Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 ؋ 10 12 viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint.

Recombinant adeno-associated viral (rAAV) vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP) is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input) used in each platform and which related impurities can be expected in final products (output). The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious), residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses), and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.

We developed a drug-free regional intravenous (r.i.) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (i.m.) delivery of the same dose of vector. We show that r.i. delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After i.m., muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although r.i. delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that r.i. is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.