Abstract
Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo, an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo. Both substitution mutations reduce AAV2’s affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo. The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.
Highlights
The non-pathogenic adeno-associated virus type 2 (AAV2) is considered endemic in the human population, with serological evidence supporting lifetime infection rates of 30%–70% worldwide.[1]
Functional Differences between AAV-NP59 and AAV2 Are Attributable to All, or a Subset of, 11 aa Substitutions A recently identified bioengineered AAV variant, AAV-NP59, functionally transduces primary human hepatocytes in a xenograft model of human liver with significantly higher efficiency than that for AAV2.45 Interestingly, sequence analysis at the DNA level revealed that NP59 differed from AAV2 at 51 positions, 37 of which were silent (Figures S1A and S1B)
Alternative Substitutions Attenuating Heparin Binding Improve In Vivo Functional Transduction of Primary Human Hepatocytes with AAV2 Given the high performance of the bioengineered vectors selected from the shuffled AAV library used by Paulk et al.,[45] we investigated whether other functional capsid variants present in that same library could provide additional insights into the relationship between capsid sequence and function on primary human hepatocytes
Summary
The non-pathogenic adeno-associated virus type 2 (AAV2) is considered endemic in the human population, with serological evidence supporting lifetime infection rates of 30%–70% worldwide.[1]. The genome of AAV2 contains two genes, the 50 (rep open reading frame [ORF]) encodes four non-structural proteins (Rep[78], Rep[68], Rep[52], and Rep40), important for genome replication and packaging into the virus capsid and acting as a transcriptional activator and repressor. The 30 part (cap ORF) of the genome encodes three overlapping capsid viral proteins (referred to as VP1, VP2, and VP3).[3] The AAV capsid is assembled from 60 copies of VP1/VP2/ VP3 in a reported 1:1:10 ratio, respectively.[4,5] In addition to the two main rep and cap ORFs, three additional nested ORFs within the cap gene have subsequently been discovered; one encodes the assembly-activating protein (AAP), the second encodes the X gene, and third encodes the membrane-associated accessory protein
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