You are here

Gersbach, Charles

Positions:

Rooney Family Associate Professor of Biomedical Engineering

Biomedical Engineering
Pratt School of Engineering

Associate Professor of Biomedical Engineering

Biomedical Engineering
Pratt School of Engineering

Associate Professor of Orthopaedic Surgery

Orthopaedics
School of Medicine

Affiliate of the Duke Initiative for Science & Society

Duke Science & Society
Institutes and Provost's Academic Units

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 2001

B.S. — Georgia Institute of Technology

Ph.D. 2006

Ph.D. — Georgia Institute of Technology

News:

Grants:

CRISPR/Cas9-Based Gene Editing for the Correction of Duchenne Muscular Dystrophy

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2016
End Date
March 31, 2021

Human EGFRvIII-specific BiTE for the treatment of Glioblastoma

Administered By
Neurosurgery
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
July 01, 2015
End Date
June 30, 2020

Investigating Autophagy in GSD-Ia

Administered By
Pediatrics, Medical Genetics
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
December 15, 2015
End Date
November 30, 2019

Treating Duchenne Cardiomyopathy in the Mouse Model by Gene Repair

Administered By
Biomedical Engineering
AwardedBy
University of Missouri
Role
Principal Investigator
Start Date
August 01, 2016
End Date
July 31, 2019

Systemic EGFRvIII-targeted bispecific antibody as immunotherapy for glioblastoma

Administered By
Neurosurgery
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
March 01, 2015
End Date
February 28, 2019

Defining the Mechanisms of Gene Regulation and Drug Resistance in Cancer

Administered By
Biomedical Engineering
AwardedBy
Thorek Memorial Foundation
Role
Principal Investigator
Start Date
February 01, 2017
End Date
January 31, 2019

Genome engineering for Duchenne muscular dystrophy

Administered By
Biomedical Engineering
AwardedBy
American Heart Association
Role
Principal Investigator
Start Date
January 01, 2017
End Date
December 31, 2018

Trans-regulatory mechanisms of glucocorticoid-mediated transcriptional repression

Administered By
Biostatistics & Bioinformatics
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
September 01, 2016
End Date
August 31, 2018

Engineering Novel Genome Engineering Systems

Administered By
Biomedical Engineering
AwardedBy
Locus Biosciences, Inc.
Role
Principal Investigator
Start Date
July 01, 2016
End Date
June 30, 2018

Engineering Targeted Epigenetic Modifiers for Precise Control of Gene Regulation

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 15, 2013
End Date
May 31, 2018

In Vivo Epigenome Editing with CRISPR-Based Histone Acetyltransferase Transgenic Mice

Administered By
Neurobiology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
April 01, 2016
End Date
March 31, 2018

Decoding and Reprogramming the Corticosteroid Transcriptional Regulatory Network

Administered By
Biostatistics & Bioinformatics
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
January 05, 2015
End Date
November 30, 2017

A Platform Technology for High-Throughput Screening of Gene Regulatory Elements

Administered By
Biomedical Engineering
AwardedBy
Element Genomics, Inc.
Role
Principal Investigator
Start Date
November 15, 2016
End Date
November 14, 2017

Functional Tissue Engineering of Cartilage Using Induced Pluripotent Stem Cells

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 01, 2015
End Date
August 31, 2017

MD140071 Multiplex CRISPR/Cas9-Based Genome Engineering for the Genetic Correction of Duchenne Muscular Dystrophy

Administered By
Biomedical Engineering
AwardedBy
United States Army Medical Research and Materiel Command
Role
Principal Investigator
Start Date
September 01, 2015
End Date
August 31, 2017

CRISPR/Cas9-based genome editing in mouse models of Duchenne muscular dystrophy

Administered By
Biomedical Engineering
AwardedBy
Hartwell Foundation
Role
Principal Investigator
Start Date
July 01, 2015
End Date
June 30, 2017

Scaffold-Mediated Gene Delivery for Engineering of Osteochondral Tissues

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2015
End Date
June 30, 2017

Circulatory system and integrated muscle tissue for drug and tissue toxicity

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
July 24, 2012
End Date
June 30, 2017

University Training Program in Biomolecular and Tissue Engineering

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1994
End Date
June 30, 2017

Gertude B. Elion Mentored Medical Research Award

Administered By
School of Medicine
AwardedBy
Triangle Community Foundation
Role
Principal Investigator
Start Date
June 01, 2016
End Date
June 01, 2017

Genome Editing of Stem Cells for Analysis of Osteoarthritis Causal Variants

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
April 01, 2014
End Date
March 31, 2017

CAREER: Photoregulated Gene Expression for Spatiotemporal Control of Morphogenesis

Administered By
Biomedical Engineering
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
April 01, 2012
End Date
March 31, 2017

Engineering Morphogenetic Factors for Enhanced Genetic Reprogramming

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2011
End Date
June 30, 2016

Engineering New Biological Therapies for Arthritis

Administered By
Orthopaedics
AwardedBy
Arthritis Foundation
Role
Collaborator
Start Date
May 01, 2014
End Date
December 31, 2015

Live-Animal Micro-CT System

Administered By
Orthopaedics
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2012
End Date
May 14, 2014

Spatially Controlled Gene Delivery of Morphogenetic Factors from Woven Scaffolds

Administered By
Biomedical Engineering
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2011
End Date
March 31, 2014
Show More

Publications:

Expanding the CRISPR Toolbox: Targeting RNA with Cas13b

© 2017 Elsevier Inc.In this issue of Molecular Cell, Smargon et al. (2017) unearth Cas13b from type VI-B CRISPR-Cas immune systems and characterize its RNA-guided, RNA-targeting activity, including regulation by the novel co-factors Csx27 and Csx28, as well as non-specific collateral RNA damage.

Authors
Barrangou, R; Gersbach, CA
MLA Citation
Barrangou, R, and Gersbach, CA. "Expanding the CRISPR Toolbox: Targeting RNA with Cas13b." Molecular Cell 65.4 (February 16, 2017): 582-584.
Source
scopus
Published In
Molecular Cell
Volume
65
Issue
4
Publish Date
2017
Start Page
582
End Page
584
DOI
10.1016/j.molcel.2017.02.002

CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments.

Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and pro-inflammatory signaling, in cell and pathway specific manners are needed. In this study we demonstrate the use of CRISPR-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investigated the functional outcomes of repression of these genes. Efficient signaling regulation was demonstrated in engineered hADSCs, as activity of the downstream transcription factor NF-κB was significantly reduced or maintained at baseline levels in the presence of TNF-α or IL-1β. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-α or IL-1β, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-α or IL-1β, as shown by superior production of glycosaminglycans in this inflammatory environment. Overall this work demonstrates a novel method for modulating cell response to inflammatory signaling that has applications in engineering cells delivered to inflammatory environments, and as a direct gene therapy to protect endogenous cells exposed to chronic inflammation, as observed in a broad spectrum of degenerative musculoskeletal pathology.

Authors
Farhang, N; Brunger, JM; Stover, JD; Thakore, PI; Lawrence, B; Guilak, F; Gersbach, CA; Setton, LA; Bowles, RD
MLA Citation
Farhang, N, Brunger, JM, Stover, JD, Thakore, PI, Lawrence, B, Guilak, F, Gersbach, CA, Setton, LA, and Bowles, RD. "CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments." Tissue engineering. Part A (January 17, 2017).
PMID
28095751
Source
epmc
Published In
Tissue Engineering, Part A
Publish Date
2017
DOI
10.1089/ten.tea.2016.0441

Genetic engineering: Chemical control for CRISPR editing

Authors
Hilton, IB; Gersbach, CA
MLA Citation
Hilton, IB, and Gersbach, CA. "Genetic engineering: Chemical control for CRISPR editing." Nature Chemical Biology 13.1 (November 7, 2016): 2-3.
Source
crossref
Published In
Nature Chemical Biology
Volume
13
Issue
1
Publish Date
2016
Start Page
2
End Page
3
DOI
10.1038/nchembio.2243

CRISPR/Cas9 editing of induced pluripotent stem cells for engineering inflammation-resistant tissues.

Pro-inflammatory cytokines such as interleukin 1 (IL-1) are elevated in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. The goals of this study were to engineer inflammation-resistant induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues.Targeted deletion of the interleukin 1 receptor 1 (Il1r1) gene in murine iPSCs was achieved using the RNA-guided, site-specific CRISPR/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histological, and biomechanical assays after a three day treatment with 1 ng/ml IL-1α.Three of 41 clones isolated possessed the Il1r1(+/-) genotype. Four clones possessed the Il1r1(-/-) genotype, and flow cytometry confirmed loss of Il1r1 on the surface of these cells and led to an absence of NF-κB transcriptional activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. By contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade.This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to pro-inflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering. This article is protected by copyright. All rights reserved.

Authors
Brunger, JM; Zutshi, A; Willard, VP; Gersbach, CA; Guilak, F
MLA Citation
Brunger, JM, Zutshi, A, Willard, VP, Gersbach, CA, and Guilak, F. "CRISPR/Cas9 editing of induced pluripotent stem cells for engineering inflammation-resistant tissues." Arthritis & rheumatology (Hoboken, N.J.) (November 3, 2016).
PMID
27813286
Source
epmc
Published In
Arthritis and Rheumatology
Publish Date
2016
DOI
10.1002/art.39982

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

Authors
Housden, BE; Muhar, M; Gemberling, M; Gersbach, CA; Stainier, DYR; Seydoux, G; Mohr, SE; Zuber, J; Perrimon, N
MLA Citation
Housden, BE, Muhar, M, Gemberling, M, Gersbach, CA, Stainier, DYR, Seydoux, G, Mohr, SE, Zuber, J, and Perrimon, N. "Loss-of-function genetic tools for animal models: cross-species and cross-platform differences." Nature Reviews Genetics 18.1 (October 31, 2016): 24-40.
Source
crossref
Published In
Nature Reviews Genetics
Volume
18
Issue
1
Publish Date
2016
Start Page
24
End Page
40
DOI
10.1038/nrg.2016.118

Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells.

Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Authors
Black, JB; Adler, AF; Wang, H-G; D'Ippolito, AM; Hutchinson, HA; Reddy, TE; Pitt, GS; Leong, KW; Gersbach, CA
MLA Citation
Black, JB, Adler, AF, Wang, H-G, D'Ippolito, AM, Hutchinson, HA, Reddy, TE, Pitt, GS, Leong, KW, and Gersbach, CA. "Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells." Cell stem cell 19.3 (September 2016): 406-414.
PMID
27524438
Source
epmc
Published In
Cell Stem Cell
Volume
19
Issue
3
Publish Date
2016
Start Page
406
End Page
414
DOI
10.1016/j.stem.2016.07.001

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

Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy

Authors
Robinson-Hamm, JN; Gersbach, CA
MLA Citation
Robinson-Hamm, JN, and Gersbach, CA. "Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy." Human Genetics 135.9 (September 2016): 1029-1040.
Source
crossref
Published In
Human Genetics
Volume
135
Issue
9
Publish Date
2016
Start Page
1029
End Page
1040
DOI
10.1007/s00439-016-1725-z

Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing.

Biological resurfacing of entire articular surfaces represents an important but challenging strategy for treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. The goal of this study was to use adult stem cells to engineer anatomically shaped, functional cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). Large (22-mm-diameter) hemispherical scaffolds were fabricated from 3D woven poly(ε-caprolactone) (PCL) fibers into two different configurations and seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing eGFP or IL-1Ra transgenes were immobilized to the PCL to transduce ASCs upon seeding, and constructs were cultured in chondrogenic conditions for 28 d. Constructs showed biomimetic cartilage properties and uniform tissue growth while maintaining their anatomic shape throughout culture. IL-1Ra-expressing constructs produced nearly 1 µg/mL of IL-1Ra upon controlled induction with dox. Treatment with IL-1 significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra-expressing constructs. Our findings show that advanced textile manufacturing combined with scaffold-mediated gene delivery can be used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function.

Authors
Moutos, FT; Glass, KA; Compton, SA; Ross, AK; Gersbach, CA; Guilak, F; Estes, BT
MLA Citation
Moutos, FT, Glass, KA, Compton, SA, Ross, AK, Gersbach, CA, Guilak, F, and Estes, BT. "Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing." Proceedings of the National Academy of Sciences of the United States of America 113.31 (August 2016): E4513-E4522.
PMID
27432980
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
31
Publish Date
2016
Start Page
E4513
End Page
E4522
DOI
10.1073/pnas.1601639113

N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions.

Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues.

Authors
Hwang, PY; Jing, L; Chen, J; Lim, F-L; Tang, R; Choi, H; Cheung, KM; Risbud, MV; Gersbach, CA; Guilak, F; Leung, VY; Setton, LA
MLA Citation
Hwang, PY, Jing, L, Chen, J, Lim, F-L, Tang, R, Choi, H, Cheung, KM, Risbud, MV, Gersbach, CA, Guilak, F, Leung, VY, and Setton, LA. "N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions." Scientific reports 6 (June 13, 2016): 28038-.
PMID
27292569
Source
epmc
Published In
Scientific Reports
Volume
6
Publish Date
2016
Start Page
28038
DOI
10.1038/srep28038

Engineering Delivery Vehicles for Genome Editing

Authors
Nelson, CE; Gersbach, CA
MLA Citation
Nelson, CE, and Gersbach, CA. "Engineering Delivery Vehicles for Genome Editing." Annual Review of Chemical and Biomolecular Engineering 7.1 (June 7, 2016): 637-662.
Source
crossref
Published In
Annual Review of Chemical and Biomolecular Engineering
Volume
7
Issue
1
Publish Date
2016
Start Page
637
End Page
662
DOI
10.1146/annurev-chembioeng-080615-034711

In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA.

Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia.

Authors
Landau, DJ; Brooks, ED; Perez-Pinera, P; Amarasekara, H; Mefferd, A; Li, S; Bird, A; Gersbach, CA; Koeberl, DD
MLA Citation
Landau, DJ, Brooks, ED, Perez-Pinera, P, Amarasekara, H, Mefferd, A, Li, S, Bird, A, Gersbach, CA, and Koeberl, DD. "In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA." Molecular therapy : the journal of the American Society of Gene Therapy 24.4 (April 2016): 697-706.
PMID
26865405
Source
epmc
Published In
Molecular Therapy
Volume
24
Issue
4
Publish Date
2016
Start Page
697
End Page
706
DOI
10.1038/mt.2016.35

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.

Authors
Josephs, EA; Kocak, DD; Fitzgibbon, CJ; McMenemy, J; Gersbach, CA; Marszalek, PE
MLA Citation
Josephs, EA, Kocak, DD, Fitzgibbon, CJ, McMenemy, J, Gersbach, CA, and Marszalek, PE. "Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage." Nucleic acids research 44.5 (March 2016): 2474-.
PMID
26578578
Source
epmc
Published In
Nucleic Acids Research
Volume
44
Issue
5
Publish Date
2016
Start Page
2474
DOI
10.1093/nar/gkv1293

Genome-editing Technologies for Gene and Cell Therapy.

Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

Authors
Maeder, ML; Gersbach, CA
MLA Citation
Maeder, ML, and Gersbach, CA. "Genome-editing Technologies for Gene and Cell Therapy." Molecular therapy : the journal of the American Society of Gene Therapy 24.3 (March 2016): 430-446. (Review)
PMID
26755333
Source
epmc
Published In
Molecular Therapy
Volume
24
Issue
3
Publish Date
2016
Start Page
430
End Page
446
DOI
10.1038/mt.2016.10

Cas9 loosens its grip on off-target sites.

Authors
Nelson, CE; Gersbach, CA
MLA Citation
Nelson, CE, and Gersbach, CA. "Cas9 loosens its grip on off-target sites." Nature biotechnology 34.3 (March 2016): 298-299.
PMID
26963555
Source
epmc
Published In
Nature Biotechnology
Volume
34
Issue
3
Publish Date
2016
Start Page
298
End Page
299
DOI
10.1038/nbt.3501

Editing the epigenome: technologies for programmable transcription and epigenetic modulation

Authors
Thakore, PI; Black, JB; Hilton, IB; Gersbach, CA
MLA Citation
Thakore, PI, Black, JB, Hilton, IB, and Gersbach, CA. "Editing the epigenome: technologies for programmable transcription and epigenetic modulation." Nature Methods 13.2 (January 28, 2016): 127-137.
Source
crossref
Published In
Nature Methods
Volume
13
Issue
2
Publish Date
2016
Start Page
127
End Page
137
DOI
10.1038/nmeth.3733

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

Authors
Thakore, PI; Gersbach, CA
MLA Citation
Thakore, PI, and Gersbach, CA. "Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors." Methods in molecular biology (Clifton, N.J.) 1338 (January 2016): 71-88.
PMID
26443215
Source
epmc
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
1338
Publish Date
2016
Start Page
71
End Page
88
DOI
10.1007/978-1-4939-2932-0_7

The Development of TALE Nucleases for Biotechnology.

The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

Authors
Ousterout, DG; Gersbach, CA
MLA Citation
Ousterout, DG, and Gersbach, CA. "The Development of TALE Nucleases for Biotechnology." Methods in molecular biology (Clifton, N.J.) 1338 (January 2016): 27-42.
PMID
26443211
Source
epmc
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
1338
Publish Date
2016
Start Page
27
End Page
42
DOI
10.1007/978-1-4939-2932-0_3

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.

Authors
Nelson, CE; Hakim, CH; Ousterout, DG; Thakore, PI; Moreb, EA; Castellanos Rivera, RM; Madhavan, S; Pan, X; Ran, FA; Yan, WX; Asokan, A; Zhang, F; Duan, D; Gersbach, CA
MLA Citation
Nelson, CE, Hakim, CH, Ousterout, DG, Thakore, PI, Moreb, EA, Castellanos Rivera, RM, Madhavan, S, Pan, X, Ran, FA, Yan, WX, Asokan, A, Zhang, F, Duan, D, and Gersbach, CA. "In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy." Science (New York, N.Y.) 351.6271 (January 2016): 403-407.
PMID
26721684
Source
epmc
Published In
Science
Volume
351
Issue
6271
Publish Date
2016
Start Page
403
End Page
407
DOI
10.1126/science.aad5143

Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

Authors
Thakore, PI; D'Ippolito, AM; Song, L; Safi, A; Shivakumar, NK; Kabadi, AM; Reddy, TE; Crawford, GE; Gersbach, CA
MLA Citation
Thakore, PI, D'Ippolito, AM, Song, L, Safi, A, Shivakumar, NK, Kabadi, AM, Reddy, TE, Crawford, GE, and Gersbach, CA. "Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements." Nature methods 12.12 (December 2015): 1143-1149.
PMID
26501517
Source
epmc
Published In
Nature Methods
Volume
12
Issue
12
Publish Date
2015
Start Page
1143
End Page
1149
DOI
10.1038/nmeth.3630

Genome editing: the end of the beginning

Authors
Doudna, JA; Gersbach, CA
MLA Citation
Doudna, JA, and Gersbach, CA. "Genome editing: the end of the beginning." Genome Biology 16.1 (December 2015).
Source
crossref
Published In
Genome Biology
Volume
16
Issue
1
Publish Date
2015
DOI
10.1186/s13059-015-0860-5

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage.

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single 'guide RNA' molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a 'conformational gating' mechanism driven by the interactions between the guide RNA and the 14th-17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.

Authors
Josephs, EA; Kocak, DD; Fitzgibbon, CJ; McMenemy, J; Gersbach, CA; Marszalek, PE
MLA Citation
Josephs, EA, Kocak, DD, Fitzgibbon, CJ, McMenemy, J, Gersbach, CA, and Marszalek, PE. "Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage." Nucleic acids research 43.18 (October 2015): 8924-8941.
PMID
26384421
Source
epmc
Published In
Nucleic Acids Research
Volume
43
Issue
18
Publish Date
2015
Start Page
8924
End Page
8941
DOI
10.1093/nar/gkv892

Enabling functional genomics with genome engineering.

Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances.

Authors
Hilton, IB; Gersbach, CA
MLA Citation
Hilton, IB, and Gersbach, CA. "Enabling functional genomics with genome engineering." Genome research 25.10 (October 2015): 1442-1455. (Review)
PMID
26430154
Source
epmc
Published In
Genome research
Volume
25
Issue
10
Publish Date
2015
Start Page
1442
End Page
1455
DOI
10.1101/gr.190124.115

Anatomically-Shaped Tissue-Engineered Cartilage with Tunable and Inducible Anti-Inflammatory Capabilities

Authors
Glass, KA; Ross, AK; Compton, SA; Gersbach, CA; Moutos, FT; Estes, BT; Guilak, F
MLA Citation
Glass, KA, Ross, AK, Compton, SA, Gersbach, CA, Moutos, FT, Estes, BT, and Guilak, F. "Anatomically-Shaped Tissue-Engineered Cartilage with Tunable and Inducible Anti-Inflammatory Capabilities." September 1, 2015.
Source
wos-lite
Published In
Tissue Engineering, Part A
Volume
21
Publish Date
2015
Start Page
S330
End Page
S330

CRISPRi Immunomodulation for Tissue Engineering/Stem Cell Therapies Targeting Intervertebral Disc Degeneration

Authors
Farhang, N; Brunger, JM; Stover, JD; Thakore, PI; Lawrence, BD; Guilak, F; Gersbach, CA; Setton, LA; Bowles, RD
MLA Citation
Farhang, N, Brunger, JM, Stover, JD, Thakore, PI, Lawrence, BD, Guilak, F, Gersbach, CA, Setton, LA, and Bowles, RD. "CRISPRi Immunomodulation for Tissue Engineering/Stem Cell Therapies Targeting Intervertebral Disc Degeneration." September 1, 2015.
Source
wos-lite
Published In
Tissue Engineering, Part A
Volume
21
Publish Date
2015
Start Page
S170
End Page
S170

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

Authors
Polstein, LR; Perez-Pinera, P; Kocak, DD; Vockley, CM; Bledsoe, P; Song, L; Safi, A; Crawford, GE; Reddy, TE; Gersbach, CA
MLA Citation
Polstein, LR, Perez-Pinera, P, Kocak, DD, Vockley, CM, Bledsoe, P, Song, L, Safi, A, Crawford, GE, Reddy, TE, and Gersbach, CA. "Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators." Genome research 25.8 (August 2015): 1158-1169.
PMID
26025803
Source
epmc
Published In
Genome research
Volume
25
Issue
8
Publish Date
2015
Start Page
1158
End Page
1169
DOI
10.1101/gr.179044.114

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

Authors
Polstein, LR; Perez-Pinera, P; Kocak, DD; Vockley, CM; Bledsoe, P; Song, L; Safi, A; Crawford, GE; Reddy, TE; Gersbach, CA
MLA Citation
Polstein, LR, Perez-Pinera, P, Kocak, DD, Vockley, CM, Bledsoe, P, Song, L, Safi, A, Crawford, GE, Reddy, TE, and Gersbach, CA. "Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators." GENOME RESEARCH 25.8 (August 2015): 1158-1169.
Source
wos-lite
Published In
Genome research
Volume
25
Issue
8
Publish Date
2015
Start Page
1158
End Page
1169
DOI
10.1101/gr.179044.114

Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

Authors
Kabadi, AM; Thakore, PI; Vockley, CM; Ousterout, DG; Gibson, TM; Guilak, F; Reddy, TE; Gersbach, CA
MLA Citation
Kabadi, AM, Thakore, PI, Vockley, CM, Ousterout, DG, Gibson, TM, Guilak, F, Reddy, TE, and Gersbach, CA. "Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain." ACS synthetic biology 4.6 (June 2015): 689-699.
PMID
25494287
Source
epmc
Published In
ACS Synthetic Biology
Volume
4
Issue
6
Publish Date
2015
Start Page
689
End Page
699
DOI
10.1021/sb500322u

Single-molecule analysis of myocyte differentiation reveals bimodal lineage commitment.

Cell differentiation is the foundation for tissue development and regeneration, disease modeling, and cell-based therapies. Although the differentiation of cell populations has been extensively studied in many systems, much less is known about the distribution of decision making of single cells within these populations. To characterize the differentiation of single skeletal muscle cells, we used single-molecule mRNA fluorescence in situ hybridization (smFISH) to precisely quantify the expression levels of the master myogenic regulatory factors MyoD and myogenin in individual myoblasts. We identified distinct cell states characterized by the number of myogenin transcripts expressed by a cell, with myoblasts stochastically transitioning to a myogenin-high state during differentiation. We also used MyoD overexpression to force the transdifferentiation of C3H10T1/2 cells into an induced myoblast phenotype. These reprogrammed cells revealed the presence of a critical threshold of MyoD expression required to initiate myogenin expression. These results provide quantitative single-molecule data to support the model of switch-like cell decision making and lineage specification.

Authors
Gibson, TM; Gersbach, CA
MLA Citation
Gibson, TM, and Gersbach, CA. "Single-molecule analysis of myocyte differentiation reveals bimodal lineage commitment." Integrative biology : quantitative biosciences from nano to macro 7.6 (June 2015): 663-671.
PMID
25953198
Source
epmc
Published In
Integrative Biology
Volume
7
Issue
6
Publish Date
2015
Start Page
663
End Page
671
DOI
10.1039/c5ib00057b

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

Authors
Hilton, IB; D'Ippolito, AM; Vockley, CM; Thakore, PI; Crawford, GE; Reddy, TE; Gersbach, CA
MLA Citation
Hilton, IB, D'Ippolito, AM, Vockley, CM, Thakore, PI, Crawford, GE, Reddy, TE, and Gersbach, CA. "Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers." Nature biotechnology 33.5 (May 2015): 510-517.
PMID
25849900
Source
epmc
Published In
Nature Biotechnology
Volume
33
Issue
5
Publish Date
2015
Start Page
510
End Page
517
DOI
10.1038/nbt.3199

Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum.

To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated, and verified, using H3K27ac ChIP-seq, reporter gene assays and CRISPR-mediated activation, that many of these regions function as neuronal enhancers. Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns. Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

Authors
Frank, CL; Liu, F; Wijayatunge, R; Song, L; Biegler, MT; Yang, MG; Vockley, CM; Safi, A; Gersbach, CA; Crawford, GE; West, AE
MLA Citation
Frank, CL, Liu, F, Wijayatunge, R, Song, L, Biegler, MT, Yang, MG, Vockley, CM, Safi, A, Gersbach, CA, Crawford, GE, and West, AE. "Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum." Nature neuroscience 18.5 (May 2015): 647-656.
PMID
25849986
Source
epmc
Published In
Nature Neuroscience
Volume
18
Issue
5
Publish Date
2015
Start Page
647
End Page
656
DOI
10.1038/nn.3995

Multiplex Gene Activation by CRISPR/Cas9-Based Transcription Factors for the Direct Conversion of Fibroblasts to a Neuronal Phenotype

Authors
Black, J; Adler, A; Hutchinson, H; Wang, H; Pitt, G; Leong, K; Gersbach, C
MLA Citation
Black, J, Adler, A, Hutchinson, H, Wang, H, Pitt, G, Leong, K, and Gersbach, C. "Multiplex Gene Activation by CRISPR/Cas9-Based Transcription Factors for the Direct Conversion of Fibroblasts to a Neuronal Phenotype." May 2015.
Source
wos-lite
Published In
Molecular Therapy
Volume
23
Publish Date
2015
Start Page
S26
End Page
S26

Knockdown of the cell cycle inhibitor p21 enhances cartilage formation by induced pluripotent stem cells.

The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering.

Authors
Diekman, BO; Thakore, PI; O'Connor, SK; Willard, VP; Brunger, JM; Christoforou, N; Leong, KW; Gersbach, CA; Guilak, F
MLA Citation
Diekman, BO, Thakore, PI, O'Connor, SK, Willard, VP, Brunger, JM, Christoforou, N, Leong, KW, Gersbach, CA, and Guilak, F. "Knockdown of the cell cycle inhibitor p21 enhances cartilage formation by induced pluripotent stem cells." Tissue engineering. Part A 21.7-8 (April 2015): 1261-1274.
PMID
25517798
Source
epmc
Published In
Tissue Engineering, Part A
Volume
21
Issue
7-8
Publish Date
2015
Start Page
1261
End Page
1274
DOI
10.1089/ten.tea.2014.0240

Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.

Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

Authors
Ousterout, DG; Kabadi, AM; Thakore, PI; Perez-Pinera, P; Brown, MT; Majoros, WH; Reddy, TE; Gersbach, CA
MLA Citation
Ousterout, DG, Kabadi, AM, Thakore, PI, Perez-Pinera, P, Brown, MT, Majoros, WH, Reddy, TE, and Gersbach, CA. "Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases." Molecular therapy : the journal of the American Society of Gene Therapy 23.3 (March 2015): 523-532.
PMID
25492562
Source
epmc
Published In
Molecular Therapy
Volume
23
Issue
3
Publish Date
2015
Start Page
523
End Page
532
DOI
10.1038/mt.2014.234

A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.

Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.

Authors
Polstein, LR; Gersbach, CA
MLA Citation
Polstein, LR, and Gersbach, CA. "A light-inducible CRISPR-Cas9 system for control of endogenous gene activation." Nature chemical biology 11.3 (March 2015): 198-200.
PMID
25664691
Source
epmc
Published In
Nature Chemical Biology
Volume
11
Issue
3
Publish Date
2015
Start Page
198
End Page
200
DOI
10.1038/nchembio.1753

Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

Authors
Ousterout, DG; Kabadi, AM; Thakore, PI; Perez-Pinera, P; Brown, MT; Majoros, WH; Reddy, TE; Gersbach, CA
MLA Citation
Ousterout, DG, Kabadi, AM, Thakore, PI, Perez-Pinera, P, Brown, MT, Majoros, WH, Reddy, TE, and Gersbach, CA. "Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases." MOLECULAR THERAPY 23.3 (March 2015): 523-532.
Source
wos-lite
Published In
Molecular Therapy
Volume
23
Issue
3
Publish Date
2015
Start Page
523
End Page
532
DOI
10.1038/mt.2074.234

Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.

The CRISPR/Cas9 genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene-editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations.

Authors
Ousterout, DG; Kabadi, AM; Thakore, PI; Majoros, WH; Reddy, TE; Gersbach, CA
MLA Citation
Ousterout, DG, Kabadi, AM, Thakore, PI, Majoros, WH, Reddy, TE, and Gersbach, CA. "Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy." Nature communications 6 (February 18, 2015): 6244-.
PMID
25692716
Source
epmc
Published In
Nature Communications
Volume
6
Publish Date
2015
Start Page
6244
DOI
10.1038/ncomms7244

Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases

© The American Society of Gene & Cell Therapy.Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ∼13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

Authors
Ousterout, DG; Kabadi, AM; Thakore, PI; Perez-Pinera, P; Brown, MT; Majoros, WH; Reddy, TE; Gersbach, CA
MLA Citation
Ousterout, DG, Kabadi, AM, Thakore, PI, Perez-Pinera, P, Brown, MT, Majoros, WH, Reddy, TE, and Gersbach, CA. "Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases." Molecular Therapy 23.3 (January 1, 2015): 523-532.
Source
scopus
Published In
Molecular Therapy
Volume
23
Issue
3
Publish Date
2015
Start Page
523
End Page
532
DOI
10.1038/mt.2014.234

A light-inducible CRISPR-Cas9 system for control of endogenous gene activation

© 2015 Nature America, Inc. All rights reserved.Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.

Authors
Polstein, LR; Gersbach, CA
MLA Citation
Polstein, LR, and Gersbach, CA. "A light-inducible CRISPR-Cas9 system for control of endogenous gene activation." Nature Chemical Biology 11.3 (January 1, 2015): 198-200.
Source
scopus
Published In
Nature Chemical Biology
Volume
11
Issue
3
Publish Date
2015
Start Page
198
End Page
200
DOI
10.1038/nchembio.1753

Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis.

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.

Authors
Chakraborty, S; Ji, H; Chen, J; Gersbach, CA; Leong, KW
MLA Citation
Chakraborty, S, Ji, H, Chen, J, Gersbach, CA, and Leong, KW. "Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis." Scientific reports 4 (December 10, 2014): 7403-.
PMID
25492703
Source
epmc
Published In
Scientific Reports
Volume
4
Publish Date
2014
Start Page
7403
DOI
10.1038/srep07403

A CRISPR/Cas9-based system for reprogramming cell lineage specification.

Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. We show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

Authors
Chakraborty, S; Ji, H; Kabadi, AM; Gersbach, CA; Christoforou, N; Leong, KW
MLA Citation
Chakraborty, S, Ji, H, Kabadi, AM, Gersbach, CA, Christoforou, N, and Leong, KW. "A CRISPR/Cas9-based system for reprogramming cell lineage specification." Stem cell reports 3.6 (December 2014): 940-947.
PMID
25448066
Source
epmc
Published In
Stem Cell Reports
Volume
3
Issue
6
Publish Date
2014
Start Page
940
End Page
947
DOI
10.1016/j.stemcr.2014.09.013

Genome Engineering for Therapeutic Applications

© 2015 Elsevier Inc. All rights reserved..Modern genome engineering technologies have made the targeted modification of human genes possible for a multitude of therapeutic applications. This chapter discusses the three main platforms that have been developed for targeting specific DNA sequences: zinc finger proteins, transcription activator-like effectors and, most recently, the CRISPR/Cas system. These technologies allow the rapid generation of custom genome engineering strategies aimed at treating a wide range of diseases from Duchenne muscular dystrophy to HIV infection to cancer. Synthetic nucleases based on these platforms have been used to facilitate targeted gene addition, correct disease-causing mutations, and create therapeutic gene knockouts. DNA-binding domains can also be linked to regulators of gene expression to create synthetic transcription factors. Targeted transcriptional activators have been employed to treat disease by upregulating compensatory genes or activating therapeutic targets, such as tumor suppressors. Synthetic repressors have been developed to treat dominant negative diseases and to silence oncogenes. With the potential to control the expression of any gene or modify any DNA sequence in the human genome, these technologies have immense potential to advance gene therapy and regenerative medicine.

Authors
Thakore, PI; Gersbach, CA
MLA Citation
Thakore, PI, and Gersbach, CA. "Genome Engineering for Therapeutic Applications." Translating Gene Therapy to the Clinic: Techniques and Approaches. November 17, 2014. 27-43.
Source
scopus
Publish Date
2014
Start Page
27
End Page
43
DOI
10.1016/B978-0-12-800563-7.00003-8

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

Authors
Kabadi, AM; Ousterout, DG; Hilton, IB; Gersbach, CA
MLA Citation
Kabadi, AM, Ousterout, DG, Hilton, IB, and Gersbach, CA. "Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector." Nucleic acids research 42.19 (October 2014): e147-.
PMID
25122746
Source
epmc
Published In
Nucleic Acids Research
Volume
42
Issue
19
Publish Date
2014
Start Page
e147
DOI
10.1093/nar/gku749

Genome engineering: the next genomic revolution.

Authors
Gersbach, CA
MLA Citation
Gersbach, CA. "Genome engineering: the next genomic revolution." Nature methods 11.10 (October 2014): 1009-1011.
PMID
25264777
Source
epmc
Published In
Nature Methods
Volume
11
Issue
10
Publish Date
2014
Start Page
1009
End Page
1011
DOI
10.1038/nmeth.3113

Special issue on engineered DNA-binding proteins.

Authors
Kabadi, AM; Gersbach, CA
MLA Citation
Kabadi, AM, and Gersbach, CA. "Special issue on engineered DNA-binding proteins." ACS synthetic biology 3.10 (October 2014): 702-703.
PMID
25324185
Source
epmc
Published In
ACS Synthetic Biology
Volume
3
Issue
10
Publish Date
2014
Start Page
702
End Page
703
DOI
10.1021/sb500325e

Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression.

Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications.

Authors
Kabadi, AM; Gersbach, CA
MLA Citation
Kabadi, AM, and Gersbach, CA. "Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression." Methods (San Diego, Calif.) 69.2 (September 2014): 188-197.
PMID
25010559
Source
epmc
Published In
Methods
Volume
69
Issue
2
Publish Date
2014
Start Page
188
End Page
197
DOI
10.1016/j.ymeth.2014.06.014

Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.

The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein-DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology.

Authors
Gersbach, CA; Gaj, T; Barbas, CF
MLA Citation
Gersbach, CA, Gaj, T, and Barbas, CF. "Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies." Accounts of chemical research 47.8 (August 2014): 2309-2318.
PMID
24877793
Source
epmc
Published In
Accounts of Chemical Research
Volume
47
Issue
8
Publish Date
2014
Start Page
2309
End Page
2318
DOI
10.1021/ar500039w

Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

Authors
Gersbach, CA; Perez-Pinera, P
MLA Citation
Gersbach, CA, and Perez-Pinera, P. "Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine." Expert opinion on therapeutic targets 18.8 (August 2014): 835-839.
PMID
24917359
Source
epmc
Published In
Expert Opinion on Therapeutic Targets
Volume
18
Issue
8
Publish Date
2014
Start Page
835
End Page
839
DOI
10.1517/14728222.2014.913572

Tissue-engineered cartilage with inducible and tunable immunomodulatory properties.

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Authors
Glass, KA; Link, JM; Brunger, JM; Moutos, FT; Gersbach, CA; Guilak, F
MLA Citation
Glass, KA, Link, JM, Brunger, JM, Moutos, FT, Gersbach, CA, and Guilak, F. "Tissue-engineered cartilage with inducible and tunable immunomodulatory properties." Biomaterials 35.22 (July 2014): 5921-5931.
PMID
24767790
Source
epmc
Published In
Biomaterials
Volume
35
Issue
22
Publish Date
2014
Start Page
5921
End Page
5931
DOI
10.1016/j.biomaterials.2014.03.073

CRISPR technology for gene therapy.

Authors
High, K; Gregory, PD; Gersbach, C
MLA Citation
High, K, Gregory, PD, and Gersbach, C. "CRISPR technology for gene therapy." Nature medicine 20.5 (May 2014): 476-477.
PMID
24804755
Source
epmc
Published In
Nature Medicine
Volume
20
Issue
5
Publish Date
2014
Start Page
476
End Page
477
DOI
10.1038/nm.3566

Mutation Detection Following Non-Homologous End Joining (NHEJ): A Comparison of Different Semi Quantitative and Quantitative Methods

Authors
Schulz, E; Bergmann, T; Gebbing, M; Schildgen, V; Schildgen, O; Gersbach, C; Ehrhardt, A
MLA Citation
Schulz, E, Bergmann, T, Gebbing, M, Schildgen, V, Schildgen, O, Gersbach, C, and Ehrhardt, A. "Mutation Detection Following Non-Homologous End Joining (NHEJ): A Comparison of Different Semi Quantitative and Quantitative Methods." May 2014.
Source
wos-lite
Published In
Molecular Therapy
Volume
22
Publish Date
2014
Start Page
S126
End Page
S127

Spatiotemporal genetic control of cellular systemsEngineering of skeletal muscle regeneration: Principles, current state, and challengesThree-dimensional micropatterning of biomaterial scaffolds for tissue engineering

Authors
Polstein, LR; Gersbach, CA; Bian, W; Juhas, M; Bursac, N; Hoffmann, JC; West, JL
MLA Citation
Polstein, LR, Gersbach, CA, Bian, W, Juhas, M, Bursac, N, Hoffmann, JC, and West, JL. "Spatiotemporal genetic control of cellular systemsEngineering of skeletal muscle regeneration: Principles, current state, and challengesThree-dimensional micropatterning of biomaterial scaffolds for tissue engineering (PublishedPublishedPublished)." Tissue and Organ Regeneration: Advances in Micro- and Nanotechnology. April 30, 2014. 156-197.
Source
scopus
Publish Date
2014
Start Page
156
End Page
197
DOI
10.4032/9789814411684

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.

Authors
Brunger, JM; Huynh, NPT; Guenther, CM; Perez-Pinera, P; Moutos, FT; Sanchez-Adams, J; Gersbach, CA; Guilak, F
MLA Citation
Brunger, JM, Huynh, NPT, Guenther, CM, Perez-Pinera, P, Moutos, FT, Sanchez-Adams, J, Gersbach, CA, and Guilak, F. "Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage." Proceedings of the National Academy of Sciences of the United States of America 111.9 (March 2014): E798-E806.
PMID
24550481
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
111
Issue
9
Publish Date
2014
Start Page
E798
End Page
E806
DOI
10.1073/pnas.1321744111

Comparing Genome Editing Technologies

Authors
Gersbach, CA; Gaj, T; Barbas, CF
MLA Citation
Gersbach, CA, Gaj, T, and Barbas, CF. "Comparing Genome Editing Technologies." Genetic Engineering & Biotechnology News 34.5 (March 2014): 1, 32-34.
Source
crossref
Published In
Genetic Engineering and Biotechnology News
Volume
34
Issue
5
Publish Date
2014
Start Page
1, 32
End Page
34
DOI
10.1089/gen.34.05.02

Tissue-engineered cartilage with inducible and tunable immunomodulatory properties

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis. © 2014 Elsevier Ltd.

Authors
Glass, KA; Link, JM; Brunger, JM; Moutos, FT; Gersbach, CA; Guilak, F
MLA Citation
Glass, KA, Link, JM, Brunger, JM, Moutos, FT, Gersbach, CA, and Guilak, F. "Tissue-engineered cartilage with inducible and tunable immunomodulatory properties." Biomaterials 35.22 (January 1, 2014): 5921-5931.
Source
scopus
Published In
Biomaterials
Volume
35
Issue
22
Publish Date
2014
Start Page
5921
End Page
5931
DOI
10.1016/j.biomaterials.2014.03.073

Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression

© 2014 Elsevier Inc.Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications.

Authors
Kabadi, AM; Gersbach, CA
MLA Citation
Kabadi, AM, and Gersbach, CA. "Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression." Methods 69.2 (January 1, 2014): 188-197.
Source
scopus
Published In
Methods
Volume
69
Issue
2
Publish Date
2014
Start Page
188
End Page
197
DOI
10.1016/j.ymeth.2014.06.014

Light-inducible gene regulation with engineered zinc finger proteins.

The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.

Authors
Polstein, LR; Gersbach, CA
MLA Citation
Polstein, LR, and Gersbach, CA. "Light-inducible gene regulation with engineered zinc finger proteins." Methods in molecular biology (Clifton, N.J.) 1148 (January 2014): 89-107.
PMID
24718797
Source
epmc
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
1148
Publish Date
2014
Start Page
89
End Page
107
DOI
10.1007/978-1-4939-0470-9_7

Erratum: Reading Frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients (Molecular Therapy (2013) 21 (1718-1726) DOI: 10.1038/mt.2013.111)

Authors
Ousterout, DG; Perez-Pinera, P; Thakore, PI; Kabadi, AM; Brown, MT; Qin, X; Fedrigo, O; Mouly, V; Tremblay, JP; Gersbach, CA
MLA Citation
Ousterout, DG, Perez-Pinera, P, Thakore, PI, Kabadi, AM, Brown, MT, Qin, X, Fedrigo, O, Mouly, V, Tremblay, JP, and Gersbach, CA. "Erratum: Reading Frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients (Molecular Therapy (2013) 21 (1718-1726) DOI: 10.1038/mt.2013.111)." Molecular Therapy 21.11 (November 1, 2013): 2130-.
Source
scopus
Published In
Molecular Therapy
Volume
21
Issue
11
Publish Date
2013
Start Page
2130
DOI
10.1038/mt.2013.229

Gene Delivery into Cells and Tissues

There are significant engineering challenges in translating the remarkable medical implications of gene and nucleic acid delivery from cell and animal models into the clinic. Off-target effects and inefficient delivery to the proper intracellular compartment of the targeted cells are major obstacles to success. Systemic delivery of any viral or nonviral vector requires an appreciation of the adverse physiological barriers that exist in vivo and incorporation of well-designed vector components that limit non-specific uptake and accelerated clearance. In addition, the genetic engineer should consider the design criteria related to cell-specific action. For example, cell surface recognition can increase the therapeutic index of a DNA- or RNA-based medication. Finally, one must consider the mechanism of cellular internalization, and investigators should attempt to target pathways and incorporate vector functionalities that will mediate trafficking to the subcellular compartment that is optimal for activity of the nucleic acid cargo. This chapter discusses key aspects of biodistribution and cellular uptake of nanoparticulate vectors and vehicles. It also surveys current strategies for engineering effective viral and nonviral packaging systems, systems for both targeted, systemic delivery and controlled, local release of nucleic acids or genes from engineered scaffolds, and strategies for directing the intracellular trafficking of vehicle contents to the nucleus or cytoplasm of target cells. © 2014 Elsevier Inc. All rights reserved.

Authors
Duvall, CL; Prokop, A; Gersbach, CA; Davidson, JM
MLA Citation
Duvall, CL, Prokop, A, Gersbach, CA, and Davidson, JM. "Gene Delivery into Cells and Tissues." (November 1, 2013): 687-723. (Chapter)
Source
scopus
Publish Date
2013
Start Page
687
End Page
723
DOI
10.1016/B978-0-12-398358-9.00035-5

RNA-guided gene activation by CRISPR-Cas9-based transcription factors.

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

Authors
Perez-Pinera, P; Kocak, DD; Vockley, CM; Adler, AF; Kabadi, AM; Polstein, LR; Thakore, PI; Glass, KA; Ousterout, DG; Leong, KW; Guilak, F; Crawford, GE; Reddy, TE; Gersbach, CA
MLA Citation
Perez-Pinera, P, Kocak, DD, Vockley, CM, Adler, AF, Kabadi, AM, Polstein, LR, Thakore, PI, Glass, KA, Ousterout, DG, Leong, KW, Guilak, F, Crawford, GE, Reddy, TE, and Gersbach, CA. "RNA-guided gene activation by CRISPR-Cas9-based transcription factors." Nat Methods 10.10 (October 2013): 973-976.
PMID
23892895
Source
pubmed
Published In
Nature Methods
Volume
10
Issue
10
Publish Date
2013
Start Page
973
End Page
976
DOI
10.1038/nmeth.2600

Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients.

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.

Authors
Ousterout, DG; Perez-Pinera, P; Thakore, PI; Kabadi, AM; Brown, MT; Qin, X; Fedrigo, O; Mouly, V; Tremblay, JP; Gersbach, CA
MLA Citation
Ousterout, DG, Perez-Pinera, P, Thakore, PI, Kabadi, AM, Brown, MT, Qin, X, Fedrigo, O, Mouly, V, Tremblay, JP, and Gersbach, CA. "Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients." Mol Ther 21.9 (September 2013): 1718-1726.
PMID
23732986
Source
pubmed
Published In
Molecular Therapy
Volume
21
Issue
9
Publish Date
2013
Start Page
1718
End Page
1726
DOI
10.1038/mt.2013.111

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases.

Authors
Gaj, T; Gersbach, CA; Barbas, CF
MLA Citation
Gaj, T, Gersbach, CA, and Barbas, CF. "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering." Trends Biotechnol 31.7 (July 2013): 397-405. (Review)
PMID
23664777
Source
pubmed
Published In
Trends in Biotechnology
Volume
31
Issue
7
Publish Date
2013
Start Page
397
End Page
405
DOI
10.1016/j.tibtech.2013.04.004

How vinculin regulates force transmission.

Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculin-null cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V(H)) and tail domains. V(H) enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with VH, implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V(H) has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V(H), correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.

Authors
Dumbauld, DW; Lee, TT; Singh, A; Scrimgeour, J; Gersbach, CA; Zamir, EA; Fu, J; Chen, CS; Curtis, JE; Craig, SW; García, AJ
MLA Citation
Dumbauld, DW, Lee, TT, Singh, A, Scrimgeour, J, Gersbach, CA, Zamir, EA, Fu, J, Chen, CS, Curtis, JE, Craig, SW, and García, AJ. "How vinculin regulates force transmission." Proc Natl Acad Sci U S A 110.24 (June 11, 2013): 9788-9793.
PMID
23716647
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
24
Publish Date
2013
Start Page
9788
End Page
9793
DOI
10.1073/pnas.1216209110

Synergistic and Tunable Gene Activation by Combinations of Synthetic Transcription Factors

Authors
Perez-Pinera, P; Ousterout, DG; Brunger, JM; Farin, AM; Glass, KA; Guilak, F; Crawford, GE; Hartemink, AJ; Gersbach, CA
MLA Citation
Perez-Pinera, P, Ousterout, DG, Brunger, JM, Farin, AM, Glass, KA, Guilak, F, Crawford, GE, Hartemink, AJ, and Gersbach, CA. "Synergistic and Tunable Gene Activation by Combinations of Synthetic Transcription Factors." June 2013.
Source
wos-lite
Published In
Molecular Therapy
Volume
21
Publish Date
2013
Start Page
S93
End Page
S93

The role of single-cell analyses in understanding cell lineage commitment.

The study of cell lineage commitment is critical for improving our understanding of tissue development and regeneration, and for realizing stem cell-based therapies and engineered tissue replacements. Recently, the discovery of an unanticipated degree of variability in fundamental biological processes, including divergent responses of genetically identical cells to various stimuli, has provided mechanistic insight into cellular decision making and the collective behavior of cell populations. Therefore, the study of lineage commitment with single-cell resolution could provide greater knowledge of cellular differentiation mechanisms and the influence of noise on cellular processes. This will require the adoption of new technologies for single-cell analysis as traditional methods typically measure average values of bulk population behavior. This review discusses the recent developments in methods for analyzing the behavior of individual cells, and how these approaches are leading to a deeper understanding and better control of cellular decision making.

Authors
Gibson, TM; Gersbach, CA
MLA Citation
Gibson, TM, and Gersbach, CA. "The role of single-cell analyses in understanding cell lineage commitment." Biotechnol J 8.4 (April 2013): 397-407. (Review)
PMID
23520130
Source
pubmed
Published In
Biotechnology Journal
Volume
8
Issue
4
Publish Date
2013
Start Page
397
End Page
407
DOI
10.1002/biot.201200201

TUNABLE EXPRESSION OF IL-1RA IN GENETICALLY MODIFIED MESENCHYMAL STEM CELLS FOR CARTILAGE TISSUE ENGINEERING

Authors
Glass, KA; Brunger, JM; Gersbach, CA; Guilak, F
MLA Citation
Glass, KA, Brunger, JM, Gersbach, CA, and Guilak, F. "TUNABLE EXPRESSION OF IL-1RA IN GENETICALLY MODIFIED MESENCHYMAL STEM CELLS FOR CARTILAGE TISSUE ENGINEERING." April 2013.
Source
wos-lite
Published In
Osteoarthritis and Cartilage
Volume
21
Publish Date
2013
Start Page
S282
End Page
S283

Highly active zinc-finger nucleases by extended modular assembly.

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three- to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.

Authors
Bhakta, MS; Henry, IM; Ousterout, DG; Das, KT; Lockwood, SH; Meckler, JF; Wallen, MC; Zykovich, A; Yu, Y; Leo, H; Xu, L; Gersbach, CA; Segal, DJ
MLA Citation
Bhakta, MS, Henry, IM, Ousterout, DG, Das, KT, Lockwood, SH, Meckler, JF, Wallen, MC, Zykovich, A, Yu, Y, Leo, H, Xu, L, Gersbach, CA, and Segal, DJ. "Highly active zinc-finger nucleases by extended modular assembly." Genome Res 23.3 (March 2013): 530-538.
PMID
23222846
Source
pubmed
Published In
Genome research
Volume
23
Issue
3
Publish Date
2013
Start Page
530
End Page
538
DOI
10.1101/gr.143693.112

Synergistic and tunable human gene activation by combinations of synthetic transcription factors.

Mammalian genes are regulated by the cooperative and synergistic actions of many transcription factors. In this study we recapitulate this complex regulation in human cells by targeting endogenous gene promoters, including regions of closed chromatin upstream of silenced genes, with combinations of engineered transcription activator-like effectors (TALEs). These combinations of TALE transcription factors induced substantial gene activation and allowed tuning of gene expression levels that will broadly enable synthetic biology, gene therapy and biotechnology.

Authors
Perez-Pinera, P; Ousterout, DG; Brunger, JM; Farin, AM; Glass, KA; Guilak, F; Crawford, GE; Hartemink, AJ; Gersbach, CA
MLA Citation
Perez-Pinera, P, Ousterout, DG, Brunger, JM, Farin, AM, Glass, KA, Guilak, F, Crawford, GE, Hartemink, AJ, and Gersbach, CA. "Synergistic and tunable human gene activation by combinations of synthetic transcription factors." Nat Methods 10.3 (March 2013): 239-242.
PMID
23377379
Source
pubmed
Published In
Nature Methods
Volume
10
Issue
3
Publish Date
2013
Start Page
239
End Page
242
DOI
10.1038/nmeth.2361

Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases.

Authors
Tremblay, JP; Xiao, X; Aartsma-Rus, A; Barbas, C; Blau, HM; Bogdanove, AJ; Boycott, K; Braun, S; Breakefield, XO; Bueren, JA; Buschmann, M; Byrne, BJ; Calos, M; Cathomen, T; Chamberlain, J; Chuah, M; Cornetta, K; Davies, KE; Dickson, JG; Duchateau, P; Flotte, TR; Gaudet, D; Gersbach, CA; Gilbert, R; Glorioso, J; Herzog, RW; High, KA; Huang, W; Huard, J; Joung, JK; Liu, D; Liu, D; Lochmüller, H; Lustig, L; Martens, J; Massie, B; Mavilio, F; Mendell, JR; Nathwani, A; Ponder, K; Porteus, M et al.
MLA Citation
Tremblay, JP, Xiao, X, Aartsma-Rus, A, Barbas, C, Blau, HM, Bogdanove, AJ, Boycott, K, Braun, S, Breakefield, XO, Bueren, JA, Buschmann, M, Byrne, BJ, Calos, M, Cathomen, T, Chamberlain, J, Chuah, M, Cornetta, K, Davies, KE, Dickson, JG, Duchateau, P, Flotte, TR, Gaudet, D, Gersbach, CA, Gilbert, R, Glorioso, J, Herzog, RW, High, KA, Huang, W, Huard, J, Joung, JK, Liu, D, Liu, D, Lochmüller, H, Lustig, L, Martens, J, Massie, B, Mavilio, F, Mendell, JR, Nathwani, A, Ponder, K, and Porteus, M et al. "Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases." Mol Ther 21.2 (February 2013): 266-268. (Letter)
PMID
23369965
Source
pubmed
Published In
Molecular Therapy
Volume
21
Issue
2
Publish Date
2013
Start Page
266
End Page
268
DOI
10.1038/mt.2013.4

Reading frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons. © The American Society of Gene and Cell Therapy.

Authors
Ousterout, DG; Perez-Pinera, P; Thakore, PI; Kabadi, AM; Brown, MT; Qin, X; Fedrigo, O; Mouly, V; Tremblay, JP; Gersbach, CA
MLA Citation
Ousterout, DG, Perez-Pinera, P, Thakore, PI, Kabadi, AM, Brown, MT, Qin, X, Fedrigo, O, Mouly, V, Tremblay, JP, and Gersbach, CA. "Reading frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients." Molecular Therapy 21.9 (2013): 1718-1726.
Source
scival
Published In
Molecular Therapy
Volume
21
Issue
9
Publish Date
2013
Start Page
1718
End Page
1726
DOI
10.1038/mt.2013.111

Targeted plasmid integration into the Human Genome by engineered recombinases

The targeted integration of transgenes into cellular genomes is central to numerous applications in biotechnology, basic science, and medicine. In recent years, a variety of advances have improved upon conventional methods for site-specific transgene integration. Most of these methods involve nucleases that cleave DNA to activate DNA repair pathways including homologous recombination or integrases that fully catalyze the integration reaction but are limited in their capacity to target new sites in the genome. Recently, zinc-finger recombinases have emerged as a class of engineered enzymes that combines the strengths of both of these previous methods. Zinc-finger recombinases can fully and autonomously catalyze plasmid integration into the genome of mammalian cells without creating free DNA breaks. In addition, they can be engineered to target new genomic recognition sites by exchanging the modular and programmable DNA-binding domain and through directed evolution of the serine recombinase catalytic domain. This chapter reviews the development of the zinc-finger recombinase technology, including discussions of its strengths and weaknesses and the future directions necessary to translate this technology into routine use for transgene integration into cellular genomes. © 2013 Springer Science+Business Media B.V.

Authors
Gersbach, CA; III, CFB
MLA Citation
Gersbach, CA, and III, CFB. "Targeted plasmid integration into the Human Genome by engineered recombinases." Topics in Current Genetics 23 (2013): 267-284.
Source
scival
Published In
Topics in Current Genetics
Volume
23
Publish Date
2013
Start Page
267
End Page
284
DOI
10.1007/978-94-007-4531-5_10

The role of single-cell analyses in understanding cell lineage commitment

The study of cell lineage commitment is critical for improving our understanding of tissue development and regeneration, and for realizing stem cell-based therapies and engineered tissue replacements. Recently, the discovery of an unanticipated degree of variability in fundamental biological processes, including divergent responses of genetically identical cells to various stimuli, has provided mechanistic insight into cellular decision making and the collective behavior of cell populations. Therefore, the study of lineage commitment with single-cell resolution could provide greater knowledge of cellular differentiation mechanisms and the influence of noise on cellular processes. This will require the adoption of new technologies for single-cell analysis as traditional methods typically measure average values of bulk population behavior. This review discusses the recent developments in methods for analyzing the behavior of individual cells, and how these approaches are leading to a deeper understanding and better control of cellular decision making. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Authors
Gibson, TM; Gersbach, CA
MLA Citation
Gibson, TM, and Gersbach, CA. "The role of single-cell analyses in understanding cell lineage commitment." Biotechnology Journal 8.4 (2013): 397-407.
Source
scival
Published In
Biotechnology Journal
Volume
8
Issue
4
Publish Date
2013
Start Page
397
End Page
407
DOI
10.1002/biot.201200201

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. © 2013 Elsevier Ltd.

Authors
Gaj, T; Gersbach, CA; Barbas, CF
MLA Citation
Gaj, T, Gersbach, CA, and Barbas, CF. "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering." Trends in Biotechnology 31.7 (2013): 397-405.
Source
scival
Published In
Trends in Biotechnology
Volume
31
Issue
7
Publish Date
2013
Start Page
397
End Page
405
DOI
10.1016/j.tibtech.2013.04.004

Engineered Proteins for Controlling Gene Expression

Authors
Gersbach, CA
MLA Citation
Gersbach, CA. "Engineered Proteins for Controlling Gene Expression." Handbook of Stem Cells 1 (2013): 125-138.
Source
scival
Published In
Handbook of Stem Cells
Volume
1
Publish Date
2013
Start Page
125
End Page
138
DOI
10.1016/B978-0-12-385942-6.00013-5

RNA-guided gene activation by CRISPR-Cas9-based transcription factors

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

Authors
Perez-Pinera, P; Kocak, DD; Vockley, CM; Adler, AF; Kabadi, AM; Polstein, LR; Thakore, PI; Glass, KA; Ousterout, DG; Leong, KW; Guilak, F; Crawford, GE; Reddy, TE; Gersbach, CA
MLA Citation
Perez-Pinera, P, Kocak, DD, Vockley, CM, Adler, AF, Kabadi, AM, Polstein, LR, Thakore, PI, Glass, KA, Ousterout, DG, Leong, KW, Guilak, F, Crawford, GE, Reddy, TE, and Gersbach, CA. "RNA-guided gene activation by CRISPR-Cas9-based transcription factors." Nature Methods 10.10 (2013): 973-976.
Source
scival
Published In
Nature Methods
Volume
10
Issue
10
Publish Date
2013
Start Page
973
End Page
976
DOI
10.1038/nmeth.2600

Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors.

Advanced gene regulatory systems are necessary for scientific research, synthetic biology, and gene-based medicine. An ideal system would allow facile spatiotemporal manipulation of gene expression within a cell population that is tunable, reversible, repeatable, and can be targeted to diverse DNA sequences. To meet these criteria, a gene regulation system was engineered that combines light-sensitive proteins and programmable zinc finger transcription factors. This system, light-inducible transcription using engineered zinc finger proteins (LITEZ), uses two light-inducible dimerizing proteins from Arabidopsis thaliana, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc finger transcription factor activity in human cells. Activation of gene expression in human cells engineered with LITEZ was reversible and repeatable by modulating the duration of illumination. The level of gene expression could also be controlled by modulating light intensity. Finally, gene expression could be activated in a spatially defined pattern by illuminating the human cell culture through a photomask of arbitrary geometry. LITEZ enables new approaches for precisely regulating gene expression in biotechnology and medicine, as well as studying gene function, cell-cell interactions, and tissue morphogenesis.

Authors
Polstein, LR; Gersbach, CA
MLA Citation
Polstein, LR, and Gersbach, CA. "Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors." J Am Chem Soc 134.40 (October 10, 2012): 16480-16483.
PMID
22963237
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
134
Issue
40
Publish Date
2012
Start Page
16480
End Page
16483
DOI
10.1021/ja3065667

Advances in targeted genome editing.

New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases.

Authors
Perez-Pinera, P; Ousterout, DG; Gersbach, CA
MLA Citation
Perez-Pinera, P, Ousterout, DG, and Gersbach, CA. "Advances in targeted genome editing." Curr Opin Chem Biol 16.3-4 (August 2012): 268-277. (Review)
PMID
22819644
Source
pubmed
Published In
Current Opinion in Chemical Biology
Volume
16
Issue
3-4
Publish Date
2012
Start Page
268
End Page
277
DOI
10.1016/j.cbpa.2012.06.007

Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases.

Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.

Authors
Perez-Pinera, P; Ousterout, DG; Brown, MT; Gersbach, CA
MLA Citation
Perez-Pinera, P, Ousterout, DG, Brown, MT, and Gersbach, CA. "Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases." Nucleic Acids Res 40.8 (April 2012): 3741-3752.
PMID
22169954
Source
pubmed
Published In
Nucleic Acids Research
Volume
40
Issue
8
Publish Date
2012
Start Page
3741
End Page
3752
DOI
10.1093/nar/gkr1214

Photoregulated gene expression in human cells with light-inducible engineered transcription factors

Authors
Polstein, LR; Gersbach, CA
MLA Citation
Polstein, LR, and Gersbach, CA. "Photoregulated gene expression in human cells with light-inducible engineered transcription factors." ASME 2012 Summer Bioengineering Conference, SBC 2012 (2012): 351-352.
Source
scival
Published In
ASME 2012 Summer Bioengineering Conference, SBC 2012
Publish Date
2012
Start Page
351
End Page
352
DOI
10.1115/SBC2012-80573

Engineered bioactive molecules

Authors
Gersbach, CA
MLA Citation
Gersbach, CA. "Engineered bioactive molecules." 5 (October 1, 2011): 131-145. (Chapter)
Source
scopus
Volume
5
Publish Date
2011
Start Page
131
End Page
145

Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.

Authors
Gersbach, CA; Gaj, T; Gordley, RM; Mercer, AC; Barbas, CF
MLA Citation
Gersbach, CA, Gaj, T, Gordley, RM, Mercer, AC, and Barbas, CF. "Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase." Nucleic Acids Res 39.17 (September 1, 2011): 7868-7878.
PMID
21653554
Source
pubmed
Published In
Nucleic Acids Research
Volume
39
Issue
17
Publish Date
2011
Start Page
7868
End Page
7878
DOI
10.1093/nar/gkr421

Structure-guided reprogramming of serine recombinase DNA sequence specificity.

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences > 10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with > 80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.

Authors
Gaj, T; Mercer, AC; Gersbach, CA; Gordley, RM; Barbas, CF
MLA Citation
Gaj, T, Mercer, AC, Gersbach, CA, Gordley, RM, and Barbas, CF. "Structure-guided reprogramming of serine recombinase DNA sequence specificity." Proc Natl Acad Sci U S A 108.2 (January 11, 2011): 498-503.
PMID
21187418
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
2
Publish Date
2011
Start Page
498
End Page
503
DOI
10.1073/pnas.1014214108

Structure-guided reprogramming of serine recombinase DNA sequence specificity

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.

Authors
Gaj, T; Mercer, AC; Gersbach, CA; Gordley, RM; III, CFB
MLA Citation
Gaj, T, Mercer, AC, Gersbach, CA, Gordley, RM, and III, CFB. "Structure-guided reprogramming of serine recombinase DNA sequence specificity." Proceedings of the National Academy of Sciences of the United States of America 108.2 (2011): 510-515.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
2
Publish Date
2011
Start Page
510
End Page
515
DOI
10.1073/pnas.1016462108

Engineered Proteins for Controlling Gene Expression

This chapter describes the potential uses of natural transcriptional regulators, enhancement of these regulators through molecular engineering, and the engineering of entirely synthetic transcription factors for targeted gene regulation. HIF-1 is a heterodimeric transcription factor composed of two basic helix-loop-helix (bHLH) proteins, HIF-1α and HIF-1β. HIF-1β is constitutively expressed in an active form in the nucleus of oxygen-sensing cells and HIF-1α is highly inducible by hypoxia, primarily through post-translational regulation. Under normoxic conditions, HIF-1α is rapidly degraded through hydroxylation of proline residues in the N- and C-terminal oxygen-dependent degradation domains (NODDD and CODDD). This post-translational modification is mediated by a family of three HIF-1α prolyl hydroxylases that use oxygen as a substrate such that enzymatic activity is tightly regulated by oxygen concentration. Hydroxylated proline residues are recognized by the von Hippel Lindau tumor suppressor (VHL), which targets HIF-1α for proteosomal proteolysis via ubiquitin ligation. Artificial transcription factors based on zinc finger proteins are engineered to regulate a variety of genes relevant to regenerative medicine such as a zinc finger transcription factor is designed to regulate the gene for vascular endothelial growth factor (VEGF). VEGF is known to stimulate the formation of new blood vessels necessary for wound healing and the repair of injured cardiovascular tissues. © 2011 Elsevier Inc. All rights reserved.

Authors
Gersbach, CA
MLA Citation
Gersbach, CA. "Engineered Proteins for Controlling Gene Expression." Principles of Regenerative Medicine (2011): 159-176.
Source
scival
Published In
Principles of Regenerative Medicine
Publish Date
2011
Start Page
159
End Page
176
DOI
10.1016/B978-0-12-381422-7.10008-2

Directed evolution of recombinase specificity by split gene reassembly.

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding beta-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.

Authors
Gersbach, CA; Gaj, T; Gordley, RM; Barbas, CF
MLA Citation
Gersbach, CA, Gaj, T, Gordley, RM, and Barbas, CF. "Directed evolution of recombinase specificity by split gene reassembly." Nucleic Acids Res 38.12 (July 2010): 4198-4206.
PMID
20194120
Source
pubmed
Published In
Nucleic Acids Research
Volume
38
Issue
12
Publish Date
2010
Start Page
4198
End Page
4206
DOI
10.1093/nar/gkq125

Synthesis of programmable integrases.

Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.

Authors
Gordley, RM; Gersbach, CA; Barbas, CF
MLA Citation
Gordley, RM, Gersbach, CA, and Barbas, CF. "Synthesis of programmable integrases." Proc Natl Acad Sci U S A 106.13 (March 31, 2009): 5053-5058.
PMID
19282480
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
13
Publish Date
2009
Start Page
5053
End Page
5058
DOI
10.1073/pnas.0812502106

Identification of novel Runx2 targets in osteoblasts: cell type-specific BMP-dependent regulation of Tram2.

Runx2 is an osteoblast master transcription factor and a target for bone morphogenetic protein (BMP) signaling, but our knowledge of events downstream of Runx2 is limited. In this study, we used ChIP Display to discover seven novel genomic regions occupied by Runx2 in living MC3T3-E1 osteoblastic cells. Six of these regions are found within or up to 1-kb away from annotated genes, but only two are found within 5'-gene flanking sequences. One of the newly identified Runx2 target genes is Tram2, whose product facilitates proper folding of type I collagen. We demonstrate that Tram2 mRNA is suppressed in non-osteoblasts when Runx2 is over-expressed, and that this suppression is alleviated upon treatment with BMP-2. Moreover, we show that BMP-induced Runx2 expression in the C3H10T1/2, ST2, C2C12, and MC3T3-E1 cell lines coincides with an increase in Tram2 mRNA levels. Thus, Runx2 may regulate Tram2 expression in a BMP-dependent manner, and Tram2 may participate in the overall osteogenic function of Runx2. Among the other Runx2 target genes discovered in this study are Lnx2, an intracellular scaffolding protein that may play a role in Notch signaling, and Tnfrsf12a, a Tumor Necrosis Factor receptor family member that influences both osteoblast and osteoclast differentiation. Expanding our knowledge of Runx2 target genes, and manipulation of these genes, are warranted to better understand the regulation of osteoblast function and to provide opportunities for the development of new bone anabolics.

Authors
Pregizer, S; Barski, A; Gersbach, CA; García, AJ; Frenkel, B
MLA Citation
Pregizer, S, Barski, A, Gersbach, CA, García, AJ, and Frenkel, B. "Identification of novel Runx2 targets in osteoblasts: cell type-specific BMP-dependent regulation of Tram2." J Cell Biochem 102.6 (December 15, 2007): 1458-1471.
PMID
17486635
Source
pubmed
Published In
Journal of Cellular Biochemistry
Volume
102
Issue
6
Publish Date
2007
Start Page
1458
End Page
1471
DOI
10.1002/jcb.21366

Biomaterial-mediated retroviral gene transfer using self-assembled monolayers.

Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH(2)-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH(3)-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH(2)-SAM was further characterized as a function of retrovirus coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-l-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer.

Authors
Gersbach, CA; Coyer, SR; Le Doux, JM; García, AJ
MLA Citation
Gersbach, CA, Coyer, SR, Le Doux, JM, and García, AJ. "Biomaterial-mediated retroviral gene transfer using self-assembled monolayers." Biomaterials 28.34 (December 2007): 5121-5127.
PMID
17698189
Source
pubmed
Published In
Biomaterials
Volume
28
Issue
34
Publish Date
2007
Start Page
5121
End Page
5127
DOI
10.1016/j.biomaterials.2007.07.047

In vitro and in vivo osteoblastic differentiation of BMP-2- and Runx2-engineered skeletal myoblasts.

Genetic engineering with osteogenic factors is a promising approach for cell-based therapeutics and orthopedic regeneration. However, the relative efficacy of different strategies for inducing osteoblastic differentiation remains unclear and is further complicated by varied delivery vehicles, cell types, and evaluation criteria. In order to elucidate the effects of distinct gene-based strategies, we quantitatively evaluated osteoblastic differentiation and mineralization of primary skeletal myoblasts overexpressing either the BMP-2 growth factor or Runx2 transcription factor. Retroviral delivery of BMP-2 or Runx2 stimulated differentiation into an osteoblastic phenotype, as demonstrated by the induction of osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in monolayer culture and on collagen scaffolds both in vitro and in an intramuscular site in vivo. In general, BMP-2 stimulated osteoblastic markers faster and to a greater extent than Runx2, although we also identified experimental conditions under which these two factors produced similar effects. Additionally, Runx2-engineered cells did not utilize paracrine signaling via secreted osteogenic factors, in contrast to cells overexpressing BMP-2, as demonstrated by conditioned media studies and activation of Smad signaling. These results emphasize the complexity of gene therapy-based orthopedic therapeutics as an integrated relationship of differentiation state, construct maturation, and paracrine signaling of osteogenic cells. This study is significant in evaluating proposed therapeutic systems and defining a successful strategy for integrating gene medicine and orthopedic regeneration.

Authors
Gersbach, CA; Guldberg, RE; García, AJ
MLA Citation
Gersbach, CA, Guldberg, RE, and García, AJ. "In vitro and in vivo osteoblastic differentiation of BMP-2- and Runx2-engineered skeletal myoblasts." J Cell Biochem 100.5 (April 1, 2007): 1324-1336.
PMID
17131362
Source
pubmed
Published In
Journal of Cellular Biochemistry
Volume
100
Issue
5
Publish Date
2007
Start Page
1324
End Page
1336
DOI
10.1002/jcb.21118

Virus-based gene therapy strategies for bone regeneration.

Gene therapy has emerged as a promising strategy for the repair and regeneration of damaged musculoskeletal tissues. Application of this paradigm to bone healing has shown enhanced efficacy in preclinical animal studies compared to conventional bone grafting approaches. This review discusses current and emerging virus-based genetic engineering strategies for the delivery of therapeutic molecules which promote skeletal regeneration. Viral gene delivery vectors are discussed in the context of bone repair in order to illustrate the challenges and applications of these methods with tissue-specific examples. Moreover the concepts discussed can be broadly applied to promote healing in a wide range of tissues. We also present important considerations involved in the application of these gene therapy techniques to a variety of osteogenic (e.g. bone marrow-derived cells) and non-osteogenic (e.g. fibroblasts and skeletal myoblasts) cell types. Criteria for the selection of regenerative molecules with soluble versus intracellular modes of action and emerging combinatorial approaches are also discussed. Overall, gene transfer technologies have the potential to overcome limitations associated with existing bone grafting approaches and may enable investigators to design therapies which more closely mimic the complex spatial and temporal cascade of proteins involved in endogenous bone development and repair.

Authors
Phillips, JE; Gersbach, CA; García, AJ
MLA Citation
Phillips, JE, Gersbach, CA, and García, AJ. "Virus-based gene therapy strategies for bone regeneration." Biomaterials 28.2 (January 2007): 211-229. (Review)
PMID
16928397
Source
pubmed
Published In
Biomaterials
Volume
28
Issue
2
Publish Date
2007
Start Page
211
End Page
229
DOI
10.1016/j.biomaterials.2006.07.032

Genetic engineering for skeletal regenerative medicine.

The clinical challenges of skeletal regenerative medicine have motivated significant advances in cellular and tissue engineering in recent years. In particular, advances in molecular biology have provided the tools necessary for the design of gene-based strategies for skeletal tissue repair. Consequently, genetic engineering has emerged as a promising method to address the need for sustained and robust cellular differentiation and extracellular matrix production. As a result, gene therapy has been established as a conventional approach to enhance cellular activities for skeletal tissue repair. Recent literature clearly demonstrates that genetic engineering is a principal factor in constructing effective methods for tissue engineering approaches to bone, cartilage, and connective tissue regeneration. This review highlights this literature, including advances in the development of efficacious gene carriers, novel cell sources, successful delivery strategies, and optimal target genes. The current status of the field and the challenges impeding the clinical realization of these approaches are also discussed.

Authors
Gersbach, CA; Phillips, JE; García, AJ
MLA Citation
Gersbach, CA, Phillips, JE, and García, AJ. "Genetic engineering for skeletal regenerative medicine." Annu Rev Biomed Eng 9 (2007): 87-119. (Review)
PMID
17425467
Source
pubmed
Published In
Annual Review of Biomedical Engineering
Volume
9
Publish Date
2007
Start Page
87
End Page
119
DOI
10.1146/annurev.bioeng.9.060906.151949

Inducible regulation of Runx2-stimulated osteogenesis.

Ex vivo gene therapy is a promising approach to orthopedic regenerative medicine. These strategies typically focus on the constitutive overexpression of osteogenic factors to induce osteoblastic differentiation and matrix mineralization. However, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis. To address these limitations, this work describes a retroviral system to deliver the Runx2 osteoblastic transcription factor under control of the tetracycline-inducible (tet-off) promoter in primary skeletal myoblasts. Runx2 expression was tightly regulated by anhydrotetracyline (aTc) concentration in cell culture media. Osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization were also controlled by aTc in a dose-dependent manner. Additionally, osteoblastic differentiation was temporally regulated by adding and removing aTc from the culture media. Engineered cells were seeded onto collagen scaffolds and implanted intramuscularly in the hind limbs of syngeneic mice. In vivo mineralization by these constructs was regulated by supplementing the drinking water with aTc, as demonstrated by micro-computed tomography and histological analyses. Collectively, these results present a novel system for regulating osteoblastic differentiation of a clinically relevant autologous cell source. This system is significant to developing controlled and effective orthopedic gene therapy strategies and studying the regulation of osteoblastic differentiation.

Authors
Gersbach, CA; Le Doux, JM; Guldberg, RE; García, AJ
MLA Citation
Gersbach, CA, Le Doux, JM, Guldberg, RE, and García, AJ. "Inducible regulation of Runx2-stimulated osteogenesis." Gene Ther 13.11 (June 2006): 873-882.
PMID
16496016
Source
pubmed
Published In
Gene Therapy
Volume
13
Issue
11
Publish Date
2006
Start Page
873
End Page
882
DOI
10.1038/sj.gt.3302725

Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation.

Glucocorticoid hormones have complex stimulatory and inhibitory effects on skeletal metabolism. Endogenous glucocorticoid signaling is required for normal bone formation in vivo, and synthetic glucocorticoids, such as dexamethasone, promote osteoblastic differentiation in several in vitro model systems. The mechanism by which these hormones induce osteogenesis remains poorly understood. We demonstrate here that the coordinate action of dexamethasone and the osteogenic transcription factor Runx2/Cbfa1 synergistically induces osteocalcin and bone sialoprotein gene expression, alkaline phosphatase activity, and biological mineral deposition in primary dermal fibroblasts. Dexamethasone decreased Runx2 phosphoserine levels, particularly on Ser125, in parallel with the upregulation of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) through a glucocorticoid-receptor-mediated mechanism. Inhibition of MKP-1 abrogated the dexamethasone-induced decrease in Runx2 serine phosphorylation, suggesting that glucocorticoids modulate Runx2 phosphorylation via MKP-1. Mutation of Ser125 to glutamic acid, mimicking constitutive phosphorylation, inhibited Runx2-mediated osteoblastic differentiation, which was not rescued by dexamethasone treatment. Conversely, mutation of Ser125 to glycine, mimicking constitutive dephosphorylation, markedly increased osteoblastic differentiation, which was enhanced by, but did not require, additional dexamethasone supplementation. Collectively, these results demonstrate that dexamethasone induces osteogenesis, at least in part, by modulating the phosphorylation state of a negative-regulatory serine residue (Ser125) on Runx2. This work identifies a novel mechanism for glucocorticoid-induced osteogenic differentiation and provides insights into the role of Runx2 phosphorylation during skeletal development.

Authors
Phillips, JE; Gersbach, CA; Wojtowicz, AM; García, AJ
MLA Citation
Phillips, JE, Gersbach, CA, Wojtowicz, AM, and García, AJ. "Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation." J Cell Sci 119.Pt 3 (February 1, 2006): 581-591.
PMID
16443755
Source
pubmed
Published In
Journal of cell science
Volume
119
Issue
Pt 3
Publish Date
2006
Start Page
581
End Page
591
DOI
10.1242/jcs.02758

Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries.

Biomaterial surface properties modulate protein adsorption and cell adhesion to elicit diverse cellular responses in biomedical and biotechnological applications. We used alkanethiol self-assembled monolayers presenting well-defined chemistries (OH, CH(3), NH(2), and COOH) to analyze the effects of surface chemistry on myoblast proliferation and differentiation. Surfaces were pre-coated with equivalent densities of fibronectin. C2C12 skeletal myoblasts exhibited surface-dependent differences in cell proliferation (COOH = NH(2) > CH(3) = OH). Myogenin and troponin T gene expression levels were up-regulated on CH(3) and OH surfaces compared to other chemistries. Furthermore, immunostaining for sarcomeric myosin revealed surface chemistry-dependent differences in myogenic differentiation following the pattern OH > CH(3) > NH(2) = COOH. Immunostaining analyses of integrin subunits demonstrated surface chemistry-dependent differences in integrin binding to adsorbed fibronectin. OH and CH(3) surfaces supported selective binding of alpha(5)beta(1) integrin while the COOH and NH(2) functionalities displayed binding of both alpha(5)beta(1) and alpha(V)beta(3) Myogenic differentiation correlated with differences in integrin binding; surface chemistries that supported selective binding of alpha(5)beta(1) displayed enhanced differentiation. Finally, blocking beta(1), but not beta(3), integrins inhibited differentiation, implicating specific integrins in the differentiation process. These results demonstrate that surface chemistry modulates myoblast proliferation and differentiation via differences in integrin binding to adsorbed fibronectin.

Authors
Lan, MA; Gersbach, CA; Michael, KE; Keselowsky, BG; García, AJ
MLA Citation
Lan, MA, Gersbach, CA, Michael, KE, Keselowsky, BG, and García, AJ. "Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries." Biomaterials 26.22 (August 2005): 4523-4531.
PMID
15722121
Source
pubmed
Published In
Biomaterials
Volume
26
Issue
22
Publish Date
2005
Start Page
4523
End Page
4531
DOI
10.1016/j.biomaterials.2004.11.028

Soluble markers for the assessment of biological activity with PTK787/ZK 222584 (PTK/ZK), a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor in patients with advanced colorectal cancer from two phase I trials

Background: Plasma and serum biomarkers of angiogenesis and activated endothelial cells were evaluated to assess biological activity of PTK787/ZK 222584 (PTK/ZK), a novel oral angiogenesis inhibitor targeting all known vascular endothelial growth factor (VEGF) receptor tyrosine kinases. Patients and methods: Patients with colorectal cancer (CRC) (n = 63) were enrolled into two phase I/II dose escalation trials of PTK/ZK in 28-day cycles until discontinuation. Patients with stable disease for ≥2 months were categorized as 'non-progressors'. Plasma markers of angiogenesis, VEGF-A and basic fibroblast growth factor (bFGF), and the serum markers of activated endothelial cells, sTIE-2 and sE-Selectin, were assessed at baseline, and pre-dose on days 1, 8, 15, 22 and 28 of every cycle, with additional assessments 10h post-dose on days 1 and 15. The percentage change from baseline was subsequently correlated with AUC and Cmax. of PTK/ZK on day 1, cycle 1 and clinical outcome. Results: A dose-dependent increase in plasma VEGF-A and bFGF was observed in the first cycle of PTK/ZK treatment. The correlation of change in plasma VEGF-A with AUC and Cmax was characterized by an Emax model, suggesting that a change of ≥150% from baseline VEGF-A correlated with non-progressive disease. Change from baseline plasma VEGF-A within the first cycle of treatment was significantly correlated with clinical outcome by logistic regression analysis (P=0.027). Conclusions: In patients with CRC treated with PTK/ZK, changes in plasma VEGF-A and bFGF demonstrate biological activity of PTK/ZK, may help to establish optimal dose and correlate with outcome. © 2005 European Society for Medical Oncology.

Authors
Drevs, J; Zirrgiebel, U; Schmidt-Gersbach, CIM; Mross, K; Medinger, M; Lee, L; Pinheiro, J; Wood, J; Thomas, AL; Unger, C; Henry, A; Steward, WP; Laurent, D; Lebwohl, D; Dugan, M; Marmé, D
MLA Citation
Drevs, J, Zirrgiebel, U, Schmidt-Gersbach, CIM, Mross, K, Medinger, M, Lee, L, Pinheiro, J, Wood, J, Thomas, AL, Unger, C, Henry, A, Steward, WP, Laurent, D, Lebwohl, D, Dugan, M, and Marmé, D. "Soluble markers for the assessment of biological activity with PTK787/ZK 222584 (PTK/ZK), a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor in patients with advanced colorectal cancer from two phase I trials." Annals of Oncology 16.4 (April 1, 2005): 558-565.
PMID
15705616
Source
scopus
Published In
Annals of Oncology
Volume
16
Issue
4
Publish Date
2005
Start Page
558
End Page
565
DOI
10.1093/annonc/mdi118

Runx2-genetically engineered cells for bone tissue engineering

Authors
Gersbach, CA; Phillips, JE; Guldberg, RE; García, AJ
MLA Citation
Gersbach, CA, Phillips, JE, Guldberg, RE, and García, AJ. "Runx2-genetically engineered cells for bone tissue engineering." Proceedings of the 2005 Summer Bioengineering Conference 2005 (2005): 603-604.
Source
scival
Published In
Proceedings of the 2005 Summer Bioengineering Conference
Volume
2005
Publish Date
2005
Start Page
603
End Page
604

Runx2/Cbfa1-genetically engineered skeletal myoblasts mineralize collagen scaffolds in vitro.

Genetic engineering of progenitor and stem cells is an attractive approach to address cell sourcing limitations associated with tissue engineering applications. Bone tissue engineering represents a promising strategy to repair large bone defects, but has been limited in part by the availability of a sustained, mineralizing cell source. This study examined the in vitro mineralization potential of primary skeletal myoblasts genetically engineered to overexpress Runx2/Cbfa1, an osteoblastic transcriptional regulator essential to bone formation. These cells were viable at the periphery of 3D fibrous collagen scaffolds for 6 weeks of static culture. Exogenous Runx2 expression induced osteogenic differentiation and repressed myogenesis in these constructs relative to controls. Runx2-modified cells deposited significant amounts of mineralized matrix and hydroxyapatite, as determined by microcomputed tomography, histological analysis, and Fourier transform infrared spectroscopy, whereas scaffolds seeded with control cells exhibited no mineralized regions. Although mineralization by Runx2-engineered cells was confined to the periphery of the construct, colocalizing with cell viability, it was sufficient to increase the compressive modulus of constructs 30-fold relative to controls. This work demonstrates that Runx2 overexpression in skeletal myoblasts may address current obstacles of bone tissue engineering by providing a potent cell source for in vitro mineralization and construct maturation. Additionally, the use of genetic engineering methods to express downstream control factors and transcriptional regulators, in contrast to soluble signaling molecules, represents a robust strategy to enhance cellular activities for tissue engineering applications.

Authors
Gersbach, CA; Byers, BA; Pavlath, GK; Guldberg, RE; García, AJ
MLA Citation
Gersbach, CA, Byers, BA, Pavlath, GK, Guldberg, RE, and García, AJ. "Runx2/Cbfa1-genetically engineered skeletal myoblasts mineralize collagen scaffolds in vitro." Biotechnol Bioeng 88.3 (November 5, 2004): 369-378.
PMID
15486943
Source
pubmed
Published In
Biotechnology & Bioengineering
Volume
88
Issue
3
Publish Date
2004
Start Page
369
End Page
378
DOI
10.1002/bit.20251

Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype.

Runx2, a transcriptional activator downstream of bone morphogenetic protein (BMP) signaling, is essential to osteoblastic differentiation and bone formation and maintenance. BMPs activate complex signaling networks, utilizing numerous signaling molecules and transcription factors to induce expression of osteoblastic markers in mesenchymal cell types. However, the role of Runx2 in this process, particularly in an environment independent of the other regulatory elements modulated by BMPs, remains poorly understood. In the present study, we used retroviral gene delivery to examine the effects of sustained Runx2 expression in primary myoblasts. Runx2 inhibited myogenesis, as demonstrated by suppression of MyoD and myogenin mRNA levels and reduced myotube formation. Additionally, Runx2-stimulated osteogenesis including osteoblastic gene expression, alkaline phosphatase activity, and biological mineral deposition. Notably, these osteogenic markers were induced to significantly greater levels than those observed in BMP-2-treated controls. These results demonstrate that direct exogenous expression of the Runx2 transcription factor, only one of numerous downstream targets of BMP signaling, is sufficient to induce transdifferentiation of myogenic cells into a mineralizing osteogenic lineage. This work underscores the potency of Runx2 as a regulator of osteogenesis and cell differentiation and provides new insights into the plasticity of committed mesenchymal cells.

Authors
Gersbach, CA; Byers, BA; Pavlath, GK; García, AJ
MLA Citation
Gersbach, CA, Byers, BA, Pavlath, GK, and García, AJ. "Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype." Exp Cell Res 300.2 (November 1, 2004): 406-417.
PMID
15475005
Source
pubmed
Published In
Experimental Cell Research
Volume
300
Issue
2
Publish Date
2004
Start Page
406
End Page
417
DOI
10.1016/j.yexcr.2004.07.031

Runx2-stimulated transdifferentiation of primary skeletal myoblasts into an osteoblastic mineralizing phenotype for bone tissue engineering

The Runx2-stimulated transdifferentiation in skeletal primary myoblasts into an osteoblastic phenotype for bone tissue engineering, was investigated. The primary myoblasts were isolated from Balb/c mice and cultured in growth media. The microscopy and immunofluorescent staining exhibited reduced myotube fusion in Runx2-transduced myoblasts, which suggested inhibited myogenesis. The scaffolds seeded with Runx2-overexpressing myoblasts displayed regions of high X-ray attenuation by microCT and elevated elastic moduli by compression analysis at 42 days. The results show that the forced expression of Runx2 may bypass parallel BMP-2 stimulated regulatory pathways, leading to increased osteogenesis and mineralization.

Authors
Gersbach, CA; Byers, BA; Guldberg, RE; Pavlath, GK; Garcia, AJ
MLA Citation
Gersbach, CA, Byers, BA, Guldberg, RE, Pavlath, GK, and Garcia, AJ. "Runx2-stimulated transdifferentiation of primary skeletal myoblasts into an osteoblastic mineralizing phenotype for bone tissue engineering." Transactions - 7th World Biomaterials Congress (2004): 328--.
Source
scival
Published In
Transactions - 7th World Biomaterials Congress
Publish Date
2004
Start Page
328-

Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype

Authors
GERSBACH, CA
MLA Citation
GERSBACH, CA. "Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype." Exp. Cell Res. 300 (2004): 406-417.
Source
cinii-english
Published In
Exp. Cell Res.
Volume
300
Publish Date
2004
Start Page
406
End Page
417

Addressing cell-sourcing limitations with gene therapy.

Authors
García, AJ; Guldberg, RE; Byers, BA; Gersbach, CA; Phillips, JE
MLA Citation
García, AJ, Guldberg, RE, Byers, BA, Gersbach, CA, and Phillips, JE. "Addressing cell-sourcing limitations with gene therapy." IEEE Eng Med Biol Mag 22.5 (September 2003): 65-70.
PMID
14699938
Source
pubmed
Published In
IEEE Engineering in Medicine and Biology Magazine
Volume
22
Issue
5
Publish Date
2003
Start Page
65
End Page
70
Show More