Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored Dystrophin reading frame in myofibers, cardiomyocytes and muscle stem cells following local or systemic delivery. AAV-Dmd CRISPR-treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors. Adeno-associated virus (AAV) is a member of the genusDependovirus, which lies within the Parvoviridae family (17). An interest in this family of viruses has been stimulated because of their potential use as gene transfer vectors (14).Little is known about the biology of AAV infections, although a significant proportion of humans and nonhuman primates have antibodies in their blood that react to some of the six existing serotypes of AAV (5, 7). This suggests that primates are hosts for infection with AAV, although the clinical sequelae of these infections have yet to be identified.The study of AAV has been limited to the previously described six serotypes, of which five were isolated as contaminants in laboratory preparations of adenoviruses (1,3,16). Our lack of understanding of AAV clinical infections has complicated the search for clinical isolates of the virus. Members of our laboratory recently described a strategy for evaluating latent or persistent AAV genomes from tissues of asymptomatic nonhuman primates through the use of PCR. These studies led to the discovery of two novel AAV serotypes, called AAV7 and AAV8, that have improved properties as vectors for gene therapy (10). In nonhuman primates, AAV sequences were quite prevalent and heterogenous (9).The goal of this study was to determine if latent AAVs exist in humans, and if so, to characterize their structural, serologic, and functional properties. MATERIALS AND METHODSCollection of primate tissues. Our sources of nonhuman primate tissues were described previously (9). Human tissues were collected under two independent IRB protocols approved by the Institutional Review Board of the University of Pennsylvania from either surgical procedures, postmortem examinations, or organ donors through two major national human tissue providers, the Cooperative Human Tissue Network and the National Disease Research Interchange. The human tissues used for this study were comprised of 18 different tissue types that included the colon, liver, lung, spl...

Recombinant adeno-associated viruses (AAVs) have unique gene-transfer properties that speak to their potential as carriers for gene therapy or vaccine applications. However, the presence of neutralizing antibodies to AAV as a result of previous exposure can significantly limit effective gene transfer. In this study, we obtained 888 human serum samples from healthy volunteers in 10 countries around the world. Samples were assayed for neutralizing antibodies to AAV1, AAV2, AAV7, and AAV8, as well as to a novel, structurally distinct AAV vector, rh32.33, in an in vitro transduction inhibition assay. Our data revealed that neutralizing antibodies to AAV2 were the most prevalent antibodies in all regions, followed by antibodies to AAV1. The seroprevalences of antibodies to AAV7 and to AAV8 were lower than that for antibodies to AAV1, and neutralization of AAVrh32.33 was only rarely detected. Our data also indicate a strong linkage of seroreactivity between apparently distinct serotypes that has not been predicted previously in animal models.