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Engineered adeno-associated virus 3 vector with reduced reactivity to serum antibodies.

ABSTRACT: The natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions (S472A, S587A, and N706A) on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials.

SUBMITTER: Ito M

PROVIDER: S-EPMC8084969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recombinant adeno-associated viral (rAAV) vector mobilization is a largely theoretical process in which intact AAV vectors spread or "mobilize" from transduced cells and infect additional cells within, or external of, the initial host. This process can be helper virus-independent (vector alone) or helper virus-dependent (de novo rAAV production facilitated by superinfection of both wild-type AAV [wtAAV] and Adenovirus 5 [Ad] helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well-established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 kb), rAAV-IDUA (3.7 kb), and rAAV-Nano-dysferlin (4.9 kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and an rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be defined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed Ad-independent mobilization). In rAAV-infected cells coinfected with Ad and wtAAV, rAAV particle production was increased >50-fold compared with no Ad conditions. In addition, Ad-dependent rAAV vectors mobilized and resulted in >1,000-fold transduction upon a subsequent second-round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization-resistant vectors and the opportunity to derive better vector production systems.

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Engineered adeno-associated virus 3 vector with reduced reactivity to serum antibodies.

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