Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAVCMVLacZ) was introduced, ␤-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product.We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle

The limb girdle muscular dystrophies (LGMD) are a genetisarcoglycan-deficient hamster model (Bio14.6). We show cally and phenotypically heterogeneous group of degenerefficient delivery and widespread expression of ␦-sarcoglyative neuromuscular diseases. A subset of the genetically can after a single intramuscular injection. Importantly, rAAV recessive forms of LGMD are caused by mutations in the vector containing the human ␦-sarcoglycan cDNA restored four muscle sarcoglycan genes (␣, ␤, ␥ and ␦). The coding secondary biochemical deficiencies, with correct localizsequences of all known sarcoglycan genes are smaller ation of other sarcoglycan proteins to the muscle fiber than 2 kb, and thus can be readily packaged in recombimembrane. Interestingly, restoration of ␣-, as well as ␤-nant adeno-associated virus (rAAV) vectors. Previously, sarcoglycan was homogeneous and properly localized we have demonstrated highly efficient and sustained transthroughout transduced muscle, and appeared unaffected duction in mature muscle tissue of immunocompetent aniby dramatic overexpression of ␦-sarcoglycan in the cytomals with rAAV vectors. In this report, we utilize recombiplasm of some myofibers. These results support the feasinant AAV containing the ␦-sarcoglycan gene for genetic bility of rAAV vector's application to treat LGMD by means complementation of muscle diseases using a ␦-of direct in vivo gene transfer.

Gene therapy of hypertension requires long-term expression of a therapeutic gene to achieve stable reduction of blood pressure. Human tissue kallikrein (HK) cleaves kininogen to produce a potent vasoactive peptide kinin, which plays an important role in the regulation of the cardiovascular and renal functions. In the present study, we have delivered human kallikrein cDNA with an rAAV vector to explore the potential therapeutic effects of kallikrein on hypertension and related secondary complications. A single tail vein injection of the rAAV-HK vector into the adult spontaneously hypertensive rats resulted in a significant reduction (12.072.55 mmHg, Po0.05, n ¼ 6, ANOVA) of the systolic blood pressure from 2 weeks after vector injection, when compared with the control rAAV-lacZ vector-injected rats. Weekly blood pressure monitoring showed stable hypertension-reduction effect throughout the course of the 20-week experiments. In addition, total urine microalbumin contents decreased as a result of rAAV-HK treatment. Histological analysis of various tissues showed remarkable amelioration of cardiovascular hypertrophy, renal injury and collagen depositions in the rAAV-treated group. Finally, persistent expression of the transgene product HK was confirmed by the enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. We conclude that rAAV-mediated HK delivery rendered a long-term and stable reduction of hypertension and protected against renal injury, cardiac remodeling in the spontaneously hypertensive rat model. Further studies are warranted for the development of a gene therapy strategy for human hypertension.

Transplantation of allogeneic pancreatic islets is an effective approach to treat type 1 diabetes. To bypass the need for systemic administration of immunosuppression drugs following transplantation, approaches to genetically modify allogeneic islets to express anti-inflammatory, immunosuppressive, or antiapoptotic proteins prior to transplantation are being developed. Adeno-associated viral (AAV) based vectors have been used for gene transfer to islets, but the efficiency of functional transduction is low. Recently, doublestranded (ds) or double-copy (dc) based AAV vectors have been developed that allow for more rapid and efficient AAVmediated transgene expression following transduction. Here we demonstrate that intact human and murine islets can be transduced with dsAAV2-eGFP efficiently compared to single-stranded AAV2-eGFP. Furthermore, our results demonstrate that murine islets transduced with dsAAV2-eGFP have normal islet glucose responsiveness, viability, and islet insulin content. Transplantation of the dsAAV2-eGFP transduced islet restored normal glycemia in diabetic mice without eliciting an immune response. Significant dsAAV2-mediated eGFP expression was observed in the islet grafts for at least 6 months post-transplant. Finally, we demonstrated that dsAAV serotypes 2, 6, and 8 infect human islets efficiently. Taken together, these results suggest that dsAAV based vectors are highly appropriate for gene transfer to islets to facilitate transplantation.

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