Rapid Uncoating of Vector Genomes Is the Key toEfficient Liver Transduction with Pseudotyped Adeno-Associated VirusVectors

Abstract

Transduction of the liver with single-stranded adeno-associated virus serotype 2 (AAV2) vectors is inefficient; less than 10% of hepatocytes are permissive for stable transduction, and transgene expression is characterized by a lag phase of up to 6 weeks. AAV2-based vector genomes packaged inside AAV6 or AAV8 capsids can transduce the liver with higher efficiency, but the molecular mechanisms underlying this phenomenon have not been determined. We now show that the primary barrier to transduction of the liver with vectors based on AAV2 capsids is uncoating of vector genomes in the nucleus. The majority of AAV2 genomes persist as encapsidated single-stranded molecules within the nucleus for as long as 6 weeks after vector administration. Double-stranded vector genomes packaged inside AAV2 capsids are at least 50-fold more active than single-stranded counterparts, but these vectors also exhibit a lag phase before maximal gene expression. Vector genomes packaged inside AAV6 or AAV8 capsids do not persist as encapsidated molecules and are more biologically active than vector genomes packaged inside AAV2 capsids. Our data suggest that the rate of uncoating of vector genomes determines the ability of complementary plus and minus single-stranded genomes to anneal together and convert to stable, biologically active double-stranded molecular forms.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

“…Transgene expression from AAV2 vectors is known to increase gradually over several weeks and to persist for months or years. [25][26][27] Consistent with this, at 2 weeks postinjection, there was little detectable human TPP-I protein in the rat striatum, but by 1 month, abundant human TPP-I protein was observed. The high level, stable AAV2-mediated expression of human TPP-I in the rat striatum persisted at comparable volume and intensity at 4, 8, 12, and 18 months ( Figure 5).…”

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

“…89 However, it is possible that differences in kinetics of vector uncoating among various AAV serotypes may influence the presentation of capsid epitopes on MHC-I molecules, thereby inducing differential CTL responses toward transduced cells. 90 Vectors based on AAV serotype 2 have been reported to uncoat slowly, thus potentially allowing processed capsid peptides to be presented by MHC-I. Serotypes that uncoat quickly might not display capsid proteins for the same amount of time or avoid presentation on the MHC-I surface of transduced cells.…”

Intracellular trafficking of adeno-associated viral vectors

“…For example, a recent report has suggested that rAAV2 has a much slower rate of uncoating than rAAV6 and rAAV8 in hepatocytes. 83 Whether uncoating rates between these serotypes are a reflection of different routes of intracellular processing remains to be elucidated. A recent review has also suggested that the rate of intracellular processing and uncoating of viral vectors may critically affect host immunological response toward input capsids in the absence of new viral protein synthesis.…”