Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5' end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination.

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the ␤-galactosidase (AAV-lacZ) gene into adult BALB͞c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dosedependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

The purpose of this work was to develop an efficient Therefore the minimum set of genes required to produce method for the production of adeno-associated virus (AAV) AAV helper activity equivalent to that provided by adenvectors in the absence of helper virus. The adenovirus ovirus infection consists of, or is a subset of, the following regions that mediate AAV vector replication were identified genes: the E4orf6 gene, the 72-M r , E2A protein gene, the and assembled into a helper plasmid. These included the VA RNA genes and the E1 region. AAV vector preparations VA, E2A and E4 regions. When this helper plasmid was made with adenovirus and by the helper virus-free method cotransfected into 293 cells, along with plasmids encoding were essentially indistinguishable with respect to particle the AAV vector, and rep and cap genes, AAV vector was density, particle to infectivity ratio, capsimer ratio and produced as efficiently as when using adenovirus infection efficiency of muscle transduction in vivo. Only AAV vector as a source of help. CMV-driven constructs expressing the preparations made by the helper virus-free method were E4orf6 and the 72-M r , E2A proteins were able to funcnot reactive with anti-adenovirus sera. tionally replace the E4 and E2A regions, respectively.