Successful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

Program in Transplantation Biology, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Molecular Therapy (Impact Factor: 6.23). 06/2012; 20(8):1501-7. DOI: 10.1038/mt.2012.111
Source: PubMed


Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

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Available from: Christine L Halbert, May 08, 2014
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    • "For comparative studies involving normal dogs, we used an AAV vector consisting of a canine factor IX cDNA driven by a CMV immediate early promoter and followed by a polyadenylation signal, all flanked by AAV2 terminal repeats.10-12 For studies in dystrophic dogs, we used an AAV vector consisting of a c-μdys cDNA17 driven by a murine muscle creatine kinase promoter and followed by a polyadenylation signal, all flanked by AAV2 terminal repeats. For serum neutralization studies, we used the ARAP4 AAV vector,23 which consists of a human placental alkaline phosphatase (AP) cDNA driven by a Rous sarcoma virus promoter and followed by a polyadenylation signal, all flanked by AAV2 terminal repeats. "
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    ABSTRACT: Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.Gene Therapy advance online publication, 6 February 2014; doi:10.1038/gt.2014.4.
    Full-text · Article · Feb 2014 · Gene therapy
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    • "Dystrophin proteins were visualized by western blot analysis using method as previously described (Wang et al., 2012). 15μg of cell lysates were loaded in designated lanes. "
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    ABSTRACT: The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.
    Full-text · Article · Dec 2013 · Stem Cell Research
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    • "Adeno-associated virus-mediated in vivo gene transfer of the microdystrophin gene, a truncated but functional form of the protein, was recently shown to be feasible in dystrophic dogs.14 Although promising, this strategy requires the in vivo administration of virus and immunosuppression of the host, procedures that can lead to multiple adverse effects.14,15,16 An alternative approach to in vivo gene transfer would be to use targeted integration to safely introduce the microdystrophin gene ex vivo into cultured myoblasts or muscle progenitor cells and then to transplant these cells into the patient.17,18 "
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    ABSTRACT: Zinc finger nucleases (ZFN) can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif) receptor 5 (CCR5) locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP) or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs) and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O(6)-methylguanine-DNA methyltransferase(P140K) gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.Molecular Therapy - Nucleic Acids (2013) 2, e68; doi:10.1038/mtna.2012.55; published online 29 January 2013.
    Full-text · Article · Jan 2013 · Molecular Therapy - Nucleic Acids
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