-9.14-T10 and limit blood loss from surgically challenged animals. Our work represents the first antidote controllable antiplatelet agent, which could conceivably lead to improved medical management of patients requiring antiplatelet medication who also need surgery.

Authors
Nimjee, SM; Lohrmann, JD; Wang, H; Snyder, DJ; Cummings, TJ; Becker, RC; Oney, S; Sullenger, BA
MLA Citation
Nimjee, SM, Lohrmann, JD, Wang, H, Snyder, DJ, Cummings, TJ, Becker, RC, Oney, S, and Sullenger, BA. "Rapidly regulating platelet activity in vivo with an antidote controlled platelet inhibitor." Mol Ther 20.2 (February 2012): 391-397.
PMID
22086230
Source
pubmed
Published In
Molecular Therapy
Volume
20
Issue
2
Publish Date
2012
Start Page
391
End Page
397
DOI
10.1038/mt.2011.226

Nucleic acid-binding polymers as anti-inflammatory agents: reducing the danger of nuclear attack.

Authors
Pisetsky, DS; Lee, J; Leong, KW; Sullenger, BA
MLA Citation
Pisetsky, DS, Lee, J, Leong, KW, and Sullenger, BA. "Nucleic acid-binding polymers as anti-inflammatory agents: reducing the danger of nuclear attack." Expert Rev Clin Immunol 8.1 (January 2012): 1-3.
PMID
22149331
Source
pubmed
Published In
Expert review of clinical immunology
Volume
8
Issue
1
Publish Date
2012
Start Page
1
End Page
3
DOI
10.1586/eci.11.82

The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

Authors
Stearns, NA; Lee, J; Leong, KW; Sullenger, BA; Pisetsky, DS
MLA Citation
Stearns, NA, Lee, J, Leong, KW, Sullenger, BA, and Pisetsky, DS. "The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers." PLoS One 7.7 (2012): e40862-.
PMID
22808279
Source
pubmed
Published In
PloS one
Volume
7
Issue
7
Publish Date
2012
Start Page
e40862
DOI
10.1371/journal.pone.0040862

Potent anticoagulant aptamer directed against factor IXa blocks macromolecular substrate interaction

An aptamer targeting factor IXa has been evaluated in animal models and several clinical studies as a potential antithombotic therapy. We elucidate the molecular mechanism by which this aptamer acts as an anticoagulant. The aptamer binds tightly to factor IXa and prolongs the clotting time of human plasma. The aptamer completely blocks factor IXa activation of factor X regardless of the presence of factor VIIIa. However, the aptamer does not completely block small synthetic substrate cleavage, although it does slow the rate of cleavage. These data are consistent with the aptamer binding to the catalytic domain of factor IXa in such a way as to block an extended substrate-binding site. Therefore, unlike small molecule inhibitors, aptamers appear to be able to bind surfaces surrounding an active site and thereby sterically interfere with enzyme activity. Thus, aptamers may be useful agents to probe and block substrate-binding sites outside of the active site of an enzyme. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Sullenger, B; Woodruff, R; Monroe, DM
MLA Citation
Sullenger, B, Woodruff, R, and Monroe, DM. "Potent anticoagulant aptamer directed against factor IXa blocks macromolecular substrate interaction." Journal of Biological Chemistry 287.16 (2012): 12779-12786.
PMID
22334679
Source
scival
Published In
The Journal of biological chemistry
Volume
287
Issue
16
Publish Date
2012
Start Page
12779
End Page
12786
DOI
10.1074/jbc.M111.300772

Notch signaling in cancer stem cells.

Subpopulations of cancer cells with stem cell-like characteristics, termed cancer stem cells, have been identified in a wide range of human cancers. Cancer stem cells are defined by their ability to self-renew as well as recapitulate the original heterogeneity of cancer cells in culture and in serial xenotransplants. Not only are cancer stem cells highly tumorigenic, but these cells are implicated in tumor resistance to conventional chemotherapy and radiotherapy, thus highlighting their significance as therapeutic targets. Considerable similarities have been found between cancer stem cells and normal stem cells on their dependence on certain signaling pathways. More specifically, the core stem cell signaling pathways, such as the Wnt, Notch and Hedgehog pathways, also critically regulate the self-renewal and survival of cancer stem cells. While the oncogenic functions of Notch pathway have been well documented, its role in cancer stem cells is just emerging. In this chapter, we will discuss recent advances in cancer stem cell research and highlight the therapeutic potential of targeting Notch in cancer stem cells.

Authors
Wang, J; Sullenger, BA; Rich, JN
MLA Citation
Wang, J, Sullenger, BA, and Rich, JN. "Notch signaling in cancer stem cells." Adv Exp Med Biol 727 (2012): 174-185. (Review)
PMID
22399347
Source
pubmed
Published In
Advances in experimental medicine and biology
Volume
727
Publish Date
2012
Start Page
174
End Page
185
DOI
10.1007/978-1-4614-0899-4_13

Effects of plasminogen activator inhibitor-1-specific RNA aptamers on cell adhesion, motility, and tube formation.

The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. The extracellular matrix protein vitronectin increases at sites of vessel injury and is also present in fibrin clots. Integrins present on the cell surface bind to vitronectin and anchor the cell to the extracellular matrix. However, the binding of PAI-1 to vitronectin prevents this interaction, thereby decreasing both cell adhesion and migration. We previously developed PAI-1-specific RNA aptamers that bind to (or in the vicinity of) the vitronectin binding site of PAI-1. These aptamers prevented cancer cells from detaching from vitronectin in the presence of PAI-1, resulting in an increase in cell adhesion. In the current study, we used in vitro assays to investigate the effects that these aptamers have on human aortic smooth muscle cell (HASMC) and human umbilical vein endothelial cell (HUVEC) migration, adhesion, and proliferation. The PAI-1-specific aptamers (SM20 and WT15) increased attachment of HASMCs and HUVECs to vitronectin in the presence of PAI-1 in a dose-dependent manner. Whereas PAI-1 significantly inhibited cell migration through its interaction with vitronectin, both SM20 and WT15 restored cell migration. The PAI-1 vitronectin binding mutant (PAI-1AK) did not facilitate cell detachment or have an effect on cell migration. The effect on cell proliferation was minimal. Additionally, both SM20 and WT15 promoted tube formation on matrigel that was supplemented with vitronectin, thereby reversing the PAI-1's inhibition of tube formation. Collectively, results from this study show that SM20 and WT15 bind to the PAI-1's vitronectin binding site and interfere with its effect on cell migration, adhesion, and tube formation. By promoting smooth muscle and endothelial cell migration, these aptamers can potentially eliminate the adverse effects of elevated PAI-1 levels in the pathogenesis of vascular disease.

Authors
Brandal, S; Blake, CM; Sullenger, BA; Fortenberry, YM
MLA Citation
Brandal, S, Blake, CM, Sullenger, BA, and Fortenberry, YM. "Effects of plasminogen activator inhibitor-1-specific RNA aptamers on cell adhesion, motility, and tube formation." Nucleic Acid Ther 21.6 (December 2011): 373-381.
PMID
22103403
Source
pubmed
Published In
Nucleic Acid Therapeutics
Volume
21
Issue
6
Publish Date
2011
Start Page
373
End Page
381
DOI
10.1089/nat.2011.0320

AN RNA APTAMER IS A POTENTIAL BIOMARKER FOR PANCREATIC CANCER

Authors
Ray, P; Sullenger, BA; White, RR
MLA Citation
Ray, P, Sullenger, BA, and White, RR. "AN RNA APTAMER IS A POTENTIAL BIOMARKER FOR PANCREATIC CANCER." October 2011.
Source
wos-lite
Published In
Nucleic Acid Therapeutics
Volume
21
Issue
5
Publish Date
2011
Start Page
A40
End Page
A41

Nucleic acid-binding polymers as anti-inflammatory agents.

Dead and dying cells release nucleic acids. These extracellular RNAs and DNAs can be taken up by inflammatory cells and activate multiple nucleic acid-sensing toll-like receptors (TLR3, 7, 8, and 9). The inappropriate activation of these TLRs can engender a variety of inflammatory and autoimmune diseases. The redundancy of the TLR family encouraged us to seek materials that can neutralize the proinflammatory effects of any nucleic acid regardless of its sequence, structure or chemistry. Herein we demonstrate that certain nucleic acid-binding polymers can inhibit activation of all nucleic acid-sensing TLRs irrespective of whether they recognize ssRNA, dsRNA or hypomethylated DNA. Furthermore, systemic administration of such polymers can prevent fatal liver injury engendered by proinflammatory nucleic acids in an acute toxic shock model in mice. Therefore these polymers represent a novel class of anti-inflammatory agent that can act as molecular scavengers to neutralize the proinflammatory effects of various nucleic acids.

Authors
Lee, J; Sohn, JW; Zhang, Y; Leong, KW; Pisetsky, D; Sullenger, BA
MLA Citation
Lee, J, Sohn, JW, Zhang, Y, Leong, KW, Pisetsky, D, and Sullenger, BA. "Nucleic acid-binding polymers as anti-inflammatory agents." Proc Natl Acad Sci U S A 108.34 (August 23, 2011): 14055-14060.
PMID
21844380
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
34
Publish Date
2011
Start Page
14055
End Page
14060
DOI
10.1073/pnas.1105777108

RNA aptamer therapy for vaso-occlusion in sickle cell disease.

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, α(v)β(3) and P-selectin, were targeted by high-affinity RNA aptamers. An in vitro flow chamber assay was used to test the antiadhesion activity of α(v)β(3) aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. α(v)β(3) aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of α(v)β(3) aptamer (P=0.04). At 2 dyn/cm(2) shear stress, 30 nM α(v)β(3) aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1 dyn/cm(2) shear stress, 60 nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.

Authors
Burnette, AD; Nimjee, SM; Batchvarova, M; Zennadi, R; Telen, MJ; Nishimura, J-I; Sullenger, BA
MLA Citation
Burnette, AD, Nimjee, SM, Batchvarova, M, Zennadi, R, Telen, MJ, Nishimura, J-I, and Sullenger, BA. "RNA aptamer therapy for vaso-occlusion in sickle cell disease." Nucleic Acid Ther 21.4 (August 2011): 275-283.
PMID
21793788
Source
pubmed
Published In
Nucleic Acid Therapeutics
Volume
21
Issue
4
Publish Date
2011
Start Page
275
End Page
283
DOI
10.1089/nat.2010.0270

Individual anticoagulant aptamers uniquely impair thrombin generation and have a synergistic effect when used in combination

Authors
Bompiani, KM; Lorhmann, J; Frederiksen, J; Sullenger, BA
MLA Citation
Bompiani, KM, Lorhmann, J, Frederiksen, J, and Sullenger, BA. "Individual anticoagulant aptamers uniquely impair thrombin generation and have a synergistic effect when used in combination." July 2011.
Source
wos-lite
Published In
Journal of Thrombosis and Haemostasis
Volume
9
Publish Date
2011
Start Page
278
End Page
278

The many faces of the contact pathway and their role in thrombosis.

Understanding inherent differences between thrombosis and hemostasis in the vascular system are critical to developing safe and effective anticoagulants. To this end, constituents of the contact activated and intrinsic pathway of coagulation appear to be involved in pathological thrombus formation, but are not required for normal hemostasis. In addition to coagulation, activation of the contact system is involved in fibrinolytic, inflammatory, and angiogenic processes that can also contribute to the thrombotic environment. This review discusses the role of the contact system in these processes, and highlights the potential of FXII and FXI as safer targets for antithrombotic therapy.

Authors
Woodruff, RS; Sullenger, B; Becker, RC
MLA Citation
Woodruff, RS, Sullenger, B, and Becker, RC. "The many faces of the contact pathway and their role in thrombosis." J Thromb Thrombolysis 32.1 (July 2011): 9-20. (Review)
PMID
21404067
Source
pubmed
Published In
Journal of Thrombosis and Thrombolysis
Volume
32
Issue
1
Publish Date
2011
Start Page
9
End Page
20
DOI
10.1007/s11239-011-0578-5

Abstract 2817: An RNA aptamer is a potential biomarker for pancreatic cancer

Authors
Ray, P; Sullenger, BA; White, RR
MLA Citation
Ray, P, Sullenger, BA, and White, RR. "Abstract 2817: An RNA aptamer is a potential biomarker for pancreatic cancer." Cancer Research 71.8 Supplement (April 15, 2011): 2817-2817.
Source
crossref
Published In
Cancer Research
Volume
71
Issue
8 Supplement
Publish Date
2011
Start Page
2817
End Page
2817
DOI
10.1158/1538-7445.AM2011-2817

A reversible aptamer improves outcome and safety in murine models of stroke and hemorrhage.

Treatment of acute ischemic stroke with intravenous tissue-type plasminogen activator is underutilized partly due to the risk of life-threatening hemorrhage. In response to the clinical need for safer stroke therapy, we explored using an aptamer-based therapeutic strategy to promote cerebral reperfusion in a murine model of ischemic stroke. Aptamers are nucleic acid ligands that bind to their targets with high affinity and specificity, and can be rapidly reversed with an antidote. Here we show that a Factor IXa aptamer administered intravenously after 60 minutes of cerebral ischemia and reperfusion improved neurological function and was associated with reduced thrombin generation and decreased inflammation. Moreover, when the aptamer was administered in the setting of intracranial hemorrhage, treatment with its specific antidote reduced hematoma volume and improved survival. The ability to rapidly reverse a pharmacologic agent that improves neurological function after ischemic stroke should intracranial hemorrhage arise indicates that aptamer-antidote pairs may represent a novel, safer approach to treatment of stroke.

Authors
Blake, CM; Wang, H; Laskowitz, DT; Sullenger, BA
MLA Citation
Blake, CM, Wang, H, Laskowitz, DT, and Sullenger, BA. "A reversible aptamer improves outcome and safety in murine models of stroke and hemorrhage." Oligonucleotides 21.1 (February 2011): 11-19.
PMID
21142878
Source
pubmed
Published In
Oligonucleotides
Volume
21
Issue
1
Publish Date
2011
Start Page
11
End Page
19
DOI
10.1089/oli.2010.0262

Something old, Something new

Authors
Sullenger, B
MLA Citation
Sullenger, B. "Something old, Something new." Nucleic Acid Therapeutics 21.6 (2011): 369--.
PMID
22196368
Source
scival
Published In
Nucleic Acid Therapeutics
Volume
21
Issue
6
Publish Date
2011
Start Page
369-
DOI
10.1089/nat.2011.1504

Translating nucleic acid aptamers to antithrombotic drugs in cardiovascular medicine.

Nucleic acid aptamers offer several distinct advantages for the selective inhibition of protein targets within the coagulation cascade. A highly attractive feature of aptamers as antithrombotics is their ability to encode for complementary "controlling agents" which selectively bind to and neutralize their active counterparts via Watson-Crick base pairing or, in a less selective and clinically characterized manner, cationic polymers that can counteract the activity of an aptamer or free/protein-complexed nucleic acid. The former property allows aptamer-based antithrombotic therapies to be administered with a goal of selective, high intensity target inhibition, knowing that rapid drug reversal is readily available. In addition, by purposefully varying the ratio of active agent to a specific controlling agent administered, the intensity of antithrombotic therapy can be regulated with precision according to patient needs and the accompanying clinical conditions. REG1, currently undergoing phase 2B clinical investigation, consists of an RNA aptamer (RB006; pegnivacogin) which targets factor IXa and its complementary controlling agent (RB007; anivamersen). Aptamers directed against other serine coagulation proteases, some with and some without parallel controlling agents, have been designed. Aptamers directed against platelet surface membrane receptor targets are in preclinical development. The following review offers a contemporary summary of nucleic acid aptamers as a translatable platform for regulatable antithrombotic drugs expanding the paradigm of patient- and disease-specific treatment in clinical practice.

Authors
Povsic, TJ; Sullenger, BA; Zelenkofske, SL; Rusconi, CP; Becker, RC
MLA Citation
Povsic, TJ, Sullenger, BA, Zelenkofske, SL, Rusconi, CP, and Becker, RC. "Translating nucleic acid aptamers to antithrombotic drugs in cardiovascular medicine." J Cardiovasc Transl Res 3.6 (December 2010): 704-716. (Review)
PMID
21080135
Source
pubmed
Published In
Journal of Cardiovascular Translational Research
Volume
3
Issue
6
Publish Date
2010
Start Page
704
End Page
716
DOI
10.1007/s12265-010-9230-6

POLYMERS AS MOLECULAR SCAVENGERS TO NEUTRALIZE PRO-INFLAMMATORY NUCLEIC ACIDS

Authors
Lee, J; Sullenger, BA
MLA Citation
Lee, J, and Sullenger, BA. "POLYMERS AS MOLECULAR SCAVENGERS TO NEUTRALIZE PRO-INFLAMMATORY NUCLEIC ACIDS." September 2010.
Source
wos-lite
Published In
Inflammation Research
Volume
59
Publish Date
2010
Start Page
S293
End Page
S293

Antithrombotic therapy in acute coronary syndrome: how far up the coagulation cascade will we go?

The contribution of thrombosis to the natural history and clinical expression of advanced atherosclerotic coronary artery disease is well established. Less well understood is the biochemical and pathobiological distinction between normal hemostasis and thrombosis as the proximate cause of acute coronary syndrome. In this article, we summarize an evolving area of interest within the field of antithrombotic therapy--the contact system and the intrinsic pathway of coagulation, focusing our discussion on factors XI, XII, and IX to include their biochemical properties, relationship to arterial thrombosis phenotypes, and rational for future investigation of targeted pharmacotherapy.

Authors
Woodruff, B; Sullenger, B; Becker, RC
MLA Citation
Woodruff, B, Sullenger, B, and Becker, RC. "Antithrombotic therapy in acute coronary syndrome: how far up the coagulation cascade will we go?." Curr Cardiol Rep 12.4 (July 2010): 315-320. (Review)
PMID
20432072
Source
pubmed
Published In
Current Cardiology Reports
Volume
12
Issue
4
Publish Date
2010
Start Page
315
End Page
320
DOI
10.1007/s11886-010-0117-6

Nucleic acid aptamers as antithrombotic agents: Opportunities in extracellular therapeutics.

Antithrombotic therapy for the acute management of thrombotic disorders has been stimulated and guided actively by our current understanding of platelet biology, coagulation proteases, and vascular science. A translatable platform for coagulation, based soundly on biochemistry, enzymology and cellular events on platelets and tissue factor-baring cells, introduces fundamental constructs, mechanistic clarity, and an unparalleled opportunity for accelerating the development and clinical investigation of both disease- and patient-specific therapies. In the current review, we build upon and expand substantially our observations surrounding nucleic acids as antithrombotic agents.

Authors
Becker, RC; Povsic, T; Cohen, MG; Rusconi, CP; Sullenger, B
MLA Citation
Becker, RC, Povsic, T, Cohen, MG, Rusconi, CP, and Sullenger, B. "Nucleic acid aptamers as antithrombotic agents: Opportunities in extracellular therapeutics." Thromb Haemost 103.3 (March 2010): 586-595. (Review)
PMID
20135076
Source
pubmed
Published In
Thrombosis and haemostasis
Volume
103
Issue
3
Publish Date
2010
Start Page
586
End Page
595
DOI
10.1160/TH09-10-0716

An anticoagulant RNA aptamer that inhibits proteinase-cofactor interactions within prothrombinase.

The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA(11F7t)) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA(11F7t) inhibits thrombin formation catalyzed by prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within prothrombinase. Competition for enzyme complex assembly results from the binding of RNA(11F7t) to factor Xa with nanomolar affinity in a Ca(2+)-dependent interaction. RNA(11F7t) binds equivalently to the zymogen factor X as well as derivatives lacking gamma-carboxyglutamic acid residues. We suggest that the ability of RNA(11F7t) to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner.

Authors
Buddai, SK; Layzer, JM; Lu, G; Rusconi, CP; Sullenger, BA; Monroe, DM; Krishnaswamy, S
MLA Citation
Buddai, SK, Layzer, JM, Lu, G, Rusconi, CP, Sullenger, BA, Monroe, DM, and Krishnaswamy, S. "An anticoagulant RNA aptamer that inhibits proteinase-cofactor interactions within prothrombinase." J Biol Chem 285.8 (February 19, 2010): 5212-5223.
PMID
20022942
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
8
Publish Date
2010
Start Page
5212
End Page
5223
DOI
10.1074/jbc.M109.049833

Notch promotes radioresistance of glioma stem cells.

Radiotherapy represents the most effective nonsurgical treatments for gliomas. However, gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study, we show that inhibition of Notch pathway with gamma-secretase inhibitors (GSIs) renders the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhance radiation-induced cell death and impair clonogenic survival of glioma stem cells but not non-stem glioma cells. Expression of the constitutively active intracellular domains of Notch1 or Notch2 protect glioma stem cells against radiation. Notch inhibition with GSIs does not alter the DNA damage response of glioma stem cells after radiation but rather reduces Akt activity and Mcl-1 levels. Finally, knockdown of Notch1 or Notch2 sensitizes glioma stem cells to radiation and impairs xenograft tumor formation. Taken together, our results suggest a critical role of Notch signaling to regulate radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment.

Authors
Wang, J; Wakeman, TP; Lathia, JD; Hjelmeland, AB; Wang, X-F; White, RR; Rich, JN; Sullenger, BA
MLA Citation
Wang, J, Wakeman, TP, Lathia, JD, Hjelmeland, AB, Wang, X-F, White, RR, Rich, JN, and Sullenger, BA. "Notch promotes radioresistance of glioma stem cells." Stem Cells 28.1 (January 2010): 17-28.
PMID
19921751
Source
pubmed
Published In
Stem Cells
Volume
28
Issue
1
Publish Date
2010
Start Page
17
End Page
28
DOI
10.1002/stem.261

In vivo selection of tumor-targeting RNA motifs.

In an effort to target the in vivo context of tumor-specific moieties, we screened a large library of nuclease-resistant RNA oligonucleotides in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to p68, an RNA helicase that has been shown to be upregulated in colorectal cancer.

Authors
Mi, J; Liu, Y; Rabbani, ZN; Yang, Z; Urban, JH; Sullenger, BA; Clary, BM
MLA Citation
Mi, J, Liu, Y, Rabbani, ZN, Yang, Z, Urban, JH, Sullenger, BA, and Clary, BM. "In vivo selection of tumor-targeting RNA motifs." Nat Chem Biol 6.1 (January 2010): 22-24.
PMID
19946274
Source
pubmed
Published In
Nature Chemical Biology
Volume
6
Issue
1
Publish Date
2010
Start Page
22
End Page
24
DOI
10.1038/nchembio.277

The role of carrier size in the pharmacodynamics of antisense and siRNA oligonucleotides

Effective therapeutic utilization of antisense and siRNA oligonucleotides represents a major challenge to drug delivery science. Although many strategies and technologies have been applied to oligonucleotide delivery, a key issue remains the role of molecular or carrier size. In this brief review, we address some of the size-related parameters that regulate the biodistribution of oligonucleotides. We also reprise several recent studies that have examined the inter-relationship of size and shape in influencing delivery. © 2010 Informa UK, Ltd.

Authors
Huang, L; Sullenger, B; Juliano, R
MLA Citation
Huang, L, Sullenger, B, and Juliano, R. "The role of carrier size in the pharmacodynamics of antisense and siRNA oligonucleotides." Journal of Drug Targeting 18.8 (2010): 567-574.
PMID
20367081
Source
scival
Published In
Journal of Drug Targeting (Informa)
Volume
18
Issue
8
Publish Date
2010
Start Page
567
End Page
574
DOI
10.3109/10611861003734019

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin.

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.

Authors
Nimjee, SM; Oney, S; Volovyk, Z; Bompiani, KM; Long, SB; Hoffman, M; Sullenger, BA
MLA Citation
Nimjee, SM, Oney, S, Volovyk, Z, Bompiani, KM, Long, SB, Hoffman, M, and Sullenger, BA. "Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin." RNA 15.12 (December 2009): 2105-2111.
PMID
19846574
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
15
Issue
12
Publish Date
2009
Start Page
2105
End Page
2111
DOI
10.1261/rna.1240109

Effect of PAI-1 Specific RNA Aptamers On Cell Adhesion and Motility

Authors
Fortenberry, Y; Blake, CM; Sullenger, BA
MLA Citation
Fortenberry, Y, Blake, CM, and Sullenger, BA. "Effect of PAI-1 Specific RNA Aptamers On Cell Adhesion and Motility." November 20, 2009.
Source
wos-lite
Published In
Blood
Volume
114
Issue
22
Publish Date
2009
Start Page
840
End Page
840

Development of universal antidotes to control aptamer activity.

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

Authors
Oney, S; Lam, RTS; Bompiani, KM; Blake, CM; Quick, G; Heidel, JD; Liu, JY-C; Mack, BC; Davis, ME; Leong, KW; Sullenger, BA
MLA Citation
Oney, S, Lam, RTS, Bompiani, KM, Blake, CM, Quick, G, Heidel, JD, Liu, JY-C, Mack, BC, Davis, ME, Leong, KW, and Sullenger, BA. "Development of universal antidotes to control aptamer activity." Nat Med 15.10 (October 2009): 1224-1228.
Website
http://hdl.handle.net/10161/6931
PMID
19801990
Source
pubmed
Published In
Nature Medicine
Volume
15
Issue
10
Publish Date
2009
Start Page
1224
End Page
1228
DOI
10.1038/nm.1990

A hybrid DNA aptamer-dendrimer nanomaterial for targeted cell labeling.

Antibodies are natural nanomaterials and have been widely used for targeted cell labeling. However, the applications of antibodies are often limited by their large size and instability. The purpose of this study is to develop a new type of multifunctional nanomaterial that is comprised of a nucleic acid aptamer and a dendrimer, both of which are stable. This nanomaterial is approximately 8 nm in size. Moreover, it could not only carry multiple signal molecules, but also bind to target cancer cells with high affinity and specificity. This sub-10 nm multifunctional nanomaterial is expected to be useful in basic biomedical research and clinical medicine.

Authors
Zhou, J; Soontornworajit, B; Martin, J; Sullenger, BA; Gilboa, E; Wang, Y
MLA Citation
Zhou, J, Soontornworajit, B, Martin, J, Sullenger, BA, Gilboa, E, and Wang, Y. "A hybrid DNA aptamer-dendrimer nanomaterial for targeted cell labeling." Macromol Biosci 9.9 (September 9, 2009): 831-835.
PMID
19434675
Source
pubmed
Published In
Macromolecular Bioscience
Volume
9
Issue
9
Publish Date
2009
Start Page
831
End Page
835
DOI
10.1002/mabi.200900046

Antidote-controlled antithrombotic therapy targeting factor IXa and von Willebrand factor.

Thrombotic disorders and their common clinical phenotypes of acute myocardial infarction, ischemic stroke, and venous thromboembolism are the proximate cause of substantial morbidity, mortality, and health care expenditures worldwide. Accordingly, therapies designed to attenuate thrombus initiation and propagation, reflecting integrated platelet-mediated and coagulation protease-mediated events, respectively, represent a standard of care. Unfortunately, there are numerous inherent limitations of existing therapies that include target nonselectivity, variable onset and offset of pharmacodynamic effects, a narrow efficacy-safety profile, and the absence of a safe and reliable platform for either accurate titration, based on existing patient-specific, disease-specific, and clinical conditions, or active reversibility. Herein, we summarize our experience with oligonucleotide antithrombotic agents and their complementary antidotes, targeting the platelet adhesive protein von Willebrand factor and the pivotal coagulation protease factor IXa.

Authors
Becker, RC; Oney, S; Becker, KCD; Sullenger, B
MLA Citation
Becker, RC, Oney, S, Becker, KCD, and Sullenger, B. "Antidote-controlled antithrombotic therapy targeting factor IXa and von Willebrand factor." Ann N Y Acad Sci 1175 (September 2009): 61-70.
PMID
19796078
Source
pubmed
Published In
Annals of the New York Academy of Sciences
Volume
1175
Publish Date
2009
Start Page
61
End Page
70
DOI
10.1111/j.1749-6632.2009.05017.x

Antimetastatic potential of PAI-1-specific RNA aptamers.

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.

Authors
Blake, CM; Sullenger, BA; Lawrence, DA; Fortenberry, YM
MLA Citation
Blake, CM, Sullenger, BA, Lawrence, DA, and Fortenberry, YM. "Antimetastatic potential of PAI-1-specific RNA aptamers." Oligonucleotides 19.2 (June 2009): 117-128.
PMID
19284310
Source
pubmed
Published In
Oligonucleotides
Volume
19
Issue
2
Publish Date
2009
Start Page
117
End Page
128
DOI
10.1089/oli.2008.0177

Oral delivery of RNA aptamer as reversible antagonist of coagulation factor IXa

Authors
Lam, RTS; Oney, S; Sullenger, BA; Leong, KW
MLA Citation
Lam, RTS, Oney, S, Sullenger, BA, and Leong, KW. "Oral delivery of RNA aptamer as reversible antagonist of coagulation factor IXa." March 22, 2009.
Source
wos-lite
Published In
ACS National Meeting Book of Abstracts
Volume
237
Publish Date
2009
Start Page
517
End Page
517

Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.

Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33x10(-9) m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

Authors
Liu, Y; Kuan, C-T; Mi, J; Zhang, X; Clary, BM; Bigner, DD; Sullenger, BA
MLA Citation
Liu, Y, Kuan, C-T, Mi, J, Zhang, X, Clary, BM, Bigner, DD, and Sullenger, BA. "Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis." Biol Chem 390.2 (February 2009): 137-144.
PMID
19040357
Source
pubmed
Published In
Biological Chemistry
Volume
390
Issue
2
Publish Date
2009
Start Page
137
End Page
144
DOI
10.1515/BC.2009.022

Aptamers in the clinic

In 1990, an RNA molecule was designed that bound to a nucleic acid binding protein to act as a decoy, thereby preventing HIV replication. That same year, two research groups identified a high-throughput method to select for nucleic acids to protein targets and the field of aptamer therapeutics was born. Over the last 20 years, numerous aptamers to therapeutic targets have been isolated and undergone in vitro and ex vivo analysis before in vivo testing. Aptamer therapeutics represents a promising new class of agents to treat disease. The capability to extensively modify these compounds provides a potential broader spectrum of clinical applications than antibodies and small molecules. A brief historical review of aptamers is presented and the therapeutic aptamers currently in clinical development are described. Copyright © 2009 Prous Science, S.A.U. or its licensors. All rights reserved.

Authors
Nimjee, SM; Sullenger, BA
MLA Citation
Nimjee, SM, and Sullenger, BA. "Aptamers in the clinic." Drugs of the Future 34.11 (January 1, 2009): 897-901. (Review)
Source
scopus
Published In
Drugs of the future
Volume
34
Issue
11
Publish Date
2009
Start Page
897
End Page
901
DOI
10.1358/dof.2009.034.11.1436007

RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells.

Osteopontin (OPN) is a secreted phosphoprotein which mediates tumorigenesis, local growth, and metastasis in a variety of cancers. It is a potential therapeutic target for the regulation of cancer metastasis. RNA aptamer technology targeting OPN may represent a clinically viable therapy. In this study, we characterize the critical sequence of an RNA aptamer, termed OPN-R3, directed against human OPN. It has a K(d) of 18 nmol/l and binds specifically to human OPN as determined by RNA electrophoretic mobility assays. In MDA-MB231 human breast cancer cells examined under fluorescence microscopy, OPN-R3 ablates cell surface binding of OPN to its cell surface CD44 and alpha(v)beta(3) integrin receptors. Critical enzymatic components of the OPN signal transduction pathways, PI3K, JNK1/2, Src and Akt, and mediators of extracellular matrix degradation, matrix metalloproteinase 2 (MMP2) and uroplasminogen activator (uPA), are significantly decreased following exposure to OPN-R3. OPN-R3 inhibits MDA-MB231 in vitro adhesion, migration, and invasion characteristics by 60, 50, and 65%, respectively. In an in vivo xenograft model of breast cancer, OPN-R3 significantly decreases local progression and distant metastases. On the basis of this "proof-of-concept" study, we conclude that RNA aptamer targeting of OPN has biologically relevance for modifying tumor growth and metastasis.

Authors
Mi, Z; Guo, H; Russell, MB; Liu, Y; Sullenger, BA; Kuo, PC
MLA Citation
Mi, Z, Guo, H, Russell, MB, Liu, Y, Sullenger, BA, and Kuo, PC. "RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells." Mol Ther 17.1 (January 2009): 153-161.
PMID
18985031
Source
pubmed
Published In
Molecular Therapy
Volume
17
Issue
1
Publish Date
2009
Start Page
153
End Page
161
DOI
10.1038/mt.2008.235

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking.

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli-expressed glutathione-S-transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein-GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.

Authors
Liu, Y; Sun, Q-A; Chen, Q; Lee, TH; Huang, Y; Wetsel, WC; Michelotti, GA; Sullenger, BA; Zhang, X
MLA Citation
Liu, Y, Sun, Q-A, Chen, Q, Lee, TH, Huang, Y, Wetsel, WC, Michelotti, GA, Sullenger, BA, and Zhang, X. "Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking." J Neurochem 108.1 (January 2009): 147-157.
PMID
19046328
Source
pubmed
Published In
Journal of Neurochemistry
Volume
108
Issue
1
Publish Date
2009
Start Page
147
End Page
157
DOI
10.1111/j.1471-4159.2008.05748.x

Aptamers in the clinic

In 1990, an RNA molecule was designed that bound to a nucleic acid binding protein to act as a decoy, thereby preventing HIV replication. That same year, two research groups identified a high-throughput method to select for nucleic acids to protein targets and the field of aptamer therapeutics was born. Over the last 20 years, numerous aptamers to therapeutic targets have been isolated and undergone in vitro and ex vivo analysis before in vivo testing. Aptamer therapeutics represents a promising new class of agents to treat disease. The capability to extensively modify these compounds provides a potential broader spectrum of clinical applications than antibodies and small molecules. A brief historical review of aptamers is presented and the therapeutic aptamers currently in clinical development are described. Copyright © 2009 Prous Science, S.A.U. or its licensors. All rights reserved.

Authors
Nimjee, SM; Sullenger, BA
MLA Citation
Nimjee, SM, and Sullenger, BA. "Aptamers in the clinic." Drugs of the Future 34.11 (2009): 897-901.
Source
scival
Published In
Drugs of the future
Volume
34
Issue
11
Publish Date
2009
Start Page
897
End Page
901
DOI
10.1358/dof.2009.34.11.1436007

Crystal structure of an RNA aptamer bound to thrombin.

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.

Authors
Long, SB; Long, MB; White, RR; Sullenger, BA
MLA Citation
Long, SB, Long, MB, White, RR, and Sullenger, BA. "Crystal structure of an RNA aptamer bound to thrombin." RNA 14.12 (December 2008): 2504-2512.
PMID
18971322
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
14
Issue
12
Publish Date
2008
Start Page
2504
End Page
2512
DOI
10.1261/rna.1239308

Activated B cells modified by electroporation of multiple mRNAs encoding immune stimulatory molecules are comparable to mature dendritic cells in inducing in vitro antigen-specific T-cell responses.

Ex-vivo-activated B cells are an alternative source of antigen-presenting cells (APCs) and a potential replacement for dendritic cells (DCs) in immunotherapy. However, the ability of ex-vivo-activated B cells to function as potent APCs has been a concern, especially when compared to DCs. Our study investigated whether modification of activated B cells with immune stimulatory molecules could enhance the ability of activated B cells to stimulate T cells. We show that murine splenic B cells, activated with a combination of Toll-like receptor agonist and agonistic anti-CD40, stimulated antigen-specific CD8+ T cells more efficiently than cells activated with Toll-like receptor agonist or anti-CD40 alone, probably by down-regulation of the immune regulatory cytokine interleukin-10 (IL-10). However, the activated B cells were still poor T-cell stimulators compared to mature DCs. Therefore, we modified the activated B cells by simultaneous electroporation of multiple messenger RNAs encoding costimulatory molecules (OX40L and 4-1BBL), cytokines (IL-12p35 and IL-12p40) and antigen. We found that de novo expression or overexpression of OX40L, 4-1BBL and IL-12p70 on activated B cells synergistically enhanced proliferation as well as IL-2 and interferon-gamma production by CD8+ T cells. Furthermore, the RNA-modified activated B cells induced antigen-specific cytotoxic T lymphocyte responses as efficiently as mature DCs in vitro. Unexpectedly, modified activated B cells were inferior to mature DCs at in vivo induction of CD8+ T-cell responses. In summary, activated B cells modified to express immune stimulatory molecules are a potent alternative to DCs in immunotherapy.

Authors
Lee, J; Dollins, CM; Boczkowski, D; Sullenger, BA; Nair, S
MLA Citation
Lee, J, Dollins, CM, Boczkowski, D, Sullenger, BA, and Nair, S. "Activated B cells modified by electroporation of multiple mRNAs encoding immune stimulatory molecules are comparable to mature dendritic cells in inducing in vitro antigen-specific T-cell responses." Immunology 125.2 (October 2008): 229-240.
PMID
18393968
Source
pubmed
Published In
Immunology
Volume
125
Issue
2
Publish Date
2008
Start Page
229
End Page
240
DOI
10.1111/j.1365-2567.2008.02833.x

Assembling OX40 aptamers on a molecular scaffold to create a receptor-activating aptamer.

We show that a molecular scaffold can be utilized to convert a receptor binding aptamer into a receptor agonist. Many receptors (including tumor necrosis receptor family members) are activated when they are multimerized on the cell surface. Molecular scaffolds have been utilized to assemble multiple receptor binding peptide ligands to generate activators of such receptors. We demonstrate that an RNA aptamer that recognizes OX40, a member of the tumor necrosis factor receptor superfamily, can be converted into a receptor-activating aptamer by assembling two copies on an olignucleotide-based scaffold. The OX40 receptor-activating aptamer is able to induce nuclear localization of nuclear factor-kappaB, cytokine production, and cell proliferation, as well as enhance the potency of dendritic cell-based tumor vaccines when systemically delivered to mice.

Authors
Dollins, CM; Nair, S; Boczkowski, D; Lee, J; Layzer, JM; Gilboa, E; Sullenger, BA
MLA Citation
Dollins, CM, Nair, S, Boczkowski, D, Lee, J, Layzer, JM, Gilboa, E, and Sullenger, BA. "Assembling OX40 aptamers on a molecular scaffold to create a receptor-activating aptamer." Chem Biol 15.7 (July 21, 2008): 675-682.
PMID
18635004
Source
pubmed
Published In
Chemistry & Biology
Volume
15
Issue
7
Publish Date
2008
Start Page
675
End Page
682
DOI
10.1016/j.chembiol.2008.05.016

Proximity-dependent and proximity-independent trans-splicing in mammalian cells.

Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed trans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates for trans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs to trans-splice with them is consistent with the observation that pre-mRNA cis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve as trans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to trans-splice while others do not.

Authors
Viles, KD; Sullenger, BA
MLA Citation
Viles, KD, and Sullenger, BA. "Proximity-dependent and proximity-independent trans-splicing in mammalian cells." RNA 14.6 (June 2008): 1081-1094.
PMID
18441053
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
14
Issue
6
Publish Date
2008
Start Page
1081
End Page
1094
DOI
10.1261/rna.384808

Inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin 2-specific RNA aptamer.

BACKGROUND: Cellular events mediated by the Tie2 receptor are important to tumor neovascularization. Despite the complex interplay of the best-characterized Tie2 ligands, angiopoietins 1 and 2, Ang2 is purportedly "proangiogenic" in the presence of vascular endothelial growth factor. We examined whether in vivo administration of an RNA aptamer that specifically blocks Ang 2 would inhibit tumor angiogenesis and growth. METHODS: Ang2-mediated Tie2 receptor phosphorylation was assessed in vitro in the absence and presence of aptamer coupled to polyethylene glycol. IN VIVO ANGIOGENESIS ASSAY: CT26 murine colon carcinoma cells expressing green fluorescent protein were delivered into mouse dorsal skinfold window chambers. Animals received daily intraperitoneal injections of phosphate-buffered saline, low-dose (Ang2 aptamer-LD; 1 mg/kg/d), or high-dose aptamer (Ang2 aptamer-HD; 10 mg/kg/d). Vascular length density was measured under fluorescence microscopy. PRIMARY TUMOR GROWTH: CT26 cells expressing luciferase were injected into flanks of BALB/c mice to allow tumor growth monitoring by bioluminescence imaging. Animals received continuous phosphate-buffered saline or aptamer (1 mg/kg/d) via ALZET pumps. Tumors were assessed for CD31/PECAM-1 immunostaining and Hoechst dye uptake. RESULTS: Pegylated aptamer inhibited Tie2 phosphorylation. Systemic aptamer administration reduced vascular length density (P < or = 0.03) and decreased bioluminescence emission (P < 0.04), corresponding to 50% decrease in tumor volume (P = 0.04). Control tumors displayed abundant vascular marker staining, in contrast to tumors from aptamer-treated animals. CONCLUSIONS: in vivo administration of a clinically relevant, pegylated RNA aptamer specifically designed against Ang2 inhibited tumor angiogenesis and growth. These findings support targeted Ang2 inhibition as a relevant anti-angiogenic, anti-neoplastic strategy.

Authors
Sarraf-Yazdi, S; Mi, J; Moeller, BJ; Niu, X; White, RR; Kontos, CD; Sullenger, BA; Dewhirst, MW; Clary, BM
MLA Citation
Sarraf-Yazdi, S, Mi, J, Moeller, BJ, Niu, X, White, RR, Kontos, CD, Sullenger, BA, Dewhirst, MW, and Clary, BM. "Inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin 2-specific RNA aptamer." J Surg Res 146.1 (May 1, 2008): 16-23.
PMID
17950331
Source
pubmed
Published In
Journal of Surgical Research
Volume
146
Issue
1
Publish Date
2008
Start Page
16
End Page
23
DOI
10.1016/j.jss.2007.04.028

Aptamers in immunotherapy.

Authors
Dollins, CM; Nair, S; Sullenger, BA
MLA Citation
Dollins, CM, Nair, S, and Sullenger, BA. "Aptamers in immunotherapy." Hum Gene Ther 19.5 (May 2008): 443-450. (Review)
PMID
18473674
Source
pubmed
Published In
Human Gene Therapy
Volume
19
Issue
5
Publish Date
2008
Start Page
443
End Page
450
DOI
10.1089/hum.2008.045

Aptamers and siRNAs in cardiovascular disease

© 2008 Springer-Verlag Berlin Heidelberg. Cardiovascular disease is the most common cause of death in the United States, and is a leading cause of morbidity. In addition, many drugs used to treat various arenas of cardiovascular disease have untoward side effects and less than desirable safety profiles. To meet the challenge of developing safe and effective treatments, aptamers and siRNAs are being developed that display promising potential. Aptamers are relatively small, synthetic nucleic acid ligands that possess high affinity and specificity for their target proteins. Also, antidote oligonucleotides may be rationally designed against any aptamer, thus introducing an additional level of safety. Similarly, siRNAs may be rationally designed to cleave any messenger RNA through sequence complimentarity, thus silencing gene expression. Also, their effects have been shown to be long lasting, reducing the need for daily treatment. Here, we present background information on both aptamer- and siRNA-based therapeutics, and in particular focus on their applications to many areas of cardiovascular disease, namely anticoagulation, intimal hyperplasia, hypertension, myocardial ischemia, atherosclerosis and heart failure. In addition, potential challenges to both fields are discussed, and ideas on how to overcome t hese hurdles are offered. Overall, both aptamers and siRNAs represent attractive, novel nucleic acid therapies for the prevention and treatment of cardiovascular disease.

Authors
Blake, CM; Oney, S; Nimjee, SM; Sullenger, BA
MLA Citation
Blake, CM, Oney, S, Nimjee, SM, and Sullenger, BA. "Aptamers and siRNAs in cardiovascular disease." RNA Technologies in Cardiovascular Medicine and Research. January 1, 2008. 255-287.
Source
scopus
Publish Date
2008
Start Page
255
End Page
287
DOI
10.1007/978-3-540-78709-9_13

RNA Aptamer-targeted Inhibition of NF-κB Suppresses Non-small Cell Lung Cancer Resistance to Doxorubicin.

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-κB (NF-κB) activation. By achieving selective, targeted, and early inhibition of NF-κB activity, we demonstrate that NF-κB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-κB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-κB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

Authors
Mi, J; Zhang, X; Rabbani, ZN; Liu, Y; Reddy, SK; Su, Z; Salahuddin, FK; Viles, K; Giangrande, PH; Dewhirst, MW; Sullenger, BA; Kontos, CD; Clary, BM
MLA Citation
Mi, J, Zhang, X, Rabbani, ZN, Liu, Y, Reddy, SK, Su, Z, Salahuddin, FK, Viles, K, Giangrande, PH, Dewhirst, MW, Sullenger, BA, Kontos, CD, and Clary, BM. "RNA Aptamer-targeted Inhibition of NF-κB Suppresses Non-small Cell Lung Cancer Resistance to Doxorubicin." Molecular therapy : the journal of the American Society of Gene Therapy 16.1 (January 2008): 66-73.
PMID
28192708
Source
epmc
Published In
Molecular Therapy
Volume
16
Issue
1
Publish Date
2008
Start Page
66
End Page
73
DOI
10.1038/sj.mt.6300320

In vivo reprogramming of hTERT by trans-splicing ribozyme to target tumor cells.

We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells.

Authors
Hong, S-H; Jeong, J-S; Lee, Y-J; Jung, H-I; Cho, K-S; Kim, C-M; Kwon, B-S; Sullenger, BA; Lee, S-W; Kim, I-H
MLA Citation
Hong, S-H, Jeong, J-S, Lee, Y-J, Jung, H-I, Cho, K-S, Kim, C-M, Kwon, B-S, Sullenger, BA, Lee, S-W, and Kim, I-H. "In vivo reprogramming of hTERT by trans-splicing ribozyme to target tumor cells." Mol Ther 16.1 (January 2008): 74-80.
PMID
17700543
Source
pubmed
Published In
Molecular Therapy
Volume
16
Issue
1
Publish Date
2008
Start Page
74
End Page
80
DOI
10.1038/sj.mt.6300282

RNA aptamer-targeted inhibition of NF-kappa B suppresses non-small cell lung cancer resistance to doxorubicin.

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-kappaB (NF-kappaB) activation. By achieving selective, targeted, and early inhibition of NF-kappaB activity, we demonstrate that NF-kappaB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-kappaB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-kappaB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1alpha/vascular endothelial growth factor (HIF-1alpha/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

Authors
Mi, J; Zhang, X; Rabbani, ZN; Liu, Y; Reddy, SK; Su, Z; Salahuddin, FK; Viles, K; Giangrande, PH; Dewhirst, MW; Sullenger, BA; Kontos, CD; Clary, BM
MLA Citation
Mi, J, Zhang, X, Rabbani, ZN, Liu, Y, Reddy, SK, Su, Z, Salahuddin, FK, Viles, K, Giangrande, PH, Dewhirst, MW, Sullenger, BA, Kontos, CD, and Clary, BM. "RNA aptamer-targeted inhibition of NF-kappa B suppresses non-small cell lung cancer resistance to doxorubicin." Mol Ther 16.1 (January 2008): 66-73.
PMID
17912235
Source
pubmed
Published In
Molecular Therapy
Volume
16
Issue
1
Publish Date
2008
Start Page
66
End Page
73
DOI
10.1038/sj.mt.6300320

Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice.

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system.

Authors
McNamara, JO; Kolonias, D; Pastor, F; Mittler, RS; Chen, L; Giangrande, PH; Sullenger, B; Gilboa, E
MLA Citation
McNamara, JO, Kolonias, D, Pastor, F, Mittler, RS, Chen, L, Giangrande, PH, Sullenger, B, and Gilboa, E. "Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice." J Clin Invest 118.1 (January 2008): 376-386.
PMID
18060045
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
118
Issue
1
Publish Date
2008
Start Page
376
End Page
386
DOI
10.1172/JCI33365

A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function.

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2'-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K ( d ) 2.8 nM) but not Ang2 (K ( d ) > 1 microM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.

Authors
White, RR; Roy, JA; Viles, KD; Sullenger, BA; Kontos, CD
MLA Citation
White, RR, Roy, JA, Viles, KD, Sullenger, BA, and Kontos, CD. "A nuclease-resistant RNA aptamer specifically inhibits angiopoietin-1-mediated Tie2 activation and function." Angiogenesis 11.4 (2008): 395-401.
PMID
19037734
Source
pubmed
Published In
Angiogenesis
Volume
11
Issue
4
Publish Date
2008
Start Page
395
End Page
401
DOI
10.1007/s10456-008-9122-4

Developing aptamers into tumor diagnostics and therapeutics

Aptamers are small single-stranded nucleic acid molecules that bind a target protein with high affinity and specificity. Due to their stability, low toxicity and immunogenicity, as well as improved safety, aptamers are attractive alternatives to antibody and are therefore suitable for in vivo applications. Aptamers are typically isolated, through a process termed SELEX (systematic evolution of ligands by exponential enrichment), from combinatorial libraries with desired proteins. In the present review, the recent non-conventional aptamer selection process will be discussed together with an overview on the aptamer application in cancer diagnosis and therapy.

Authors
Mi, J; Sullenger, BA; Clary, BM
MLA Citation
Mi, J, Sullenger, BA, and Clary, BM. "Developing aptamers into tumor diagnostics and therapeutics." Chinese Journal of Cancer Biotherapy 15.4 (2008): 301-304.
Source
scival
Published In
Chinese Journal of Cancer Biotherapy
Volume
15
Issue
4
Publish Date
2008
Start Page
301
End Page
304
DOI
10.3872/j.issn.1007-385X.2008.04.001

Nucleic acid aptamers and their complimentary antidotes. Entering an era of antithrombotic pharmacobiologic therapy.

The translation of fundamental science-based constructs to the preemptive identification and optimal management of individuals with or those at risk for thrombotic disorders of the cardiovascular system has taken a step closer to being realized with the development of molecular technologies that include nucleic acid aptamers and their complimentary oligonucleotide antidotes. Herein, we summarize our experience with factor IX and von Willebrand factor aptamers, and introduce the era of antithrombotic pharmacobiologic therapy.

Authors
Becker, RC; Oney, S; Becker, KCD; Rusconi, CP; Sullenger, B
MLA Citation
Becker, RC, Oney, S, Becker, KCD, Rusconi, CP, and Sullenger, B. "Nucleic acid aptamers and their complimentary antidotes. Entering an era of antithrombotic pharmacobiologic therapy." Hamostaseologie 27.5 (December 2007): 378-382.
PMID
18060250
Source
pubmed
Published In
Hamostaseologie
Volume
27
Issue
5
Publish Date
2007
Start Page
378
End Page
382

Blocking adhesion of sickle erythrocytes to endothelial P-selectin using an RNAaptamer

Authors
Nishimura, J-I; Burnette, AD; Oney, S; Batchvarova, M; Delahunty, M; Zennadi, R; Sullenger, BA; Telen, MJ
MLA Citation
Nishimura, J-I, Burnette, AD, Oney, S, Batchvarova, M, Delahunty, M, Zennadi, R, Sullenger, BA, and Telen, MJ. "Blocking adhesion of sickle erythrocytes to endothelial P-selectin using an RNAaptamer." November 16, 2007.
Source
wos-lite
Published In
Blood
Volume
110
Issue
11
Publish Date
2007
Start Page
51A
End Page
51A

Distinct roles of E2F proteins in vascular smooth muscle cell proliferation and intimal hyperplasia.

Intimal hyperplasia (IH) and restenosis limit the long-term utility of bypass surgery and angioplasty due to pathological proliferation and migration of vascular smooth muscle cells (VSMCs) into the intima of treated vessels. Consequently, much attention has been focused on developing inhibitory agents that reduce this pathogenic process. The E2F transcription factors are key cell cycle regulators that play important roles in modulating cell proliferation and cell fate. Nonselective E2F inhibitors have thus been extensively evaluated for this purpose. Surprisingly, these E2F inhibitors have failed to reduce IH. These findings prompted us to evaluate the roles of different E2Fs during IH to determine how selective targeting of E2F isoforms impacts VSMC proliferation. Importantly, we show that E2F3 promotes proliferation of VSMCs leading to increased IH, whereas E2F4 inhibits this pathological response. Furthermore, we use RNA probes to show that selective inhibition of E2F3, not global inhibition of E2F activity, significantly reduces VSMC proliferation and limits IH in murine bypass grafts.

Authors
Giangrande, PH; Zhang, J; Tanner, A; Eckhart, AD; Rempel, RE; Andrechek, ER; Layzer, JM; Keys, JR; Hagen, P-O; Nevins, JR; Koch, WJ; Sullenger, BA
MLA Citation
Giangrande, PH, Zhang, J, Tanner, A, Eckhart, AD, Rempel, RE, Andrechek, ER, Layzer, JM, Keys, JR, Hagen, P-O, Nevins, JR, Koch, WJ, and Sullenger, BA. "Distinct roles of E2F proteins in vascular smooth muscle cell proliferation and intimal hyperplasia." Proc Natl Acad Sci U S A 104.32 (August 7, 2007): 12988-12993.
PMID
17652516
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
32
Publish Date
2007
Start Page
12988
End Page
12993
DOI
10.1073/pnas.0704754104

NF-kappaB inhibition by an adenovirus expressed aptamer sensitizes TNFalpha-induced apoptosis.

Prolonged activation of NF-kappaB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-kappaB activation is considered to be a main mechanism opposing TNFalpha-induced apoptosis. We investigated whether inhibition of NF-kappaB could sensitize tumor and endothelial cells to TNFalpha-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-kappaB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFalpha-induced apoptosis through the multiple pathways regulated by NF-kappaB, including Bcl-XL, HIF-1alpha, and VEGF. Our findings also suggest a new mechanism of HIF-1alpha regulation by NF-kappaB in the normoxic environment. RNA aptamer inhibition of NF-kappaB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFalpha-induced apoptosis.

Authors
Mi, J; Zhang, X; Liu, Y; Reddy, SK; Rabbani, ZN; Sullenger, BA; Clary, BM
MLA Citation
Mi, J, Zhang, X, Liu, Y, Reddy, SK, Rabbani, ZN, Sullenger, BA, and Clary, BM. "NF-kappaB inhibition by an adenovirus expressed aptamer sensitizes TNFalpha-induced apoptosis." Biochem Biophys Res Commun 359.3 (August 3, 2007): 475-480.
PMID
17560552
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
359
Issue
3
Publish Date
2007
Start Page
475
End Page
480
DOI
10.1016/j.bbrc.2007.05.125

Using 5'-PTMs to repair mutant beta-globin transcripts.

Trans-splicing has been used to repair mutant RNA transcripts via competition for the spliceosome using pre-trans-splicing molecules, or "PTMs." Previous studies have demonstrated that functional PTMs can be designed for either 3'- or 5'-exon replacement, with a vast majority of the work to date focusing on repair of mutations within internal exons and via 3'-exon replacement. Here, we describe the first use of trans-splicing to target the first exon and intron of a therapeutically relevant gene and repair the mutant RNA by 5'-exon replacement. Our results show that 5'-PTMs can be designed to repair mutations in the beta-globin transcript involved in sickle cell anemia and beta-thalassemia while providing insight into considerations for competition between trans- versus cis-splicing in mammalian cells. Target transcripts with impaired cis-splicing capabilities, like those produced in some forms of beta-thalassemia, are more efficiently repaired via trans-splicing than targets in which cis-splicing is unaffected as with sickle beta-globin. This study reveals desirable characteristics in substrate RNAs for trans-splicing therapeutics as well as provides an opportunity for further exploration into general splicing mechanisms via 5'-PTMs.

Authors
Kierlin-Duncan, MN; Sullenger, BA
MLA Citation
Kierlin-Duncan, MN, and Sullenger, BA. "Using 5'-PTMs to repair mutant beta-globin transcripts." RNA 13.8 (August 2007): 1317-1327.
PMID
17556711
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
13
Issue
8
Publish Date
2007
Start Page
1317
End Page
1327
DOI
10.1261/rna.525607

Gene therapy progress and prospects: RNA aptamers.

Aptamers are oligonucleotides evolved in vitro or in nature to bind target ligands with high affinity and specificity. They are emerging as powerful tools in the fields of therapeutics, drug development, target validation and diagnostics. Aptamers are attractive alternatives to antibody- and small-molecule-based therapeutics owing to their stability, low toxicity, low immunogenicity and improved safety. With the recent approval of the first aptamer drug Macugen by the US FDA, there is great impetus to develop therapeutic aptamers that can target a wide array of disease states. The recent demonstration that aptamer activity can be reversed by the administration of a simple antidote greatly enhances the potential value of aptamers as therapeutic agents.

Authors
Que-Gewirth, NS; Sullenger, BA
MLA Citation
Que-Gewirth, NS, and Sullenger, BA. "Gene therapy progress and prospects: RNA aptamers." Gene Ther 14.4 (February 2007): 283-291. (Review)
PMID
17279100
Source
pubmed
Published In
Gene Therapy
Volume
14
Issue
4
Publish Date
2007
Start Page
283
End Page
291
DOI
10.1038/sj.gt.3302900

Antidote-controlled platelet inhibition targeting von Willebrand factor with aptamers.

Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.

Authors
Oney, S; Nimjee, SM; Layzer, J; Que-Gewirth, N; Ginsburg, D; Becker, RC; Arepally, G; Sullenger, BA
MLA Citation
Oney, S, Nimjee, SM, Layzer, J, Que-Gewirth, N, Ginsburg, D, Becker, RC, Arepally, G, and Sullenger, BA. "Antidote-controlled platelet inhibition targeting von Willebrand factor with aptamers." Oligonucleotides 17.3 (2007): 265-274.
PMID
17854267
Source
pubmed
Published In
Oligonucleotides
Volume
17
Issue
3
Publish Date
2007
Start Page
265
End Page
274
DOI
10.1089/oli.2007.0089

Simultaneous generation of aptamers to multiple gamma-carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection.

By using the in vitro selection method SELEX against the complex mixture of GLA proteins and utilizing methods to deconvolute the resulting ligands, we were able to successfully generate 2'-ribo purine, 2'-fluoro pyrimidine aptamers to various individual targets in the GLA protein proteome that ranged in concentration from 10 nM to 1.4 microM in plasma. Perhaps not unexpectedly, the majority of the aptamers isolated following SELEX bind the most abundant protein in the mixture, prothrombin (FII), with high affinity. We show that by deselecting the dominant prothrombin aptamer the selection can be redirected. By using this DeSELEX approach, we were able to shift the selection toward other sequences and to less abundant protein targets and obtained an aptamer to Factor IX (FIX). We also demonstrate that by using an RNA library that is focused around a proteome, purified protein targets can then be used to rapidly generate aptamers to the protein targets that are rare in the initial mixture such as Factor VII (FVII) and Factor X (FX). Moreover, for all four proteins targeted (FII, FVII, FIX, and FX), aptamers were identified that could inhibit the individual protein's activitity in coagulation assays. Thus, by applying the concepts of DeSELEX and focused library selection, aptamers specific for any protein in a particular proteome can theoretically be generated, even when the proteins in the mixture are present at very different concentrations.

Authors
Layzer, JM; Sullenger, BA
MLA Citation
Layzer, JM, and Sullenger, BA. "Simultaneous generation of aptamers to multiple gamma-carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection." Oligonucleotides 17.1 (2007): 1-11.
PMID
17461758
Source
pubmed
Published In
Oligonucleotides
Volume
17
Issue
1
Publish Date
2007
Start Page
1
End Page
11
DOI
10.1089/oli.2006.0059

Blocking adhesion of sickle erythrocytes to endothelial alpha V beta 3 using RNA aptamer.

Authors
Nishimura, J-I; Burnette, AD; Batchvarova, M; Nimjee, SM; Zennadi, R; Sullenger, BA; Telen, MJ
MLA Citation
Nishimura, J-I, Burnette, AD, Batchvarova, M, Nimjee, SM, Zennadi, R, Sullenger, BA, and Telen, MJ. "Blocking adhesion of sickle erythrocytes to endothelial alpha V beta 3 using RNA aptamer." BLOOD 108.11 (November 16, 2006): 206A-207A.
Source
wos-lite
Published In
Blood
Volume
108
Issue
11
Publish Date
2006
Start Page
206A
End Page
207A

Aptamer to factor IXa and matched antidote improve cardiac function after cardiopulmonary bypass in rats; an alternative to heparin and protamine

Authors
de Lange, F; Nimjee, SM; Pitoc, GA; Grocott, HP; Mackensen, GB; Sullenger, BA
MLA Citation
de Lange, F, Nimjee, SM, Pitoc, GA, Grocott, HP, Mackensen, GB, and Sullenger, BA. "Aptamer to factor IXa and matched antidote improve cardiac function after cardiopulmonary bypass in rats; an alternative to heparin and protamine." October 31, 2006.
Source
wos-lite
Published In
Circulation
Volume
114
Issue
18
Publish Date
2006
Start Page
104
End Page
104

A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Authors
Nimjee, SM; Keys, JR; Pitoc, GA; Quick, G; Rusconi, CP; Sullenger, BA
MLA Citation
Nimjee, SM, Keys, JR, Pitoc, GA, Quick, G, Rusconi, CP, and Sullenger, BA. "A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery." Mol Ther 14.3 (September 2006): 408-415.
PMID
16765093
Source
pubmed
Published In
Molecular Therapy
Volume
14
Issue
3
Publish Date
2006
Start Page
408
End Page
415
DOI
10.1016/j.ymthe.2006.04.006

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Authors
McNamara, JO; Andrechek, ER; Wang, Y; Viles, KD; Rempel, RE; Gilboa, E; Sullenger, BA; Giangrande, PH
MLA Citation
McNamara, JO, Andrechek, ER, Wang, Y, Viles, KD, Rempel, RE, Gilboa, E, Sullenger, BA, and Giangrande, PH. "Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras." Nat Biotechnol 24.8 (August 2006): 1005-1015.
PMID
16823371
Source
pubmed
Published In
Nature Biotechnology
Volume
24
Issue
8
Publish Date
2006
Start Page
1005
End Page
1015
DOI
10.1038/nbt1223

Aptamers to Proteins

Authors
Nimjee, SM; Rusconi, CP; Sullenger, BA
MLA Citation
Nimjee, SM, Rusconi, CP, and Sullenger, BA. "Aptamers to Proteins." The Aptamer Handbook: Functional Oligonucleotides and Their Applications. June 6, 2006. 131-166.
Source
scopus
Publish Date
2006
Start Page
131
End Page
166
DOI
10.1002/3527608192.ch6

Cell-Type Specific Delivery of siRNAs with Aptamer-siRNA Chimeras

Authors
Giangrande, PH; McNamara, JO; Andrecheck, E; Gilboa, E; Sullenger, BA
MLA Citation
Giangrande, PH, McNamara, JO, Andrecheck, E, Gilboa, E, and Sullenger, BA. "Cell-Type Specific Delivery of siRNAs with Aptamer-siRNA Chimeras." May 2006.
Source
wos-lite
Published In
Molecular Therapy
Volume
13
Publish Date
2006
Start Page
S35
End Page
S35

Use of Antisense Oligonucleotides To Restore Activity in Mutant p53

Authors
Catron, DJ; Giangrande, PH; Sullenger, BA
MLA Citation
Catron, DJ, Giangrande, PH, and Sullenger, BA. "Use of Antisense Oligonucleotides To Restore Activity in Mutant p53." May 2006.
Source
wos-lite
Published In
Molecular Therapy
Volume
13
Publish Date
2006
Start Page
S395
End Page
S395

H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity.

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a 'pure' RNA aptamer specific for NF-kappaB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-kappaB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.

Authors
Mi, J; Zhang, X; Rabbani, ZN; Liu, Y; Su, Z; Vujaskovic, Z; Kontos, CD; Sullenger, BA; Clary, BM
MLA Citation
Mi, J, Zhang, X, Rabbani, ZN, Liu, Y, Su, Z, Vujaskovic, Z, Kontos, CD, Sullenger, BA, and Clary, BM. "H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity. (Published online)" Nucleic Acids Res 34.12 (2006): 3577-3584.
PMID
16855294
Source
pubmed
Published In
Nucleic Acids Research
Volume
34
Issue
12
Publish Date
2006
Start Page
3577
End Page
3584
DOI
10.1093/nar/gkl482

Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival.

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

Authors
Mi, J; Zhang, X; Giangrande, PH; McNamara, JO; Nimjee, SM; Sarraf-Yazdi, S; Sullenger, BA; Clary, BM
MLA Citation
Mi, J, Zhang, X, Giangrande, PH, McNamara, JO, Nimjee, SM, Sarraf-Yazdi, S, Sullenger, BA, and Clary, BM. "Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival." Biochem Biophys Res Commun 338.2 (December 16, 2005): 956-963.
PMID
16256939
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
338
Issue
2
Publish Date
2005
Start Page
956
End Page
963
DOI
10.1016/j.bbrc.2005.10.043

Blocking complement-mediated hemolysis using RNA aptamers that bind complement component C8

Authors
Nishimura, JI; Nimjee, SM; Jiang, HX; Howard, TA; Pitoc, GA; Rusconi, CP; Omine, M; Chao, NJ; Frank, MM; Sullenger, BA; Ware, RE
MLA Citation
Nishimura, JI, Nimjee, SM, Jiang, HX, Howard, TA, Pitoc, GA, Rusconi, CP, Omine, M, Chao, NJ, Frank, MM, Sullenger, BA, and Ware, RE. "Blocking complement-mediated hemolysis using RNA aptamers that bind complement component C8." November 16, 2005.
Source
wos-lite
Published In
Blood
Volume
106
Issue
11
Publish Date
2005
Start Page
57A
End Page
58A

Retargeting mobile group II introns to repair mutant genes.

Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications. Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth-promoting gene can engender pathological consequences. For example, retroviral vector-mediated gene transfer induced leukemia in 2 of 11 children treated for severe combined immunodeficiency, raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences. Here, we examine the use of a mobile retroposable genetic element that can be targeted to introduce therapeutic sequences site specifically into mutant genes. The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ (approved gene symbol GLB1) and beta-globin (approved gene symbol HBB) genes with high efficiency and fidelity in model systems in bacteria. These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair.

Authors
Jones, JP; Kierlin, MN; Coon, RG; Perutka, J; Lambowitz, AM; Sullenger, BA
MLA Citation
Jones, JP, Kierlin, MN, Coon, RG, Perutka, J, Lambowitz, AM, and Sullenger, BA. "Retargeting mobile group II introns to repair mutant genes." Mol Ther 11.5 (May 2005): 687-694.
PMID
15851007
Source
pubmed
Published In
Molecular Therapy
Volume
11
Issue
5
Publish Date
2005
Start Page
687
End Page
694
DOI
10.1016/j.ymthe.2005.01.014

The potential of aptamers as anticoagulants.

Useful additional options for anticoagulant therapy have been introduced over the last 15 years, including low-molecular-weight heparins and direct thrombin inhibitors. Despite these impressive advances, a need for safer effective anticoagulants remains. Aptamers represent a therapeutic modality that has the potential to address this unmet need. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. Aptamers are delivered by parenteral administration, can be formulated to possess a very short or sustained half-life, and are purported to be nonimmunogenic. Perhaps most relevant to the development of safer anticoagulant therapies, recent studies have shown that antidotes can be rationally designed to control the pharmacologic effects of aptamers in vivo, paving the way for a new class of antidote-controlled therapeutics. This review discusses the limitations of current anticoagulant therapies, the properties of aptamers and how these properties can be exploited to address the unmet needs within this therapeutic class, and the progress to date in developing new aptamer-based anticoagulant therapies.

Authors
Nimjee, SM; Rusconi, CP; Harrington, RA; Sullenger, BA
MLA Citation
Nimjee, SM, Rusconi, CP, Harrington, RA, and Sullenger, BA. "The potential of aptamers as anticoagulants." Trends Cardiovasc Med 15.1 (January 2005): 41-45. (Review)
PMID
15795162
Source
pubmed
Published In
Trends in Cardiovascular Medicine
Volume
15
Issue
1
Publish Date
2005
Start Page
41
End Page
45
DOI
10.1016/j.tcm.2005.01.002

Aptamers: an emerging class of therapeutics.

Numerous nucleic acid ligands, also termed decoys or aptamers, have been developed during the past 15 years that can inhibit the activity of many pathogenic proteins. Two of them, Macugen and E2F decoy, are in phase III clinical trials. Several properties of aptamers make them an attractive class of therapeutic compounds. Their affinity and specificity for a given protein make it possible to isolate a ligand to virtually any target, and adjusting their bioavailability expands their clinical utility. The ability to develop aptamers that retain activity in multiple organisms facilitates preclinical development. Antidote control of aptamer activity enables safe, tightly controlled therapeutics. Aptamers may prove useful in the treatment of a wide variety of human maladies, including infectious diseases, cancer, and cardiovascular disease. We review the observations that facilitated the development of this emerging class of therapeutics, summarize progress to date, and speculate on the eventual utility of such agents in the clinic.

Authors
Nimjee, SM; Rusconi, CP; Sullenger, BA
MLA Citation
Nimjee, SM, Rusconi, CP, and Sullenger, BA. "Aptamers: an emerging class of therapeutics." Annu Rev Med 56 (2005): 555-583. (Review)
PMID
15660527
Source
pubmed
Published In
Annual Review of Medicine
Volume
56
Publish Date
2005
Start Page
555
End Page
583
DOI
10.1146/annurev.med.56.062904.144915

Nucleic acid aptamers in therapeutic anticoagulation. Technology, development and clinical application

The evolution of anticoagulant therapy for the prevention and treatment of thrombotic disorders has progressed at a relatively modest pace considering the scope of the problem and our current understanding of platelet biology, coagulation proteases, and vascular science as they apply to protective haemostasis and pathologic thrombosis. Recent observations, dedicated to cellular-based models of coagulation, provide fundamental constructs, mechanistic clarity, and potentially unparalleled opportunity for accelerating the development and wide-scale clinical use of safe, effective, regulatable and patient-specific therapies. The following review introduces a novel domain of anticoagulant therapy referred to as aptamers (derived from the Latin aptus - to fit), considering their history, development, and potential application in patient care arenas. © 2005 Schattauer GmbH, Stuttgart.

Authors
Becker, RC; Rusconi, C; Sullenger, B
MLA Citation
Becker, RC, Rusconi, C, and Sullenger, B. "Nucleic acid aptamers in therapeutic anticoagulation. Technology, development and clinical application." Thrombosis and Haemostasis 93.6 (2005): 1014-1020.
PMID
15968382
Source
scival
Published In
Thrombosis and Haemostasis
Volume
93
Issue
6
Publish Date
2005
Start Page
1014
End Page
1020
DOI
10.1160/TH04-12-0790

Riboswitches--to kill or save the messenger.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Riboswitches--to kill or save the messenger." N Engl J Med 351.26 (December 23, 2004): 2759-2760.
PMID
15616213
Source
pubmed
Published In
The New England journal of medicine
Volume
351
Issue
26
Publish Date
2004
Start Page
2759
End Page
2760
DOI
10.1056/NEJMcibr043522

Blocking complement-mediated hemolysis of PNH erythrocytes by RNA aptamers to C8 and C9.

Authors
Nishimura, JI; Nimjee, S; Jiang, HX; Kawaguchi, T; Nakakuma, H; Omine, M; Frank, MM; Ware, RE; Sullenger, BA
MLA Citation
Nishimura, JI, Nimjee, S, Jiang, HX, Kawaguchi, T, Nakakuma, H, Omine, M, Frank, MM, Ware, RE, and Sullenger, BA. "Blocking complement-mediated hemolysis of PNH erythrocytes by RNA aptamers to C8 and C9." November 16, 2004.
Source
wos-lite
Published In
Blood
Volume
104
Issue
11
Publish Date
2004
Start Page
772A
End Page
772A

Antidote-mediated control of an anticoagulant aptamer in vivo.

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.

Authors
Rusconi, CP; Roberts, JD; Pitoc, GA; Nimjee, SM; White, RR; Quick, G; Scardino, E; Fay, WP; Sullenger, BA
MLA Citation
Rusconi, CP, Roberts, JD, Pitoc, GA, Nimjee, SM, White, RR, Quick, G, Scardino, E, Fay, WP, and Sullenger, BA. "Antidote-mediated control of an anticoagulant aptamer in vivo." Nat Biotechnol 22.11 (November 2004): 1423-1428.
PMID
15502817
Source
pubmed
Published In
Nature Biotechnology
Volume
22
Issue
11
Publish Date
2004
Start Page
1423
End Page
1428
DOI
10.1038/nbt1023

Regulatable aptamers in medicine: focus on antithrombotic strategies.

Proteins generally execute the key physiological activities required for normal growth and homeostasis. As such, many different classes of proteins, including proteases, kinases, cellular receptors and signalling proteins, represent attractive targets for diagnosis or therapy. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. After a decade of intensive research, technology development and initial clinical evaluation, aptamers have now demonstrated broad potential as direct protein ligands and inhibitors, and thus represent an exciting class of compounds for the development of new therapeutic and diagnostic agents. This review will discuss the basic properties and isolation of aptamers, their use in animals and the clinic, and describe an exciting recent advance in the development of antidotes to certain aptamers, which will add a repertoire of new agents with regulatable activity for clinical use.

Authors
Potti, A; Rusconi, CP; Sullenger, BA; Ortel, TL
MLA Citation
Potti, A, Rusconi, CP, Sullenger, BA, and Ortel, TL. "Regulatable aptamers in medicine: focus on antithrombotic strategies." Expert Opin Biol Ther 4.10 (October 2004): 1641-1647. (Review)
PMID
15461575
Source
pubmed
Published In
Expert Opinion on Biological Therapy
Volume
4
Issue
10
Publish Date
2004
Start Page
1641
End Page
1647
DOI
10.1517/14712598.4.10.1641

Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA.

A variety of mutations in the p53 tumor suppressor gene have been found in over half of human tumors. Thus, restoration of wild-type p53 activity by repair of mutant RNA has been previously suggested as an approach to cancer treatment. To explore the potential utility of RNA repair for cancer therapy, we developed a group I intron-based ribozyme that can replace mutant p53 RNA with a wild-type RNA sequence attached to the 3' end of the ribozyme by trans-splicing reaction. First, RNA mapping analysis demonstrated that the leader sequences upstream of the AUG start codon in the mutant p53 RNA appeared to be particularly accessible to the ribozymes. Then, the trans-splicing ribozyme specifically recognizing the most accessible sequence induced functional p53 activity, resulting in an 8- and a 2.6-fold induction of transactivation of p53-responsive promoters in two mutant p53-related ovarian cancer cell lines, SKOV3 cells and 2774 cells, respectively, by repairing defective p53 RNA. The repair efficiency of the mutant p53 RNA was almost 10% in 2774 cells. Moreover, the ribozyme activated the expression level of endogenous p21 and Bax genes in the cells. Furthermore, apoptosis was efficiently triggered in the human cancer cells transfected with the specific ribozyme. These results suggest that a trans-splicing ribozyme could be a potent anti-cancer agent that can revert the defective p53-related neoplastic phenotype.

Authors
Shin, K-S; Sullenger, BA; Lee, S-W
MLA Citation
Shin, K-S, Sullenger, BA, and Lee, S-W. "Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA." Mol Ther 10.2 (August 2004): 365-372.
PMID
15294183
Source
pubmed
Published In
Molecular Therapy
Volume
10
Issue
2
Publish Date
2004
Start Page
365
End Page
372
DOI
10.1016/j.ymthe.2004.05.007

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins.

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.

Authors
Jeter, ML; Ly, LV; Fortenberry, YM; Whinna, HC; White, RR; Rusconi, CP; Sullenger, BA; Church, FC
MLA Citation
Jeter, ML, Ly, LV, Fortenberry, YM, Whinna, HC, White, RR, Rusconi, CP, Sullenger, BA, and Church, FC. "RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins." FEBS Lett 568.1-3 (June 18, 2004): 10-14.
PMID
15196911
Source
pubmed
Published In
FEBS Letters
Volume
568
Issue
1-3
Publish Date
2004
Start Page
10
End Page
14
DOI
10.1016/j.febslet.2004.04.087

In vivo activity of nuclease-resistant siRNAs.

Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.

Authors
Layzer, JM; McCaffrey, AP; Tanner, AK; Huang, Z; Kay, MA; Sullenger, BA
MLA Citation
Layzer, JM, McCaffrey, AP, Tanner, AK, Huang, Z, Kay, MA, and Sullenger, BA. "In vivo activity of nuclease-resistant siRNAs." RNA 10.5 (May 2004): 766-771.
PMID
15100431
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
10
Issue
5
Publish Date
2004
Start Page
766
End Page
771

Aptamer to factor IXa and its matched antidote in cardiopulmonary bypass: An alternative to heparin and protamine

Authors
Nimjee, SM; Keys, JR; Pitoc, GA; Quick, G; Rusconi, CP; Sullenger, BA
MLA Citation
Nimjee, SM, Keys, JR, Pitoc, GA, Quick, G, Rusconi, CP, and Sullenger, BA. "Aptamer to factor IXa and its matched antidote in cardiopulmonary bypass: An alternative to heparin and protamine." May 2004.
Source
wos-lite
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
24
Issue
5
Publish Date
2004
Start Page
E7
End Page
E7

Group I intron-mediated trans-splicing revision of mutant p53 RNA transcripts

Authors
Catron, DJ; Watanabe, T; Shin, KS; Toloza, EM; Lee, SW; Sullenger, BA
MLA Citation
Catron, DJ, Watanabe, T, Shin, KS, Toloza, EM, Lee, SW, and Sullenger, BA. "Group I intron-mediated trans-splicing revision of mutant p53 RNA transcripts." May 2004.
Source
wos-lite
Published In
Molecular Therapy
Volume
9
Publish Date
2004
Start Page
S231
End Page
S231

The Sabiston vision of basic science in a department of surgery.

Authors
Nimjee, SM; Sullenger, BA
MLA Citation
Nimjee, SM, and Sullenger, BA. "The Sabiston vision of basic science in a department of surgery." Ann Surg 238.6 Suppl (December 2003): S28-S32.
PMID
14703741
Source
pubmed
Published In
Annals of Surgery
Volume
238
Issue
6 Suppl
Publish Date
2003
Start Page
S28
End Page
S32

Complement-mediated hemolysis of erythrocytes is blocked by RNA aptamers to complement components C5 and C8.

Authors
Nishimura, J; Nimjee, SM; Jiang, HX; Pitoc, G; Howard, TA; Rusconi, CP; Omine, M; Frank, MM; Sullenger, BA; Ware, RE
MLA Citation
Nishimura, J, Nimjee, SM, Jiang, HX, Pitoc, G, Howard, TA, Rusconi, CP, Omine, M, Frank, MM, Sullenger, BA, and Ware, RE. "Complement-mediated hemolysis of erythrocytes is blocked by RNA aptamers to complement components C5 and C8." November 16, 2003.
Source
wos-lite
Published In
Blood
Volume
102
Issue
11
Publish Date
2003
Start Page
510A
End Page
510A

Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity.

The potency of cancer immunotherapy can be enhanced by administration of high-avidity ligands specific to receptors expressed on T cells. Antibodies or cytokines are the main agents used in such capacity. Antibody-mediated inhibition of cytotoxic T cell antigen-4 (CTLA-4) function in mice augments antitumor immunity and could serve as an important adjunct in cancer immunotherapy. However, antibody-based therapy used in the setting of chronic diseases such as cancer poses significant cost, manufacturing, and regulatory challenges. Here we describe the development of RNA aptamers that bind CTLA-4 with high affinity and specificity. These aptamers inhibit CTLA-4 function in vitro and enhance tumor immunity in mice. Moreover, assembly of the aptamers into tetrameric forms significantly enhances their bioactivity in vitro and in vivo. These results demonstrate that aptamers can be used to manipulate the immune system for therapeutic applications and that multivalent versions of aptamers may be particularly potent agents in vivo.

Authors
Santulli-Marotto, S; Nair, SK; Rusconi, C; Sullenger, B; Gilboa, E
MLA Citation
Santulli-Marotto, S, Nair, SK, Rusconi, C, Sullenger, B, and Gilboa, E. "Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity." Cancer Res 63.21 (November 1, 2003): 7483-7489.
PMID
14612549
Source
pubmed
Published In
Cancer Research
Volume
63
Issue
21
Publish Date
2003
Start Page
7483
End Page
7489

In Vivo applications of an antidote-controlled anticoagulant targeting coagulation factor IXa

Authors
Rusconi, CP; Roberts, JD; Pitoc, GA; Nimjee, SM; White, RR; Fay, WP; Sullenger, BA
MLA Citation
Rusconi, CP, Roberts, JD, Pitoc, GA, Nimjee, SM, White, RR, Fay, WP, and Sullenger, BA. "In Vivo applications of an antidote-controlled anticoagulant targeting coagulation factor IXa." October 28, 2003.
Source
wos-lite
Published In
Circulation
Volume
108
Issue
17
Publish Date
2003
Start Page
138
End Page
138

Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes.

Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.

Authors
Byun, J; Lan, N; Long, M; Sullenger, BA
MLA Citation
Byun, J, Lan, N, Long, M, and Sullenger, BA. "Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes." RNA 9.10 (October 2003): 1254-1263.
PMID
13130139
Source
pubmed
Published In
RNA (New York, N.Y.)
Volume
9
Issue
10
Publish Date
2003
Start Page
1254
End Page
1263

Ribozyme-mediated revision of RNA and DNA.

Authors
Long, MB; Jones, JP; Sullenger, BA; Byun, J
MLA Citation
Long, MB, Jones, JP, Sullenger, BA, and Byun, J. "Ribozyme-mediated revision of RNA and DNA." J Clin Invest 112.3 (August 2003): 312-318. (Review)
PMID
12897196
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
112
Issue
3
Publish Date
2003
Start Page
312
End Page
318
DOI
10.1172/JCI19386

Targeted genetic repair: an emerging approach to genetic therapy.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Targeted genetic repair: an emerging approach to genetic therapy." J Clin Invest 112.3 (August 2003): 310-311. (Review)
PMID
12897195
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
112
Issue
3
Publish Date
2003
Start Page
310
End Page
311
DOI
10.1172/JCI19419

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2.

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.

Authors
White, RR; Shan, S; Rusconi, CP; Shetty, G; Dewhirst, MW; Kontos, CD; Sullenger, BA
MLA Citation
White, RR, Shan, S, Rusconi, CP, Shetty, G, Dewhirst, MW, Kontos, CD, and Sullenger, BA. "Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2." Proc Natl Acad Sci U S A 100.9 (April 29, 2003): 5028-5033.
PMID
12692304
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
9
Publish Date
2003
Start Page
5028
End Page
5033
DOI
10.1073/pnas.0831159100

Therapeutic aptamers and antidotes: a novel approach to safer drug design.

Authors
Sullenger, BA; White, RR; Rusconi, CP
MLA Citation
Sullenger, BA, White, RR, and Rusconi, CP. "Therapeutic aptamers and antidotes: a novel approach to safer drug design." Ernst Schering Res Found Workshop 43 (2003): 217-223. (Review)
PMID
12894459
Source
pubmed
Published In
Ernst Schering Research Foundation workshop
Issue
43
Publish Date
2003
Start Page
217
End Page
223

Functional repair of a mutant chloride channel using a trans-splicing ribozyme.

RNA repair has been proposed as a novel gene-based therapeutic strategy. Modified Tetrahymena group I intron ribozymes have been used to mediate trans-splicing of therapeutically relevant RNA transcripts, but the efficiency of the ribozyme-mediated RNA repair process has not been determined precisely and subsequent restoration of protein function has been demonstrated only by indirect means. We engineered a ribozyme that targets the mRNA of a mutant canine skeletal muscle chloride channel (cClC-1) (mutation T268M in ClC-1 causing myotonia congenita) and replaces the mutant-containing 3' portion by trans-splicing the corresponding 4-kb wild-type sequence. Repair efficiency assessed by quantitative RT-PCR was 1.2% +/- 0.1% in a population of treated cells. However, when chloride channel function was examined in single cells, a wide range of electrophysiological activity was observed, with 18% of cells exhibiting significant functional restoration and some cells exhibiting complete rescue of the biophysical phenotype. These results indicate that RNA repair can restore wild-type protein activity and reveal considerable cell-to-cell variability in ribozyme-mediated trans-splicing reaction efficiency.

Authors
Rogers, CS; Vanoye, CG; Sullenger, BA; George, AL
MLA Citation
Rogers, CS, Vanoye, CG, Sullenger, BA, and George, AL. "Functional repair of a mutant chloride channel using a trans-splicing ribozyme." J Clin Invest 110.12 (December 2002): 1783-1789.
PMID
12488428
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
110
Issue
12
Publish Date
2002
Start Page
1783
End Page
1789
DOI
10.1172/JCI16481

RNA aptamers as reversible antagonists of coagulation factor IXa.

Many therapeutic agents are associated with adverse effects in patients. Anticoagulants can engender acute complications such as significant bleeding that increases patient morbidity and mortality. Antidote control provides the safest means to regulate drug action. For this reason, despite its known limitations and toxicities, heparin use remains high because it is the only anticoagulant that can be controlled by an antidote, the polypeptide protamine. To date, no generalizable strategy for developing drug-antidote pairs has been described. We investigated whether drug-antidote pairs could be rationally designed by taking advantage of properties inherent to nucleic acids to make antidote-controlled anticoagulant agents. Here we show that protein-binding oligonucleotides (aptamers) against coagulation factor IXa are potent anticoagulants. We also show that oligonucleotides complementary to these aptamers can act as antidotes capable of efficiently reversing the activity of these new anticoagulants in plasma from healthy volunteers and from patients who cannot tolerate heparin. This generalizable strategy for rationally designing a drug-antidote pair thus opens up the way for developing safer regulatable therapeutics.

Authors
Rusconi, CP; Scardino, E; Layzer, J; Pitoc, GA; Ortel, TL; Monroe, D; Sullenger, BA
MLA Citation
Rusconi, CP, Scardino, E, Layzer, J, Pitoc, GA, Ortel, TL, Monroe, D, and Sullenger, BA. "RNA aptamers as reversible antagonists of coagulation factor IXa." Nature 419.6902 (September 5, 2002): 90-94.
PMID
12214238
Source
pubmed
Published In
Nature
Volume
419
Issue
6902
Publish Date
2002
Start Page
90
End Page
94
DOI
10.1038/nature00963

Emerging clinical applications of RNA.

RNA is a versatile biological macromolecule that is crucial in mobilizing and interpreting our genetic information. It is not surprising then that researchers have sought to exploit the inherent properties of RNAs so as to interfere with or repair dysfunctional nucleic acids or proteins and to stimulate the production of therapeutic gene products in a variety of pathological situations. The first generation of the resulting RNA therapeutics are now being evaluated in clinical trials, raising significant interest in this emerging area of medical research.

Authors
Sullenger, BA; Gilboa, E
MLA Citation
Sullenger, BA, and Gilboa, E. "Emerging clinical applications of RNA." Nature 418.6894 (July 11, 2002): 252-258. (Review)
PMID
12110902
Source
pubmed
Published In
Nature
Volume
418
Issue
6894
Publish Date
2002
Start Page
252
End Page
258
DOI
10.1038/418252a

Optimizing aptamer activity for gene therapy applications using expression cassette SELEX.

RNA aptamers against a variety of clinically relevant target proteins have been generated. For example, we previously isolated an RNA aptamer that inhibits the function of the E2F family of transcription factors that play a critical role in the control of cell proliferation. However, the development of this and other aptamers for gene therapy applications has been complicated by the fact that expression of RNA aptamers in the context of flanking sequences can inhibit the ability of an aptamer to fold into its functional conformation. Insertion of the E2F aptamer into a tRNA expression cassette resulted in the production of high levels of chimeric tRNA that contains a misfolded and inactive aptamer in transfected mammalian cells. To overcome this problem, we randomized the sequence flanking the aptamer and selected for chimeric tRNAs that retained high affinity binding to E2F1. This expression cassette SELEX strategy yielded RNAs that bind E2F with high affinity (IC50 of 15 nM) and which can be expressed at high levels in mammalian cells. Moreover, these chimeric tRNA-E2F aptamers are functional and can inhibit E2F-mediated transactivation by up to 80% in human 293 cells. Expression cassette SELEX should greatly facilitate the use of aptamers for a variety of gene therapy applications.

Authors
Martell, RE; Nevins, JR; Sullenger, BA
MLA Citation
Martell, RE, Nevins, JR, and Sullenger, BA. "Optimizing aptamer activity for gene therapy applications using expression cassette SELEX." Mol Ther 6.1 (July 2002): 30-34.
PMID
12095300
Source
pubmed
Published In
Molecular Therapy
Volume
6
Issue
1
Publish Date
2002
Start Page
30
End Page
34

Regulation of thrombin-serpin inhibition reactions by a thrombin-specific RNA aptamer

Authors
Jeter, ML; White, RR; Sullenger, BA; Rusconi, CP; Church, FC
MLA Citation
Jeter, ML, White, RR, Sullenger, BA, Rusconi, CP, and Church, FC. "Regulation of thrombin-serpin inhibition reactions by a thrombin-specific RNA aptamer." March 22, 2002.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
16
Issue
5
Publish Date
2002
Start Page
A1193
End Page
A1193

Generation of species cross-reactive aptamers using "toggle" SELEX.

Species cross-reactivity facilitates the preclinical evaluation of potentially therapeutic molecules in animal models. Here we describe an in vitro selection strategy in which RNA ligands (aptamers) that bind both human and porcine thrombin were selected by "toggling" the protein target between human and porcine thrombin during alternating rounds of selection. The "toggle" selection process yielded a family of aptamers, all of which bound both human and porcine thrombin with high affinity. Toggle-25, a characteristic member, inhibited two of thrombin's most important functions: plasma clot formation and platelet activation. If appropriate targets are available, the toggle strategy is a simple measure that promotes cross-reactivity and may be generalizable to related proteins of the same species as well as to other combinatorial library screening strategies. This strategy should facilitate the isolation of ligands with needed properties for gene therapy and other therapeutic and diagnostic applications.

Authors
White, R; Rusconi, C; Scardino, E; Wolberg, A; Lawson, J; Hoffman, M; Sullenger, B
MLA Citation
White, R, Rusconi, C, Scardino, E, Wolberg, A, Lawson, J, Hoffman, M, and Sullenger, B. "Generation of species cross-reactive aptamers using "toggle" SELEX." Mol Ther 4.6 (December 2001): 567-573.
PMID
11735341
Source
pubmed
Published In
Molecular Therapy
Volume
4
Issue
6
Publish Date
2001
Start Page
567
End Page
573
DOI
10.1006/mthe.2001.0495

A novel inhibitor of Angiopoietin-1 disrupts Tie2-mediated endothelial cell survival

Authors
White, RR; Rusconi, CP; Sullenger, BA; Kontos, CD
MLA Citation
White, RR, Rusconi, CP, Sullenger, BA, and Kontos, CD. "A novel inhibitor of Angiopoietin-1 disrupts Tie2-mediated endothelial cell survival." October 23, 2001.
Source
wos-lite
Published In
Circulation
Volume
104
Issue
17
Publish Date
2001
Start Page
315
End Page
315

RNA repair: a novel approach to gene therapy.

Treatment of genetic disorders by gene therapy has conventionally been attempted through the transfer of a wild type version of a gene to the cells of a patient harboring defective copies of a disease associated gene. Despite significant advances using this paradigm, several technical hurdles must still be overcome before this 'gene replacement' approach will become useful in the treatment of a variety of genetic maladies. Such limitations have led a number of researchers to begin to investigate alternative strategies to genetic therapy. Repair of mutant genetic instructions represents a fundamentally different approach to genetic therapy that may have significant advantages over gene replacement. Herein, we will discuss recent advances using repair of mutant RNAs as a novel means to correct genetic deficiencies.

Authors
Watanabe, T; Sullenger, BA
MLA Citation
Watanabe, T, and Sullenger, BA. "RNA repair: a novel approach to gene therapy." Adv Drug Deliv Rev 44.2-3 (November 15, 2000): 109-118. (Review)
PMID
11072109
Source
pubmed
Published In
Advanced Drug Delivery Reviews
Volume
44
Issue
2-3
Publish Date
2000
Start Page
109
End Page
118

Blocking the initiation of coagulation by RNA aptamers to factor VIIa.

The tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factor-dependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of approximately 15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

Authors
Rusconi, CP; Yeh, A; Lyerly, HK; Lawson, JH; Sullenger, BA
MLA Citation
Rusconi, CP, Yeh, A, Lyerly, HK, Lawson, JH, and Sullenger, BA. "Blocking the initiation of coagulation by RNA aptamers to factor VIIa." Thromb Haemost 84.5 (November 2000): 841-848.
PMID
11127866
Source
pubmed
Published In
Thrombosis and haemostasis
Volume
84
Issue
5
Publish Date
2000
Start Page
841
End Page
848

Series introduction: emerging clinical applications of nucleic acids

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Series introduction: emerging clinical applications of nucleic acids." J Clin Invest 106.8 (October 2000): 921-922.
PMID
11032849
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
106
Issue
8
Publish Date
2000
Start Page
921
End Page
922
DOI
10.1172/JCI11343

Developing aptamers into therapeutics.

Authors
White, RR; Sullenger, BA; Rusconi, CP
MLA Citation
White, RR, Sullenger, BA, and Rusconi, CP. "Developing aptamers into therapeutics." J Clin Invest 106.8 (October 2000): 929-934.
PMID
11032851
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
106
Issue
8
Publish Date
2000
Start Page
929
End Page
934
DOI
10.1172/JCI11325

Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA.

Recent reports have demonstrated that trans-splicing ribozymes can be employed to repair mutant RNAs. One key factor that influences RNA repair efficiency is the accessibility of the substrate RNA for ribozyme binding, which is complicated by the fact that RNAs may assume multiple conformations and have proteins bound to them in vivo. Here we describe a strategy to map accessible sites on sickle beta-globin (beta(s)-globin) transcripts in vitro and in vivo and to use this information to enhance RNA repair efficiency. Two sites upstream of the sickle mutation were identified as accessible in some fraction of the beta-globin RNA by mapping with a ribozyme library and the accessibility of those sites was assessed by in vitro cleavage analyses. Ribozymes targeting either site could only convert a certain fraction of the beta(s)-globin RNA to product but not drive the reaction to completion. However, cleavage and splicing reactions were driven further toward completion when the two ribozymes were both added to the reactions, suggesting that the substrate RNA is present in multiple conformations in vitro. These two ribozymes were each able to repair beta(s)-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Moreover, the relative accessibility of the targeted sites in vivo is as predicted by mapping and in vitro analyses. These results demonstrate that this novel RNA mapping strategy represents an effective means to determine the accessible regions of target RNAs and that combinations of trans-splicing ribozymes can be employed to enhance RNA repair efficiency of clinically relevant transcripts such as beta(s)-globin RNA.

Authors
Lan, N; Rooney, BL; Lee, SW; Howrey, RP; Smith, CA; Sullenger, BA
MLA Citation
Lan, N, Rooney, BL, Lee, SW, Howrey, RP, Smith, CA, and Sullenger, BA. "Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA." Mol Ther 2.3 (September 2000): 245-255.
PMID
10985955
Source
pubmed
Published In
Molecular Therapy
Volume
2
Issue
3
Publish Date
2000
Start Page
245
End Page
255
DOI
10.1006/mthe.2000.0125

Group II introns designed to insert into therapeutically relevant DNA target sites in human cells.

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.

Authors
Guo, H; Karberg, M; Long, M; Jones, JP; Sullenger, B; Lambowitz, AM
MLA Citation
Guo, H, Karberg, M, Long, M, Jones, JP, Sullenger, B, and Lambowitz, AM. "Group II introns designed to insert into therapeutically relevant DNA target sites in human cells." Science 289.5478 (July 21, 2000): 452-457.
PMID
10903206
Source
pubmed
Published In
Science
Volume
289
Issue
5478
Publish Date
2000
Start Page
452
End Page
457

Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts.

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

Authors
Watanabe, T; Sullenger, BA
MLA Citation
Watanabe, T, and Sullenger, BA. "Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts." Proc Natl Acad Sci U S A 97.15 (July 18, 2000): 8490-8494.
PMID
10890910
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
15
Publish Date
2000
Start Page
8490
End Page
8494
DOI
10.1073/pnas.150104097

An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies

A variety of gene therapy strategies are under development for the treatment of sickle cell anemia and other hemoglobinopathies. A number of alternative vectors have been developed to transfer and express the β-globin gene and other therapeutic molecules, but none has resulted in efficient transduction and stable long-term expression in primary hematopoietic cells. One reason for this problem is that most vectors are initially evaluated in immortalized cell lines which may not faithfully recapitulate the biology of primary erythroid cells. In order to provide a more relevant system for efficiently evaluating alternative vector constructs for β-globin disorders, we have developed (1) a simple method for generating primary human red blood cell (RBC) precursors in liquid culture established with mononuclear cells obtained from normal donors as well as patients with Hb SC disease; (2) a high titer retroviral vector which can be easily modified to optimize gene transfer and transgene expression; and (3) methods for transducing the RBC precursors at high efficiency. The development of simple and efficient methods and reagents for generating and transducing primary human RBC precursors provides a facile and effective means for screening alternative gene therapy strategies.

Authors
Howrey, RP; El-Alfondi, M; Phillips, KL; Wilson, L; Rooney, B; Lan, N; Sullenger, B; Smith, C
MLA Citation
Howrey, RP, El-Alfondi, M, Phillips, KL, Wilson, L, Rooney, B, Lan, N, Sullenger, B, and Smith, C. "An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies." Gene Therapy 7.3 (2000): 215-223.
PMID
10694798
Source
scival
Published In
Gene Therapy
Volume
7
Issue
3
Publish Date
2000
Start Page
215
End Page
223

Evaluating group I intron catalytic efficiency in mammalian cells.

Recent reports have demonstrated that the group I ribozyme from Tetrahymena thermophila can perform trans-splicing reactions to repair mutant RNAs. For therapeutic use, such ribozymes must function efficiently when transcribed from genes delivered to human cells, yet it is unclear how group I splicing reactions are influenced by intracellular expression of the ribozyme. Here we evaluate the self-splicing efficiency of group I introns from transcripts expressed by RNA polymerase II in human cells to directly measure ribozyme catalysis in a therapeutically relevant setting. Intron-containing expression cassettes were transfected into a human cell line, and RNA transcripts were analyzed for intron removal. The percentage of transcripts that underwent self-splicing ranged from 0 to 50%, depending on the construct being tested. Thus, self-splicing activity is supported in the mammalian cellular environment. However, we find that the extent of self-splicing is greatly influenced by sequences flanking the intron and presumably reflects differences in the intron's ability to fold into an active conformation inside the cell. In support of this hypothesis, we show that the ability of the intron to fold and self-splice from cellular transcripts in vitro correlates well with the catalytic efficiency observed from the same transcripts expressed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozymes. The self-splicing system that we describe should facilitate these efforts as well as aid in efforts at enhancing in vivo ribozyme activity for various applications of RNA repair.

Authors
Long, MB; Sullenger, BA
MLA Citation
Long, MB, and Sullenger, BA. "Evaluating group I intron catalytic efficiency in mammalian cells." Mol Cell Biol 19.10 (October 1999): 6479-6487.
PMID
10490588
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
10
Publish Date
1999
Start Page
6479
End Page
6487

RNA aptamers as potential anticoagulants directed against factor VIIa function

Authors
Rusconi, CP; Yeh, A; Lyerly, HK; Lawson, JH; Sullenger, BA
MLA Citation
Rusconi, CP, Yeh, A, Lyerly, HK, Lawson, JH, and Sullenger, BA. "RNA aptamers as potential anticoagulants directed against factor VIIa function." August 1999.
Source
wos-lite
Published In
Thrombosis and haemostasis
Publish Date
1999
Start Page
468
End Page
468

RNA repair as a novel approach to genetic therapy.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "RNA repair as a novel approach to genetic therapy." Gene Ther 6.4 (April 1999): 461-462.
PMID
10476204
Source
pubmed
Published In
Gene Therapy
Volume
6
Issue
4
Publish Date
1999
Start Page
461
End Page
462
DOI
10.1038/sj.gt.3300903

Optimizing the substrate specificity of a group I intron ribozyme.

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.

Authors
Zarrinkar, PP; Sullenger, BA
MLA Citation
Zarrinkar, PP, and Sullenger, BA. "Optimizing the substrate specificity of a group I intron ribozyme." Biochemistry 38.11 (March 16, 1999): 3426-3432.
PMID
10079089
Source
pubmed
Published In
Biochemistry
Volume
38
Issue
11
Publish Date
1999
Start Page
3426
End Page
3432
DOI
10.1021/bi982688m

Probing the interplay between the two steps of group I intron splicing: competition of exogenous guanosine with omega G.

One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.

Authors
Zarrinkar, PP; Sullenger, BA
MLA Citation
Zarrinkar, PP, and Sullenger, BA. "Probing the interplay between the two steps of group I intron splicing: competition of exogenous guanosine with omega G." Biochemistry 37.51 (December 22, 1998): 18056-18063.
PMID
9922174
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
51
Publish Date
1998
Start Page
18056
End Page
18063

Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors.

Sickle cell anemia is the most common heritable hematological disease, yet no curative treatment exists for this disorder. Moreover, the intricacies of globin gene expression have made the development of treatments for hemoglobinopathies based on gene therapy difficult. An alternative genetic approach to sickle cell therapy is based on RNA repair. A trans-splicing group I ribozyme was used to alter mutant beta-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Sickle beta-globin transcripts were converted into messenger RNAs encoding the anti-sickling protein gamma-globin. These results suggest that RNA repair may become a useful approach in the treatment of genetic disorders.

Authors
Lan, N; Howrey, RP; Lee, SW; Smith, CA; Sullenger, BA
MLA Citation
Lan, N, Howrey, RP, Lee, SW, Smith, CA, and Sullenger, BA. "Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors." Science 280.5369 (June 5, 1998): 1593-1596.
PMID
9616120
Source
pubmed
Published In
Science
Volume
280
Issue
5369
Publish Date
1998
Start Page
1593
End Page
1596

Evaluating and enhancing ribozyme reaction efficiency in mammalian cells.

The ability of ribozymes to cleave specific transcripts and repair defective RNAs in the test tube has engendered speculation about their potential clinical utility. Therapeutic development has been hindered by an inability to evaluate and optimize the efficiency of RNA catalysis in vivo. We describe an experimental system that has allowed us to assess and enhance the efficiency with which a trans-splicing group I ribozyme reacts with a targeted RNA in mammalian cells. These results demonstrate that the ribozyme can convert up to 49% of a specific substrate RNA to product in the cellular environment and that the efficiency of this reaction is apparently a function of the ribozyme's ability to find and bind to the substrate RNA in the cell. These observations suggest that trans-splicing ribozymes may become useful reagents to repair a therapeutically significant fraction of mutant RNAs associated with a variety of genetic diseases.

Authors
Jones, JT; Sullenger, BA
MLA Citation
Jones, JT, and Sullenger, BA. "Evaluating and enhancing ribozyme reaction efficiency in mammalian cells." Nat Biotechnol 15.9 (September 1997): 902-905.
PMID
9306408
Source
pubmed
Published In
Nature Biotechnology
Volume
15
Issue
9
Publish Date
1997
Start Page
902
End Page
905
DOI
10.1038/nbt0997-902

Isolation of a nuclease-resistant decoy RNA that can protect human acetylcholine receptors from myasthenic antibodies.

The muscular weakness and fatigability associated with myasthenia gravis are engendered by autoantibodies directed against acetylcholine receptors on muscle cells at neuromuscular junctions. The pathogenic consequences of this immune response can potentially be modulated by molecules that bind such autoantibodies and block their interaction with these receptors. We report the isolation of a small nuclease-resistant RNA molecule that binds both a rat monoclonal antibody that recognizes the main immunogenic region on the acetylcholine receptor, and autoantibodies from patients with myasthenia gravis. Moreover, this RNA can act as a decoy and protect acetylcholine receptors on human cells from the effects of these antibodies.

Authors
Lee, SW; Sullenger, BA
MLA Citation
Lee, SW, and Sullenger, BA. "Isolation of a nuclease-resistant decoy RNA that can protect human acetylcholine receptors from myasthenic antibodies." Nat Biotechnol 15.1 (January 1997): 41-45.
PMID
9035104
Source
pubmed
Published In
Nature Biotechnology
Volume
15
Issue
1
Publish Date
1997
Start Page
41
End Page
45
DOI
10.1038/nbt0197-41

Trans-splicing reactions by ribozymes.

Authors
Jones, JT; Lee, SW; Sullenger, BA
MLA Citation
Jones, JT, Lee, SW, and Sullenger, BA. "Trans-splicing reactions by ribozymes." Methods Mol Biol 74 (1997): 341-348.
PMID
9204448
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
74
Publish Date
1997
Start Page
341
End Page
348
DOI
10.1385/0-89603-389-9:341

Inhibition of cell proliferation by an RNA ligand that selectively blocks E2F function.

The control of cell proliferation is of central importance to the proper development of a multicellular organism, the homeostatic maintenance of tissues, and the ability of certain cell types to respond appropriately to environmental cues. Disruption of normal cell growth control underlies many pathological conditions, including endothelial proliferative disorders in cardiovascular disease as well as the development of malignant tumors. Particularly critical for the control of cell growth is the pathway involving the G1 cyclin-dependent kinases that regulate the Rb family of proteins, which in turn control E2F transcription factor activity. Because E2F is critical for regulation of cell proliferation, we sought to identify and to develop specific inhibitors of E2F function that might also be useful in the control of cellular proliferation. Moreover, because the control of E2F activity appears to be the end result of G1 regulatory cascades, the ability to inhibit E2F may be particularly effective in impeding a wide variety of proliferative events. We have used in vitro selection to isolate several unique RNA species from high complexity RNA libraries that avidly bind to the E2F family of proteins. These RNAs also inhibit the DNA binding capacity of the E2F proteins. We also show that an E2F RNA ligand can block the induction of S phase in quiescent cells stimulated by serum addition. As such, these data demonstrate the critical role for E2F activity in cell proliferation and suggest that such RNA molecules may be effective as therapeutic entities to control cellular proliferation.

Authors
Ishizaki, J; Nevins, JR; Sullenger, BA
MLA Citation
Ishizaki, J, Nevins, JR, and Sullenger, BA. "Inhibition of cell proliferation by an RNA ligand that selectively blocks E2F function." Nat Med 2.12 (December 1996): 1386-1389.
PMID
8946842
Source
pubmed
Published In
Nature Medicine
Volume
2
Issue
12
Publish Date
1996
Start Page
1386
End Page
1389

Ribozyme-mediated repair of RNAs encoding mutant tumor suppressors.

The observation that a number of genetic mutations can result in neoplastic transformation has led many investigators to speculate that gene therapy may represent a useful approach to treat cancer. Conceptually, this application of gene therapy seems quite simple: to treat cancer, restore the correctly regulated expression of the needed tumor suppressor genes inside tumor cells and revert the transformed phenotype of such cells. However, such regulated expression has been difficult to achieve in practice. Here we describe recent efforts at such restoration via a novel approach to gene therapy that involves ribozyme-mediated repair of mutant RNA transcripts.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Ribozyme-mediated repair of RNAs encoding mutant tumor suppressors." Cytokines Mol Ther 2.3 (September 1996): 201-205. (Review)
PMID
9384705
Source
pubmed
Published In
Cytokines and Molecular Therapy
Volume
2
Issue
3
Publish Date
1996
Start Page
201
End Page
205

Isolation of a nuclease-resistant decoy RNA that selectively blocks autoantibody binding to insulin receptors on human lymphocytes.

An RNA containing 2'-amino pyrimidines has been isolated using in vitro selection techniques that specifically and avidly (apparent Kd approximately 30 nM) binds a mouse monoclonal antibody called MA20. This 2'-amino-derivatized RNA is at least 10,000-fold more stable than unmodified RNA in serum, and can act as a decoy and block MA20 binding to its natural antigen, the human insulin receptor, on lymphocytes. Furthermore, this RNA decoy can inhibit MA20-mediated downmodulation of insulin receptor expression on human lymphocytes in culture by up to 90%. Surprisingly, the decoy RNA cross-reacts with autoantibodies from patients with extreme insulin resistance and can inhibit these antiinsulin receptor antibodies from downmodulating insulin receptor expression by up to 80% without impeding insulin binding to its receptor. These results suggest that in vitro-selected decoy RNAs may be able to specifically and selectively block oligoclonal autoimmune responses to self-antigens in patients with autoimmune diseases.

Authors
Lee, SW; Sullenger, BA
MLA Citation
Lee, SW, and Sullenger, BA. "Isolation of a nuclease-resistant decoy RNA that selectively blocks autoantibody binding to insulin receptors on human lymphocytes." J Exp Med 184.2 (August 1, 1996): 315-324.
PMID
8760785
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
184
Issue
2
Publish Date
1996
Start Page
315
End Page
324

Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells.

In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases.

Authors
Jones, JT; Lee, SW; Sullenger, BA
MLA Citation
Jones, JT, Lee, SW, and Sullenger, BA. "Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells." Nat Med 2.6 (June 1996): 643-648.
PMID
8640554
Source
pubmed
Published In
Nature Medicine
Volume
2
Issue
6
Publish Date
1996
Start Page
643
End Page
648

Colocalizing ribozymes with substrate RNAs to increase their efficacy as gene inhibitors.

The ability to target ribozymes to specifically cleave viral RNAs in vitro has led to much speculation about their potential therapeutic value as antiviral agents in vivo. To transfer a ribozyme's potential as an antiviral agent from test tubes to cells and organisms successfully, the characteristics that distinguish these settings must be considered. In vitro, ribozymes and substrate RNAs freely diffuse in solution in test tubes, and trans-cleavage reactions are dependent on a diffusive step. In eukaryotic cells, by contrast, many RNAs do not appear to diffuse freely. Instead, they appear to be highly compartmentalized and actively sorted to specific cellular locations. Such RNA trafficking may result in localization of substrate RNAs in a different compartment than ribozymes, which would effectively reduce substrate RNA availability to ribozymes and therefore limit the effectiveness of ribozymes as gene inhibitors.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Colocalizing ribozymes with substrate RNAs to increase their efficacy as gene inhibitors." Appl Biochem Biotechnol 54.1-3 (July 1995): 57-61. (Review)
PMID
7486985
Source
pubmed
Published In
Applied Biochemistry and Biotechnology
Volume
54
Issue
1-3
Publish Date
1995
Start Page
57
End Page
61

Revising messages traveling along the cellular information superhighway.

Cells use RNA messages to carry instructions from the genome to ribosomes about the types of proteins that should be made. These messages are generally revised by splicing before translation. Engineering ribozymes that can use splicing to repair mutant transcripts may be a useful approach to gene therapy of several genetic diseases.

Authors
Sullenger, BA
MLA Citation
Sullenger, BA. "Revising messages traveling along the cellular information superhighway." Chem Biol 2.5 (May 1995): 249-253. (Review)
PMID
9383426
Source
pubmed
Published In
Chemistry & Biology
Volume
2
Issue
5
Publish Date
1995
Start Page
249
End Page
253

Selection of an RNA molecule that mimics a major autoantigenic epitope of human insulin receptor.

Autoimmunity often involves the abnormal targeting of self-antigens by antibodies, leading to tissue destruction and other pathologies. This process could potentially be disrupted by small ligands that bind specifically to autoantibodies and inhibit their interaction with the target antigen. Here we report the identification of an RNA sequence that binds a mouse monoclonal antibody specific for an autoantigenic epitope of human insulin receptor. The RNA ligand binds specifically and with high affinity (apparent Kd congruent to 2 nM) to the anti-insulin receptor antibody and not to other mouse IgGs. The RNA can also act as a decoy, blocking the antibody from binding the insulin receptor. Thus, it probably binds near the combining site on the antibody. Strikingly, the RNA cross-reacts with autoantibodies from patients with extreme insulin resistance. One simple explanation is that the selected RNA may structurally mimic the antigenic epitope on the insulin receptor protein. These results suggest that decoy RNAs may be used in the treatment of autoimmune diseases.

Authors
Doudna, JA; Cech, TR; Sullenger, BA
MLA Citation
Doudna, JA, Cech, TR, and Sullenger, BA. "Selection of an RNA molecule that mimics a major autoantigenic epitope of human insulin receptor." Proc Natl Acad Sci U S A 92.6 (March 14, 1995): 2355-2359.
PMID
7534420
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
92
Issue
6
Publish Date
1995
Start Page
2355
End Page
2359

RIBOZYME-MEDIATED REPAIR OF DEFECTIVE MESSENGER-RNAS BY TARGETED TRANSSPLICING - RNA BASED MOLECULAR SURGERY

Authors
SULLENGER, BA
MLA Citation
SULLENGER, BA. "RIBOZYME-MEDIATED REPAIR OF DEFECTIVE MESSENGER-RNAS BY TARGETED TRANSSPLICING - RNA BASED MOLECULAR SURGERY." January 5, 1995.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1995
Start Page
206
End Page
206

AIDS and HIV infection.

Authors
Smith, C; Sullenger, BA
MLA Citation
Smith, C, and Sullenger, BA. "AIDS and HIV infection." Mol Cell Biol Hum Dis Ser 5 (1995): 195-236. (Review)
PMID
9532568
Source
pubmed
Published In
Molecular and cell biology of human diseases series
Volume
5
Publish Date
1995
Start Page
195
End Page
236

Alternative approaches for the application of ribozymes as gene therapies for retroviral infections.

Authors
Campbell, TB; Sullenger, BA
MLA Citation
Campbell, TB, and Sullenger, BA. "Alternative approaches for the application of ribozymes as gene therapies for retroviral infections." Adv Pharmacol 33 (1995): 143-178. (Review)
PMID
7495669
Source
pubmed
Published In
Advances in pharmacology (San Diego, Calif.)
Volume
33
Publish Date
1995
Start Page
143
End Page
178

STRUCTURE AND MECHANISM OF A GROUP-I RIBOZYME AND APPLICATIONS IN BIOTECHNOLOGY

Authors
CECH, TR; STROBEL, SA; SULLENGER, BA
MLA Citation
CECH, TR, STROBEL, SA, and SULLENGER, BA. "STRUCTURE AND MECHANISM OF A GROUP-I RIBOZYME AND APPLICATIONS IN BIOTECHNOLOGY." PROTEIN ENGINEERING 8 (1995): 16-16.
Source
wos-lite
Published In
Protein Engineering
Volume
8
Publish Date
1995
Start Page
16
End Page
16

Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing.

Ribozymes can be targeted to cleave specific RNAs, which has led to much interest in their potential as gene inhibitors. Such trans-cleaving ribozymes join a growing list of agents that stop the flow of genetic information. Here we describe a different application of ribozymes for which they may be uniquely suited. By targeted trans-splicing, a ribozyme can replace a defective portion of RNA with a functional sequence. The self-splicing intron from Tetrahymena thermophila was previously shown to mediate trans-splicing of oligonucleotides in vitro. As a model system for messenger RNA repair, this group I intron was re-engineered to regenerate the proper coding capacity of short, truncated lacZ transcripts. Trans-splicing was efficient in vitro and proceeded in Escherichia coli to generate translatable lacZ messages. Targeted trans-splicing represents a general means of altering the sequence of specified transcripts and may provide a new approach to the treatment of many genetic diseases.

Authors
Sullenger, BA; Cech, TR
MLA Citation
Sullenger, BA, and Cech, TR. "Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing." Nature 371.6498 (October 13, 1994): 619-622.
PMID
7935797
Source
pubmed
Published In
Nature
Volume
371
Issue
6498
Publish Date
1994
Start Page
619
End Page
622
DOI
10.1038/371619a0

Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA.

Cellular compartmentalization of RNAs is thought to influence their susceptibility to ribozyme cleavage. As a test of this idea, two retroviral vectors--one encoding a hammer-head ribozyme designed to cleave lacZ transcripts and another encoding the lacZ messenger RNA--were coexpressed inside retroviral packaging cells. Because of the retroviral packaging signal, the ribozyme would be expected to colocalize with the lacZ-containing viral genomic RNA but not with the lacZ messenger RNA. The ribozyme was found to reduce the titer of infectious virus containing lacZ by 90 percent, but had no effect on translation of lacZ messenger RNA. These results indicate that sorting gene inhibitors to appropriate intracellular sites may increase their effectiveness.

Authors
Sullenger, BA; Cech, TR
MLA Citation
Sullenger, BA, and Cech, TR. "Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA." Science 262.5139 (December 3, 1993): 1566-1569.
PMID
8248806
Source
pubmed
Published In
Science
Volume
262
Issue
5139
Publish Date
1993
Start Page
1566
End Page
1569

Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells.

Overexpression of sequences corresponding to the major Rev-binding site in the Rev response element of human immunodeficiency virus type 1 (HIV-1) (RRE decoys) was used to render cells resistant to HIV-1 replication. This was accomplished by the use of a chimeric tRNA-RRE transcription unit in a double-copy murine retroviral vector to express high levels of HIV-1 RRE-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited more than 90% in cells expressing chimeric tRNA-RRE transcripts, as determined by in situ immunofluorescence analysis and a p24 antigen ELISA test. Analysis of RNA from HIV-1-infected cells suggests that expression of RRE-containing sequences in CEM SS cells inhibits HIV-1 replication by interfering with Rev function, presumably by competing for Rev binding to its physiological target. The use of a subfragment of RRE as decoy RNA reduces the likelihood that essential cellular factors will be sequestered in cells expressing the decoy RNA. Thus, use of RRE-based decoy RNA to inhibit HIV-1 replication may represent a safer alternative to the use of TAR decoy RNA.

Authors
Lee, TC; Sullenger, BA; Gallardo, HF; Ungers, GE; Gilboa, E
MLA Citation
Lee, TC, Sullenger, BA, Gallardo, HF, Ungers, GE, and Gilboa, E. "Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells." New Biol 4.1 (January 1992): 66-74.
PMID
1536832
Source
pubmed
Published In
The New biologist
Volume
4
Issue
1
Publish Date
1992
Start Page
66
End Page
74

Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS.

Authors
Sullenger, BA; Gallardo, HF; Ungers, GE; Gilboa, E
MLA Citation
Sullenger, BA, Gallardo, HF, Ungers, GE, and Gilboa, E. "Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation." J Virol 65.12 (December 1991): 6811-6816.
PMID
1942253
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
12
Publish Date
1991
Start Page
6811
End Page
6816

Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies.

Authors
Sullenger, BA; Lee, TC; Smith, CA; Ungers, GE; Gilboa, E
MLA Citation
Sullenger, BA, Lee, TC, Smith, CA, Ungers, GE, and Gilboa, E. "Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication." Mol Cell Biol 10.12 (December 1990): 6512-6523.
PMID
2247070
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
10
Issue
12
Publish Date
1990
Start Page
6512
End Page
6523

Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication.

Overexpression of TAR-containing sequences (TAR decoys) was used to render cells resistant to HIV replication. A chimeric tRNA(meti)-TAR transcription unit contained in a double copy murine retroviral vector was used to express high levels of HIV-1 TAR-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited over 99% in cells expressing chimeric tRNA-TAR transcripts, but an amphotropic murine retrovirus replicated normally in these cells. Expression of TAR sequences in CEM SS cells had no adverse effects on cell viability, indicating that essential cellular factors are not being sequestered in these cells. TAR decoy RNA-mediated HIV inhibition may also be effective against natural HIV isolates in spite of their hypervariable nature, as suggested by the fact that replication of SIVmac was also inhibited in cells expressing HIV-1 TAR decoys.

Authors
Sullenger, BA; Gallardo, HF; Ungers, GE; Gilboa, E
MLA Citation
Sullenger, BA, Gallardo, HF, Ungers, GE, and Gilboa, E. "Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication." Cell 63.3 (November 2, 1990): 601-608.
PMID
2225067
Source
pubmed
Published In
Cell
Volume
63
Issue
3
Publish Date
1990
Start Page
601
End Page
608

Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.

Authors
Hantzopoulos, PA; Sullenger, BA; Ungers, G; Gilboa, E
MLA Citation
Hantzopoulos, PA, Sullenger, BA, Ungers, G, and Gilboa, E. "Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector." Proc Natl Acad Sci U S A 86.10 (May 1989): 3519-3523.
PMID
2542934
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
10
Publish Date
1989
Start Page
3519
End Page
3523
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