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Puzzo F, Crossley MP, Goswami A, Zhang F, Pekrun K, Garzon JL, Cimprich KA, Kay MA. AAV-mediated genome editing is influenced by the formation of R-loops. Mol Ther 2024; 32:4256-4271. [PMID: 39369271 PMCID: PMC11638834 DOI: 10.1016/j.ymthe.2024.09.035] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Revised: 08/05/2024] [Accepted: 09/30/2024] [Indexed: 10/07/2024] Open
Abstract
Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.
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Affiliation(s)
- Francesco Puzzo
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Magdalena P Crossley
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
| | - Aranyak Goswami
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Feijie Zhang
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Katja Pekrun
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Jada L Garzon
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
| | - Karlene A Cimprich
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
| | - Mark A Kay
- Department of Genetics, Stanford University, Stanford, CA 94305, USA; Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
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2
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Puzzo F, Crossley MP, Goswami A, Zhang F, Pekrun K, Garzon JL, Cimprich KA, Kay MA. AAV-mediated genome editing is influenced by the formation of R-loops. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.07.592855. [PMID: 38766176 PMCID: PMC11100726 DOI: 10.1101/2024.05.07.592855] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2024]
Abstract
Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.
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Affiliation(s)
- Francesco Puzzo
- Department of Genetics, Stanford University, Stanford, CA
- Department of Pediatrics, Stanford University, Stanford, CA
| | | | - Aranyak Goswami
- Department of Genetics, Stanford University, Stanford, CA
- Department of Pediatrics, Stanford University, Stanford, CA
| | - Feijie Zhang
- Department of Genetics, Stanford University, Stanford, CA
- Department of Pediatrics, Stanford University, Stanford, CA
| | - Katja Pekrun
- Department of Genetics, Stanford University, Stanford, CA
- Department of Pediatrics, Stanford University, Stanford, CA
| | - Jada L Garzon
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA
| | - Karlene A Cimprich
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA
| | - Mark A Kay
- Department of Genetics, Stanford University, Stanford, CA
- Department of Pediatrics, Stanford University, Stanford, CA
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3
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Kasimsetty A, Sabatino DE. Integration and the risk of liver cancer-Is there a real risk? J Viral Hepat 2024; 31 Suppl 1:26-34. [PMID: 38606944 DOI: 10.1111/jvh.13915] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Revised: 12/10/2023] [Accepted: 12/15/2023] [Indexed: 04/13/2024]
Abstract
Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.
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Affiliation(s)
- Aradhana Kasimsetty
- Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Denise E Sabatino
- The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
- Division of Hematology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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4
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Simpson BP, Yrigollen CM, Izda A, Davidson BL. Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain. Mol Ther 2023; 31:760-773. [PMID: 36617193 PMCID: PMC10014281 DOI: 10.1016/j.ymthe.2023.01.004] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Revised: 12/07/2022] [Accepted: 01/04/2023] [Indexed: 01/09/2023] Open
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.
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Affiliation(s)
- Bryan P Simpson
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; Cell and Molecular Biology Graduate Group, Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, PA, USA
| | - Carolyn M Yrigollen
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Aleksandar Izda
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Beverly L Davidson
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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5
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Shakirova A, Karpov T, Komarova Y, Lepik K. In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization. Front Genome Ed 2023; 5:1068637. [PMID: 36911237 PMCID: PMC9992834 DOI: 10.3389/fgeed.2023.1068637] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Accepted: 02/08/2023] [Indexed: 02/24/2023] Open
Abstract
Gene therapy is a fast developing field of medicine with hundreds of ongoing early-stage clinical trials and numerous preclinical studies. Genome editing (GE) now is an increasingly important technology for achieving stable therapeutic effect in gene correction, with hematopoietic cells representing a key target cell population for developing novel treatments for a number of hereditary diseases, infections and cancer. By introducing a double strand break (DSB) in the defined locus of genomic DNA, GE tools allow to knockout the desired gene or to knock-in the therapeutic gene if provided with an appropriate repair template. Currently, the efficiency of methods for GE-mediated knock-in is limited. Significant efforts were focused on improving the parameters and interaction of GE nuclease proteins. However, emerging data suggests that optimal characteristics of repair templates may play an important role in the knock-in mechanisms. While viral vectors with notable example of AAVs as a donor template carrier remain the mainstay in many preclinical trials, non-viral templates, including plasmid and linear dsDNA, long ssDNA templates, single and double-stranded ODNs, represent a promising alternative. Furthermore, tuning of editing conditions for the chosen template as well as its structure, length, sequence optimization, homology arm (HA) modifications may have paramount importance for achieving highly efficient knock-in with favorable safety profile. This review outlines the current developments in optimization of templates for the GE mediated therapeutic gene correction.
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Affiliation(s)
- Alena Shakirova
- RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov University, Saint Petersburg, Russia
| | - Timofey Karpov
- RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov University, Saint Petersburg, Russia.,Peter the Great St. Petersburg Polytechnic University, Saint Petersburg, Russia
| | - Yaroslava Komarova
- RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov University, Saint Petersburg, Russia
| | - Kirill Lepik
- RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov University, Saint Petersburg, Russia
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6
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Tsuji S, Stephens CJ, Bortolussi G, Zhang F, Baj G, Jang H, de Alencastro G, Muro AF, Pekrun K, Kay MA. Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice. Nat Biotechnol 2022; 40:1285-1294. [PMID: 35393561 PMCID: PMC11648996 DOI: 10.1038/s41587-022-01240-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Accepted: 01/28/2022] [Indexed: 12/12/2022]
Abstract
Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.
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Affiliation(s)
- Shinnosuke Tsuji
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Calvin J Stephens
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Giulia Bortolussi
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
| | - Feijie Zhang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Gabriele Baj
- Light Microscopy Imaging Center, Department of Life Sciences, University of Trieste, Trieste, Italy
| | - Hagoon Jang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | | | - Andrés F Muro
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
| | - Katja Pekrun
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Mark A Kay
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA.
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7
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Bijlani S, Pang KM, Sivanandam V, Singh A, Chatterjee S. The Role of Recombinant AAV in Precise Genome Editing. Front Genome Ed 2022; 3:799722. [PMID: 35098210 PMCID: PMC8793687 DOI: 10.3389/fgeed.2021.799722] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Accepted: 12/14/2021] [Indexed: 12/14/2022] Open
Abstract
The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.
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8
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Boddu PC, Gupta AK, Kim JS, Neugebauer KM, Waldman T, Pillai MM. Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase. Commun Biol 2021; 4:1184. [PMID: 34645977 PMCID: PMC8514589 DOI: 10.1038/s42003-021-02690-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Accepted: 09/15/2021] [Indexed: 11/09/2022] Open
Abstract
Scalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.
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Affiliation(s)
- Prajwal C. Boddu
- grid.47100.320000000419368710Section of Hematology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT USA
| | - Abhishek K. Gupta
- grid.47100.320000000419368710Section of Hematology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT USA
| | - Jung-Sik Kim
- grid.213910.80000 0001 1955 1644Department of Oncology, Molecular Biology and Genetics, Lombardi Cancer Center, Georgetown University, Washington, DC USA
| | - Karla M. Neugebauer
- grid.47100.320000000419368710Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT USA
| | - Todd Waldman
- grid.213910.80000 0001 1955 1644Department of Oncology, Molecular Biology and Genetics, Lombardi Cancer Center, Georgetown University, Washington, DC USA
| | - Manoj M. Pillai
- grid.47100.320000000419368710Section of Hematology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT USA ,grid.47100.320000000419368710Department of Pathology, Yale University School of Medicine, New Haven, CT USA
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9
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Chatterjee S, Sivanandam V, Wong KKM. Adeno-Associated Virus and Hematopoietic Stem Cells: The Potential of Adeno-Associated Virus Hematopoietic Stem Cells in Genetic Medicines. Hum Gene Ther 2021; 31:542-552. [PMID: 32253938 DOI: 10.1089/hum.2020.049] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Adeno-associated virus (AAV)-based vectors have transformed into powerful elements of genetic medicine with proven therapeutic efficacy and a good safety profile. Over the years, efforts to transduce hematopoietic stem cells (HSCs) with AAV2 vectors have, however, been challenging. While there was evidence that AAV2 delivered vector genomes to primitive, quiescent, multipotential, self-renewing, in vivo engrafting HSCs, transgene expression was elusive. In this study, we review the evolution of AAV transduction of HSC, starting with AAV2 vectors leading to the isolation of a family of naturally occurring AAVs from human CD34+ HSC, the AAVHSC. The stem cell-derived AAVHSCs have turned out to have remarkable potentials for genetic therapies well beyond the hematopoietic system. AAVHSCs have tropism for a wide variety of peripheral tissues, including the liver, muscle, and the retina. They cross the blood-brain barrier and transduce cells of the central nervous system. Preclinical gene therapy studies underway using AAVHSC vectors are discussed. We review the notable ability of AAVHSCs to mediate efficient, seamless homologous recombination in the absence of exogenous nuclease activity and discuss the therapeutic implications. We also discuss early results from an AAVHSC-based clinical gene therapy trial that is underway for the treatment of phenylketonuria. Thus, the stem cell-derived AAVHSC, offer a multifaceted platform for in vivo gene therapy and genome editing for the treatment of inherited diseases.
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Affiliation(s)
- Saswati Chatterjee
- Department of Surgery, Beckman Research Institute of City of Hope Medical Center, Duarte, California, USA
| | - Venkatesh Sivanandam
- Department of Surgery, Beckman Research Institute of City of Hope Medical Center, Duarte, California, USA
| | - Kamehameha Kai-Min Wong
- Department of Hematology and Stem Cell Transplantation, City of Hope Medical Center, Duarte, California, USA
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10
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Lam AJ, Lin DTS, Gillies JK, Uday P, Pesenacker AM, Kobor MS, Levings MK. Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity. Cell Rep 2021; 36:109494. [PMID: 34348163 DOI: 10.1016/j.celrep.2021.109494] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 06/28/2021] [Accepted: 07/14/2021] [Indexed: 12/30/2022] Open
Abstract
Regulatory T cell (Treg) therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knockin in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies.
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Affiliation(s)
- Avery J Lam
- Department of Surgery, University of British Columbia, Vancouver, BC V5Z 1M9, Canada; BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada
| | - David T S Lin
- BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, BC V6H 3N1, Canada
| | - Jana K Gillies
- Department of Surgery, University of British Columbia, Vancouver, BC V5Z 1M9, Canada; BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada
| | - Prakruti Uday
- Department of Surgery, University of British Columbia, Vancouver, BC V5Z 1M9, Canada; BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada
| | - Anne M Pesenacker
- Department of Surgery, University of British Columbia, Vancouver, BC V5Z 1M9, Canada; BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada
| | - Michael S Kobor
- BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, BC V6H 3N1, Canada
| | - Megan K Levings
- Department of Surgery, University of British Columbia, Vancouver, BC V5Z 1M9, Canada; BC Children's Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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11
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Spector LP, Tiffany M, Ferraro NM, Abell NS, Montgomery SB, Kay MA. Evaluating the Genomic Parameters Governing rAAV-Mediated Homologous Recombination. Mol Ther 2021; 29:1028-1046. [PMID: 33248247 PMCID: PMC7934627 DOI: 10.1016/j.ymthe.2020.11.025] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2020] [Revised: 10/16/2020] [Accepted: 11/18/2020] [Indexed: 12/26/2022] Open
Abstract
Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1,000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors, with each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions. Targeting efficiency was enhanced by target site transcription, while chromatin accessibility was associated with an increased likelihood of targeting. ChromHMM chromatin states characterizing transcription and enhancers in wild-type K562 cells were also associated with increased AAV-HR efficiency with and without target site transcription, respectively. Furthermore, the amenability of a site to targeting was influenced by the endogenous transcriptional level of intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.
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Affiliation(s)
- Laura P Spector
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA
| | - Matthew Tiffany
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA
| | - Nicole M Ferraro
- Biomedical Informatics Program, Stanford University School of Medicine, Stanford, CA, USA
| | - Nathan S Abell
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA
| | - Stephen B Montgomery
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Mark A Kay
- Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
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12
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de Alencastro G, Puzzo F, Pavel-Dinu M, Zhang F, Pillay S, Majzoub K, Tiffany M, Jang H, Sheikali A, Cromer MK, Meetei R, Carette JE, Porteus MH, Pekrun K, Kay MA. Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex. Mol Ther 2021; 29:1016-1027. [PMID: 33678249 DOI: 10.1016/j.ymthe.2020.10.020] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 08/27/2020] [Accepted: 10/19/2020] [Indexed: 11/16/2022] Open
Abstract
Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to ∼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by ∼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by ∼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.
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Affiliation(s)
| | - Francesco Puzzo
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Mara Pavel-Dinu
- Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford, CA, USA
| | - Feijie Zhang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Sirika Pillay
- Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA
| | - Karim Majzoub
- Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA
| | - Matthew Tiffany
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Hagoon Jang
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Adam Sheikali
- Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford, CA, USA
| | - M Kyle Cromer
- Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford, CA, USA
| | - Ruhikanta Meetei
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Jan E Carette
- Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA
| | - Matthew H Porteus
- Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford, CA, USA
| | - Katja Pekrun
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA
| | - Mark A Kay
- Departments of Pediatrics and Genetics, Stanford University, Stanford, CA, USA.
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13
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Chen HM, Resendes R, Ghodssi A, Sookiasian D, Tian M, Dollive S, Adamson-Small L, Avila N, Tazearslan C, Thompson JF, Ellsworth JL, Francone O, Seymour A, Wright JB. Molecular characterization of precise in vivo targeted gene integration in human cells using AAVHSC15. PLoS One 2020; 15:e0233373. [PMID: 32453743 PMCID: PMC7250422 DOI: 10.1371/journal.pone.0233373] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Accepted: 05/04/2020] [Indexed: 02/08/2023] Open
Abstract
Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.
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Affiliation(s)
- Huei-Mei Chen
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Rachel Resendes
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Azita Ghodssi
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | | | - Michael Tian
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Serena Dollive
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | | | - Nancy Avila
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Cagdas Tazearslan
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - John F. Thompson
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Jeff L. Ellsworth
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Omar Francone
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Albert Seymour
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
| | - Jason B. Wright
- Homology Medicines Inc., Bedford, Massachusetts, United States of America
- * E-mail:
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14
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DNA methyltransferase inhibitors induce a BRCAness phenotype that sensitizes NSCLC to PARP inhibitor and ionizing radiation. Proc Natl Acad Sci U S A 2019; 116:22609-22618. [PMID: 31591209 DOI: 10.1073/pnas.1903765116] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC.
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15
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Smith LJ, Wright J, Clark G, Ul-Hasan T, Jin X, Fong A, Chandra M, St Martin T, Rubin H, Knowlton D, Ellsworth JL, Fong Y, Wong KK, Chatterjee S. Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing. Proc Natl Acad Sci U S A 2018; 115:E7379-E7388. [PMID: 30018062 PMCID: PMC6077703 DOI: 10.1073/pnas.1802343115] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+ cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.
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Affiliation(s)
- Laura J Smith
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | | | - Gabriella Clark
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | - Taihra Ul-Hasan
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | - Xiangyang Jin
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | - Abigail Fong
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | - Manasa Chandra
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | | | | | | | | | - Yuman Fong
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010
| | - Kamehameha K Wong
- Department of Hematology and Stem Cell Transplantation, City of Hope Medical Center, Duarte, CA 91010
| | - Saswati Chatterjee
- Department of Surgery, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010;
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16
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Vandemoortele G, De Sutter D, Eyckerman S. Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery. Bio Protoc 2017; 7:e2211. [PMID: 34541219 DOI: 10.21769/bioprotoc.2211] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2016] [Revised: 12/27/2016] [Accepted: 03/14/2017] [Indexed: 11/02/2022] Open
Abstract
The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol, we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line.
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Affiliation(s)
- Giel Vandemoortele
- VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.,Department of Biochemistry, Ghent University, Ghent, Belgium
| | - Delphine De Sutter
- VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.,Department of Biochemistry, Ghent University, Ghent, Belgium
| | - Sven Eyckerman
- VIB-UGent Center for Medical Biotechnology, Ghent, Belgium.,Department of Biochemistry, Ghent University, Ghent, Belgium
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17
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Combining Engineered Nucleases with Adeno-associated Viral Vectors for Therapeutic Gene Editing. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1016:29-42. [PMID: 29130152 DOI: 10.1007/978-3-319-63904-8_2] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
With the recent advent of several generations of targeted DNA nucleases, most recently CRISPR/Cas9, genome editing has become broadly accessible across the biomedical community. Importantly, the capacity of these nucleases to modify specific genomic loci associated with human disease could render new classes of genetic disease, including autosomal dominant or even idiopathic disease, accessible to gene therapy. In parallel, the emergence of adeno-associated virus (AAV) as a clinically important vector raises the possibility of integrating these two technologies towards the development of gene editing therapies. Though clear challenges exist, numerous proof-of-concept studies in preclinical models offer exciting promise for the future of gene therapy.
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18
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CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells. MOLECULAR THERAPY. NUCLEIC ACIDS 2016; 5:e393. [PMID: 27898094 PMCID: PMC5155318 DOI: 10.1038/mtna.2016.100] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/30/2016] [Accepted: 09/30/2016] [Indexed: 12/18/2022]
Abstract
Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.
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19
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Sather BD, Romano Ibarra GS, Sommer K, Curinga G, Hale M, Khan IF, Singh S, Song Y, Gwiazda K, Sahni J, Jarjour J, Astrakhan A, Wagner TA, Scharenberg AM, Rawlings DJ. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med 2016; 7:307ra156. [PMID: 26424571 DOI: 10.1126/scitranslmed.aac5530] [Citation(s) in RCA: 182] [Impact Index Per Article: 20.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.
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Affiliation(s)
- Blythe D Sather
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Guillermo S Romano Ibarra
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Karen Sommer
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Gabrielle Curinga
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Malika Hale
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Iram F Khan
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Swati Singh
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Yumei Song
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Kamila Gwiazda
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | - Jaya Sahni
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA
| | | | | | - Thor A Wagner
- Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, WA 98101, USA. Department of Pediatrics, University of Washington, Seattle, WA 98101, USA
| | - Andrew M Scharenberg
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA. Department of Pediatrics, University of Washington, Seattle, WA 98101, USA. Department of Immunology, University of Washington, Seattle, WA 98101, USA
| | - David J Rawlings
- Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA. Department of Pediatrics, University of Washington, Seattle, WA 98101, USA. Department of Immunology, University of Washington, Seattle, WA 98101, USA
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20
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Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications. Mol Ther 2015; 24:458-64. [PMID: 26373345 DOI: 10.1038/mt.2015.151] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2015] [Accepted: 07/21/2015] [Indexed: 12/18/2022] Open
Abstract
In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.
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21
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Salganik M, Hirsch ML, Samulski RJ. Adeno-associated Virus as a Mammalian DNA Vector. Microbiol Spectr 2015; 3:10.1128/microbiolspec.MDNA3-0052-2014. [PMID: 26350320 PMCID: PMC4677393 DOI: 10.1128/microbiolspec.mdna3-0052-2014] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2014] [Indexed: 12/20/2022] Open
Abstract
In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells.
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Affiliation(s)
- Max Salganik
- Gene Therapy Center, Department of Pharmacology, University of North Carolina, Chapel Hill, NC
| | - Matthew L Hirsch
- Gene Therapy Center, Department of Pharmacology, University of North Carolina, Chapel Hill, NC
| | - Richard Jude Samulski
- Gene Therapy Center, Department of Pharmacology, University of North Carolina, Chapel Hill, NC
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22
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Abstract
The ability to edit the genome of cell lines has provided valuable insights into biological processes and the contribution of specific mutations to disease biology. These techniques fall into two categories based on the DNA repair mechanism that is used to incorporate the genetic change. Nuclease-based technologies, such as Zinc-Finger Nucleases, TALENS, and Crispr/Cas9, rely on non-homologous end-joining (NHEJ) and homology directed repair (HDR) to generate a range of genetic modifications. Adeno-Associated Virus (AAV) utilizes homologous recombination to generate precise and predictable genetic modifications directly at the target locus. AAV has been used to create over 500 human isogenic cell lines comprising a wide range of genetic alterations from gene knockouts, insertions of point mutations, indels, epitope tags, and reporter genes. Here we describe the generation and use of AAV gene targeting vectors and viruses to create targeted isogenic cell lines.
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Affiliation(s)
- Rob Howes
- MedImmune, Milstein Building, Granta Park, Cambridge, CB21 6GH, UK,
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23
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Deyle DR, Hansen RS, Cornea AM, Li LB, Burt AA, Alexander IE, Sandstrom RS, Stamatoyannopoulos JA, Wei CL, Russell DW. A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol 2014; 21:969-75. [PMID: 25282150 PMCID: PMC4405182 DOI: 10.1038/nsmb.2895] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Accepted: 08/27/2014] [Indexed: 02/03/2023]
Abstract
To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. We used an adeno-associated virus vector to target identical loci introduced as transcriptionally active retroviral vectors. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats or DNase I-hypersensitive sites. Targeted sites were preferentially located within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. We determined the impact of DNA replication by mapping replication forks, which revealed a preference for recombination at target loci transcribed toward an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells and demonstrate a strong recombinogenic effect of colliding polymerases.
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Affiliation(s)
- David R Deyle
- Department of Medicine, University of Washington, Seattle, Washington, USA
| | - R Scott Hansen
- Department of Medicine, University of Washington, Seattle, Washington, USA
| | - Anda M Cornea
- Department of Molecular and Cellular Biology, University of Washington, Seattle, Washington, USA
| | - Li B Li
- Department of Medicine, University of Washington, Seattle, Washington, USA
| | - Amber A Burt
- Department of Medicine, University of Washington, Seattle, Washington, USA
| | - Ian E Alexander
- Gene Therapy Research Unit, Children's Medical Research Institute, Westmead, New South Wales, Australia
| | - Richard S Sandstrom
- Department of Genome Sciences, University of Washington, Seattle, Washington, USA
| | | | - Chia-Lin Wei
- Genomic Technologies Department, Joint Genome Institute, Walnut Creek, California, USA
| | - David W Russell
- 1] Department of Medicine, University of Washington, Seattle, Washington, USA. [2] Department of Biochemistry, University of Washington, Seattle, Washington, USA
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24
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Ohba S, Mukherjee J, See WL, Pieper RO. Mutant IDH1-driven cellular transformation increases RAD51-mediated homologous recombination and temozolomide resistance. Cancer Res 2014; 74:4836-44. [PMID: 25035396 DOI: 10.1158/0008-5472.can-14-0924] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (γ-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 μmol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma.
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Affiliation(s)
- Shigeo Ohba
- Department of Neurological Surgery, University of California, San Francisco, San Francisco, California
| | - Joydeep Mukherjee
- Department of Neurological Surgery, University of California, San Francisco, San Francisco, California
| | - Wendy L See
- Department of Neurological Surgery, University of California, San Francisco, San Francisco, California
| | - Russell O Pieper
- Department of Neurological Surgery, University of California, San Francisco, San Francisco, California
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25
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Nemo-like kinase (NLK) negatively regulates NF-kappa B activity through disrupting the interaction of TAK1 with IKKβ. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2014; 1843:1365-72. [PMID: 24721172 DOI: 10.1016/j.bbamcr.2014.03.028] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/25/2013] [Revised: 03/02/2014] [Accepted: 03/31/2014] [Indexed: 01/01/2023]
Abstract
Stringent negative regulation of the transcription factor NF-κB is essential for maintaining cellular stress responses and homeostasis. However, the tight regulation mechanisms of IKKβ are still not clear. Here, we reported that nemo-like kinase (NLK) is a suppressor of tumor necrosis factor (TNFα)-induced NF-κB signaling by inhibiting the phosphorylation of IKKβ. Overexpression of NLK largely blocked TNFα-induced NF-κB activation, p65 nuclear localization and IκBα degradation; whereas genetic inactivation of NLK showed opposing results. Mechanistically, we identified that NLK interacted with IκB kinase (IKK)-associated complex, which in turn inhibited the assembly of the TAK1/IKKβ and thereby, diminished the IκB kinase phosphorylation. Our results indicate that NLK functions as a pivotal negative regulator in TNFα-induced activation of NF-κB via disrupting the interaction of TAK1 with IKKβ.
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26
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Galli A, Cervelli T. Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae. BMC Mol Biol 2014; 15:5. [PMID: 24521444 PMCID: PMC3925961 DOI: 10.1186/1471-2199-15-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2013] [Accepted: 02/06/2014] [Indexed: 12/03/2022] Open
Abstract
Background Homologous recombination mediated gene targeting is still too inefficient to be applied extensively in genomics and gene therapy. Although sequence-specific nucleases could greatly stimulate gene targeting efficiency, the off-target cleavage sites of these nucleases highlighted the risk of this strategy. Adeno-associated virus (AAV)-based vectors are used for specific gene knockouts, since several studies indicate that these vectors are able to induce site-specific genome alterations at high frequency. Since each targeted event is accompanied by at least ten random integration events, increasing our knowledge regarding the mechanisms behind these events is necessary in order to understand the potential of AAV-mediated gene targeting for therapy application. Moreover, the role of AAV regulatory proteins (Rep) and inverted terminal repeated sequences (ITRs) in random and homologous integration is not completely known. In this study, we used the yeast Saccharomyces cerevisiae as a genetic model system to evaluate whether the presence of ITRs in the integrating plasmid has an effect on gene targeting and random integration. Results We have shown that the presence of ITRs flanking a gene targeting vector containing homology to its genomic target decreased the frequency of random integration, leading to an increase in the gene targeting/random integration ratio. On the other hand, the expression of Rep proteins, which produce a nick in the ITR, significantly increased non-homologous integration of a DNA fragment sharing no homology to the genome, but had no effect on gene targeting or random integration when the DNA fragment shared homology with the genome. Molecular analysis showed that ITRs are frequently conserved in the random integrants, and that they induce rearrangements. Conclusions Our results indicate that ITRs may be a useful tool for decreasing random integration, and consequently favor homologous gene targeting.
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Affiliation(s)
- Alvaro Galli
- Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56125 Pisa, Italy.
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Deyle DR, Li LB, Ren G, Russell DW. The effects of polymorphisms on human gene targeting. Nucleic Acids Res 2013; 42:3119-24. [PMID: 24371280 PMCID: PMC3950700 DOI: 10.1093/nar/gkt1303] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.
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Affiliation(s)
- David R Deyle
- Department of Medicine, University of Washington, Seattle, WA, 98195, USA and Department of Biochemistry, University of Washington, Seattle, WA, 98195, USA
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28
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Zhang X, Li H, Mao Y, Li Z, Wang R, Guo T, Jin L, Song R, Xu W, Zhou N, Zhang Y, Hu R, Wang X, Huang H, Lei Z, Niu G, Irwin DM, Tan H. An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology. PLoS One 2013; 8:e75493. [PMID: 24223114 PMCID: PMC3819351 DOI: 10.1371/journal.pone.0075493] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2013] [Accepted: 07/17/2013] [Indexed: 01/17/2023] Open
Abstract
Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.
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Affiliation(s)
- Xiaojing Zhang
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Hui Li
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Yiqing Mao
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Zhixin Li
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Rong Wang
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Tingting Guo
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Ling Jin
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Rongjing Song
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Wei Xu
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Na Zhou
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Yizhuang Zhang
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Ruobi Hu
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Xi Wang
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Huakang Huang
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
| | - Zhen Lei
- Department of Pharmacology, Ningxia Medical University, Yinchuan, China
| | - Gang Niu
- Beijing N&N Genetech Company, Beijing, China
| | - David M. Irwin
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
- * E-mail: (HT); (DMI)
| | - Huanran Tan
- Department of Pharmacology, Peking University, Health Science Center, Beijing, China
- * E-mail: (HT); (DMI)
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Ramamoorthi K, Curtis D, Asuri P. Advances in homology directed genetic engineering of human pluripotent and adult stem cells. World J Stem Cells 2013; 5:98-105. [PMID: 24179598 PMCID: PMC3812527 DOI: 10.4252/wjsc.v5.i4.98] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/26/2013] [Revised: 09/06/2013] [Accepted: 09/17/2013] [Indexed: 02/06/2023] Open
Abstract
The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.
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Rahman SH, Bobis-Wozowicz S, Chatterjee D, Gellhaus K, Pars K, Heilbronn R, Jacobs R, Cathomen T. The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting. Hum Gene Ther 2013; 24:67-77. [PMID: 23072634 PMCID: PMC3555098 DOI: 10.1089/hum.2012.168] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2012] [Accepted: 10/08/2012] [Indexed: 01/28/2023] Open
Abstract
Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.
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Affiliation(s)
- Shamim H Rahman
- Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany.
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31
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Paulk NK, Loza LM, Finegold MJ, Grompe M. AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo. Hum Gene Ther 2012; 23:658-65. [PMID: 22486314 DOI: 10.1089/hum.2012.038] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.
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Affiliation(s)
- Nicole K Paulk
- Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR 97239, USA
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32
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Luo Y, Kofod-Olsen E, Christensen R, Sørensen CB, Bolund L. Targeted genome editing by recombinant adeno-associated virus (rAAV) vectors for generating genetically modified pigs. J Genet Genomics 2012; 39:269-74. [PMID: 22749014 DOI: 10.1016/j.jgg.2012.05.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2012] [Accepted: 05/10/2012] [Indexed: 12/22/2022]
Abstract
Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applicability to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer (SCNT), this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.
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Affiliation(s)
- Yonglun Luo
- Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, DK-8000 Aarhus C, Denmark.
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33
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Targeted In Situ Gene Correction of Dysfunctional APOE Alleles to Produce Atheroprotective Plasma ApoE3 Protein. Cardiol Res Pract 2012; 2012:148796. [PMID: 22645694 PMCID: PMC3356902 DOI: 10.1155/2012/148796] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Accepted: 01/30/2012] [Indexed: 02/07/2023] Open
Abstract
Cardiovascular disease is the leading worldwide cause of death. Apolipoprotein E (ApoE) is a 34-kDa circulating glycoprotein, secreted by the liver and macrophages with pleiotropic antiatherogenic functions and hence a candidate to treat hypercholesterolaemia and atherosclerosis. Here, we describe atheroprotective properties of ApoE, though also potential proatherogenic actions, and the prevalence of dysfunctional isoforms, outline conventional gene transfer strategies, and then focus on gene correction therapeutics that can repair defective APOE alleles. In particular, we discuss the possibility and potential benefit of applying in combination two technical advances to repair aberrant APOE genes: (i) an engineered endonuclease to introduce a double-strand break (DSB) in exon 4, which contains the common, but dysfunctional, ε2 and ε4 alleles; (ii) an efficient and selectable template for homologous recombination (HR) repair, namely, an adeno-associated viral (AAV) vector, which harbours wild-type APOE sequence. This technology is applicable ex vivo, for example to target haematopoietic or induced pluripotent stem cells, and also for in vivo hepatic gene targeting. It is to be hoped that such emerging technology will eventually translate to patient therapy to reduce CVD risk.
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34
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Asuri P, Bartel MA, Vazin T, Jang JH, Wong TB, Schaffer DV. Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells. Mol Ther 2012; 20:329-38. [PMID: 22108859 PMCID: PMC3277219 DOI: 10.1038/mt.2011.255] [Citation(s) in RCA: 97] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2011] [Accepted: 10/27/2011] [Indexed: 12/12/2022] Open
Abstract
Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.
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Affiliation(s)
- Prashanth Asuri
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720-1462, USA
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35
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Sargent RG, Kim S, Gruenert DC. Oligo/polynucleotide-based gene modification: strategies and therapeutic potential. Oligonucleotides 2011; 21:55-75. [PMID: 21417933 DOI: 10.1089/oli.2010.0273] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Oligonucleotide- and polynucleotide-based gene modification strategies were developed as an alternative to transgene-based and classical gene targeting-based gene therapy approaches for treatment of genetic disorders. Unlike the transgene-based strategies, oligo/polynucleotide gene targeting approaches maintain gene integrity and the relationship between the protein coding and gene-specific regulatory sequences. Oligo/polynucleotide-based gene modification also has several advantages over classical vector-based homologous recombination approaches. These include essentially complete homology to the target sequence and the potential to rapidly engineer patient-specific oligo/polynucleotide gene modification reagents. Several oligo/polynucleotide-based approaches have been shown to successfully mediate sequence-specific modification of genomic DNA in mammalian cells. The strategies involve the use of polynucleotide small DNA fragments, triplex-forming oligonucleotides, and single-stranded oligodeoxynucleotides to mediate homologous exchange. The primary focus of this review will be on the mechanistic aspects of the small fragment homologous replacement, triplex-forming oligonucleotide-mediated, and single-stranded oligodeoxynucleotide-mediated gene modification strategies as it relates to their therapeutic potential.
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Affiliation(s)
- R Geoffrey Sargent
- Department of Otolaryngology-Head and Neck Surgery, University of California , San Francisco, California 94115, USA
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36
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Jensen NM, Dalsgaard T, Jakobsen M, Nielsen RR, Sørensen CB, Bolund L, Jensen TG. An update on targeted gene repair in mammalian cells: methods and mechanisms. J Biomed Sci 2011; 18:10. [PMID: 21284895 PMCID: PMC3042377 DOI: 10.1186/1423-0127-18-10] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2010] [Accepted: 02/02/2011] [Indexed: 11/10/2022] Open
Abstract
Transfer of full-length genes including regulatory elements has been the preferred gene therapy strategy for clinical applications. However, with significant drawbacks emerging, targeted gene alteration (TGA) has recently become a promising alternative to this method. By means of TGA, endogenous DNA repair pathways of the cell are activated leading to specific genetic correction of single-base mutations in the genome. This strategy can be implemented using single-stranded oligodeoxyribonucleotides (ssODNs), small DNA fragments (SDFs), triplex-forming oligonucleotides (TFOs), adeno-associated virus vectors (AAVs) and zinc-finger nucleases (ZFNs). Despite difficulties in the use of TGA, including lack of knowledge on the repair mechanisms stimulated by the individual methods, the field holds great promise for the future. The objective of this review is to summarize and evaluate the different methods that exist within this particular area of human gene therapy research.
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Affiliation(s)
- Nanna M Jensen
- Institute of Human Genetics, The Bartholin Building, University of Aarhus, 8000 Aarhus C, Denmark
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Gellhaus K, Cornu TI, Heilbronn R, Cathomen T. Fate of Recombinant Adeno-Associated Viral Vector Genomes During DNA Double-Strand Break-Induced Gene Targeting in Human Cells. Hum Gene Ther 2010; 21:543-53. [DOI: 10.1089/hum.2009.167] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Affiliation(s)
- Katharina Gellhaus
- Institute of Virology, Charité Medical School, Campus Benjamin Franklin, D-12203 Berlin, Germany
- Present address: Epiontis GmbH, D-12489 Berlin, Germany
| | - Tatjana I. Cornu
- Institute of Virology, Charité Medical School, Campus Benjamin Franklin, D-12203 Berlin, Germany
- Present address: Epiontis GmbH, D-12489 Berlin, Germany
| | - Regine Heilbronn
- Institute of Virology, Charité Medical School, Campus Benjamin Franklin, D-12203 Berlin, Germany
| | - Toni Cathomen
- Institute of Virology, Charité Medical School, Campus Benjamin Franklin, D-12203 Berlin, Germany
- Department of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany
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Ordinario EC, Yabuki M, Handa P, Cummings WJ, Maizels N. RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions. BMC Mol Biol 2009; 10:98. [PMID: 19863810 PMCID: PMC2774322 DOI: 10.1186/1471-2199-10-98] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2009] [Accepted: 10/28/2009] [Indexed: 01/08/2023] Open
Abstract
Background Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences. This allows gene conversion and recombinational repair to be studied using the chicken DT40 B cell line, which carries out constitutive gene conversion and provides a robust and physiological model for homology-directed repair in vertebrate cells. Results We show that DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of a DT40 derivative in which the rearranged and diversifying immunoglobulin λR light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-λR, showed that RAD51D and XRCC2 localize to the diversifying λR gene. Colocalizations correlate both functionally and physically with active immunoglobulin gene conversion. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants. Conclusion These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy to promote gene correction in other cell types.
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Affiliation(s)
- Ellen C Ordinario
- Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195-7650, USA.
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Adelfalk C, Janschek J, Revenkova E, Blei C, Liebe B, Göb E, Alsheimer M, Benavente R, de Boer E, Novak I, Höög C, Scherthan H, Jessberger R. Cohesin SMC1beta protects telomeres in meiocytes. J Cell Biol 2009; 187:185-99. [PMID: 19841137 PMCID: PMC2768837 DOI: 10.1083/jcb.200808016] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2008] [Accepted: 09/17/2009] [Indexed: 12/29/2022] Open
Abstract
Meiosis-specific mammalian cohesin SMC1beta is required for complete sister chromatid cohesion and proper axes/loop structure of axial elements (AEs) and synaptonemal complexes (SCs). During prophase I, telomeres attach to the nuclear envelope (NE), but in Smc1beta(-/-) meiocytes, one fifth of their telomeres fail to attach. This study reveals that SMC1beta serves a specific role at telomeres, which is independent of its role in determining AE/SC length and loop extension. SMC1beta is necessary to prevent telomere shortening, and SMC3, present in all known cohesin complexes, properly localizes to telomeres only if SMC1beta is present. Very prominently, telomeres in Smc1beta(-/-) spermatocytes and oocytes loose their structural integrity and suffer a range of abnormalities. These include disconnection from SCs and formation of large telomeric protein-DNA extensions, extended telomere bridges between SCs, ring-like chromosomes, intrachromosomal telomeric repeats, and a reduction of SUN1 foci in the NE. We suggest that a telomere structure protected from DNA rearrangements depends on SMC1beta.
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Affiliation(s)
- Caroline Adelfalk
- Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, 01307 Dresden, Germany
| | - Johannes Janschek
- Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, 01307 Dresden, Germany
| | - Ekaterina Revenkova
- Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029
| | - Cornelia Blei
- Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, 01307 Dresden, Germany
| | - Bodo Liebe
- Max Planck Institute of Molecular Genetics, D-14195 Berlin, Germany
| | - Eva Göb
- Department of Cell and Developmental Biology, University of Würzburg, 97074 Würzburg, Germany
| | - Manfred Alsheimer
- Department of Cell and Developmental Biology, University of Würzburg, 97074 Würzburg, Germany
| | - Ricardo Benavente
- Department of Cell and Developmental Biology, University of Würzburg, 97074 Würzburg, Germany
| | - Esther de Boer
- Memorial Sloan-Kettering Cancer Center, New York, NY 10044
| | - Ivana Novak
- Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden
| | - Christer Höög
- Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden
| | - Harry Scherthan
- Max Planck Institute of Molecular Genetics, D-14195 Berlin, Germany
| | - Rolf Jessberger
- Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, 01307 Dresden, Germany
- Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029
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Deyle DR, Russell DW. Adeno-associated virus vector integration. CURRENT OPINION IN MOLECULAR THERAPEUTICS 2009; 11:442-447. [PMID: 19649989 PMCID: PMC2929125] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Adeno-associated virus (AAV) vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo. Although AAV vector genomes can persist within cells as episomes, vector integration has been observed in various experimental settings, either at non-homologous sites where DNA damage may have occurred or by homologous recombination. In some cases, integration is essential for the therapeutic or experimental efficacy of AAV vectors. Recently, insertional mutagenesis resulting from the integration of AAV vectors was associated with tumorigenesis in mice, a consideration that may have relevance for certain clinical applications.
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Affiliation(s)
- David R Deyle
- University of Washington, Departments of Medicine, Mailstop 357720, 1954 NE Pacific Street, Seattle, WA 98195, USA
| | - David W Russell
- University of Washington, Departments of Medicine, Mailstop 357720, 1954 NE Pacific Street, Seattle, WA 98195, USA
- University of Washington, Department of Biochemistry, Mailstop 357720, 1954 NE Pacific Street, Seattle, WA 98195, USA
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Chaurushiya MS, Weitzman MD. Viral manipulation of DNA repair and cell cycle checkpoints. DNA Repair (Amst) 2009; 8:1166-76. [PMID: 19473887 DOI: 10.1016/j.dnarep.2009.04.016] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.
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Affiliation(s)
- Mira S Chaurushiya
- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
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A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair. BMC Biotechnol 2009; 9:35. [PMID: 19379497 PMCID: PMC2676283 DOI: 10.1186/1472-6750-9-35] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2008] [Accepted: 04/20/2009] [Indexed: 12/14/2022] Open
Abstract
Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.
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Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells. Proc Natl Acad Sci U S A 2008; 105:8703-8. [PMID: 18562296 DOI: 10.1073/pnas.0712060105] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Gene targeting has two important applications. One is the inactivation of genes ("knockouts"), and the second is the correction of a mutated allele back to wild-type ("gene therapy"). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombination (HR) to mediate correct targeting; however, the specifics of this mechanism remain unknown. In this work, we attempted to generate Ku70-null human somatic cells by using a rAAV-based gene knockout strategy. Ku70 is the heterodimeric partner of Ku86, and together they constitute an end-binding activity that is required for a pathway [nonhomologous end joining (NHEJ)] of DSB repair that is believed to compete with HR. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70(+/-) cells, the frequency of gene targeting was 5- to 10-fold higher than in wild-type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70(+/-) cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. Our data suggest that gene-targeting frequencies can be significantly improved in human cells by impairing the NHEJ pathway, and we propose that Ku70 depletion can be used to facilitate both knockout and gene therapy approaches.
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Schultz BR, Chamberlain JS. Recombinant adeno-associated virus transduction and integration. Mol Ther 2008; 16:1189-99. [PMID: 18500252 DOI: 10.1038/mt.2008.103] [Citation(s) in RCA: 148] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Recombinant adeno-associated virus (rAAV) holds promise as a gene therapy vector for a multitude of genetic disorders such as hemophilia, cystic fibrosis, and the muscular dystrophies. Given the variety of applications and tissue types toward which these vectors may be targeted, an understanding of rAAV transduction is crucial for the effective application of therapy. rAAV transduction mechanisms have been the subject of much study, resulting in a body of knowledge relating to events from virus-cell attachment through to vector genome conformation in the target cell nucleus. Instead of utilizing one mechanism in each phase of vector transduction, rAAV appears to employ multiple possible pathways toward transgene expression, in part dependent on rAAV serotype, dose, and target cell type. Once inside the nucleus, the rAAV genome exists in a predominantly episomal form; therefore, nondividing cells tend to be most stably transduced. However, rAAV has a low frequency of integration into the host cell genome, often in or near genes, and can be associated with host genome mutations. This review describes the current understanding of the mechanisms and rate-limiting steps involved in rAAV transduction.
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Affiliation(s)
- Brian R Schultz
- Molecular and Cellular Biology, Senator Paul D Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington, Seattle, Washington, USA
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Fernandez SL, Russell DW, Hurlin PJ. Development of human gene reporter cell lines using rAAV mediated homologous recombination. Biol Proced Online 2007; 9:84-90. [PMID: 18464937 PMCID: PMC2374725 DOI: 10.1251/bpo136] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2007] [Revised: 10/04/2007] [Accepted: 11/19/2007] [Indexed: 11/23/2022] Open
Abstract
Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used for selection of correctly targeted alleles by taking advantage of known regulatory conditions that activate transcription of specific genes. Using this method, we describe the generation of primary human fibroblasts that express EGFP-Luciferase under the control of the c-Myc oncogene.
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Affiliation(s)
- Sandra L Fernandez
- Shriners Hospitals for Children. Department of Cell Developmental Biology, Oregon Health & Science, Portland, OR 97201, USA
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Wang Y, Huang F, Cai R, Qian C, Liu X. Targeting strategies for adeno-associated viral vector. ACTA ACUST UNITED AC 2007. [DOI: 10.1007/s11434-007-0260-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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