Adeno-associated viral vector (AAV)-mediated gene transfer represents a promising gene replacement strategy for treating Duchenne muscular dystrophy (DMD). However, recent studies demonstrated cellular immunity specific to AAV capsid proteins in animal models, which resulted in liver toxicity and elimination of transgene expression in a human trial of hemophilia B. We have recently developed immunosuppressive strategies to prevent such immunity for successful long-term transgene expression in dog muscle. Here, we describe in detail the immunosuppressive regimens employed in both normal and DMD dogs and provide methods for evaluating the efficiency of the regimens following intramuscular injection of AAV in dogs.
"CSP and MMF were used as described above.11, 18 Muscle biopsies were taken 4 weeks, 6 months (at the time of discontinuation of immunosuppression) and 7 months (one month after discontinuation of immunosuppression) after vector administration, and extensive expression of c-μdys was detected in myofibers at all time points in both dystrophic dogs without obvious inflammation and with normalized muscle histology (Figure 6). Importantly, extensive c-μdys expression was detected 1 month after cessation of immunosuppression, 7 months after vector injection. "
[Show abstract][Hide abstract] 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.
"Nevertheless, since CSP is already used as an oral drug (in our protocol and Wang et al. (2007b) protocol) we believe our method should be more convenient (both CSP and MMF as oral drugs). In terms of CSP, Wang et al. (2011) targeted a CSP trough level of 100 to 350 ng/mL using the dose of 10 mg/kg/d. In our study, we used a higher dose of 10–20 mg/kg/d to reach a higher CSP trough level of 400 ng/mL. "
[Show abstract][Hide abstract] ABSTRACT: Highly abbreviated micro-dystrophin genes have been intensively studied for Duchenne muscular dystrophy (DMD) gene therapy. Following adeno-associated virus (AAV) gene transfer, robust microgene expression is achieved in murine DMD models in the absence of immune suppression. Interestingly, a recent study suggests that AAV gene transfer in dystrophic dogs may require up to 18 weeks' immune suppression using a combination of three different immune-suppressive drugs (cyclosporine, mycophenolate mofetil, and anti-dog thymocyte globulin). Continued immune suppression is not only costly but also may cause untoward reactions. Further, some of the drugs (such as anti-dog thymocyte globulin) are not readily available. To overcome these limitations, we developed a novel 5-week immune suppression scheme using only cyclosporine and mycophenolate mofetil. AAV vectors (either AV.RSV.AP that expresses the heat-resistant human alkaline phosphatase gene, or AV.CMV.μDys that expresses the canine R16-17/H3/ΔC microgene) at 2.85×10(12) vg particles were injected into adult dystrophic dog limb muscles under the new immune suppression protocol. Sustained transduction was observed for nearly half year (the end of the study). The simplified immune suppression strategy described here may facilitate preclinical studies in the dog model.
Human gene therapy 10/2011; 23(2):202-9. DOI:10.1089/hum.2011.147 · 3.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular diseases, such as ischemic heart disease and heart failure, are the most common cause of death in the Western world and are associated with an enor- mous amount of human suffering and tremendous health care and economic cost . Great strides have been made over the past years in treating cardiac diseases, including congestive heart failure. Pharmacological intervention with beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, and aldosterone antagonists has contributed significantly towards improved therapy and both increase survival and enhance the quality of life in patients with heart failure. However, none of these treatments are curative, and heart failure is ultimately fatal [2, 3]. As a result, inno- vative and novel treatment modalities are urgently needed. One conceptually novel approach that shows considerable promise for the treatment and potential cure of cardiovascular diseases is gene therapy. In this chapter, we will discuss the potential and limitations of gen
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