Characterizing the Biopotency of Truncated Transgene Variants in rAAV8 viral vectors: Essential Considerations for Gene Therapy Applications

Abstract

Background The production of recombinant Adeno-Associated Virus (rAAV) vectors for gene therapy applications is a critical process, especially when dealing with large transgenes that challenge the 4.7kb capacity limit of rAAV. We aimed to investigate the variances in recombinant rAAV samples post-ultracentrifugation produced in HEK293, focusing on those rAAV8s with either single-stranded (ss) or self-complementary (sc) DNA. Special attention was given to partially filled variants, which might reflect incomplete transgene structures in the manufacturing of rAAV8-FVIII and rAAV8-FIX. A variety of analytical techniques were applied to thoroughly examine these fractions. Results Our data revealed that the so-called "partially empty" capsids, which were presumed to have very low biological activity, actually showed biopotency levels and a significant contribution to the total vector preparation similar to that of the complete rAAV8 fractions. Observations extended to other rAAV8 variants in our lab, including those carrying ssDNA and scDNA for human coagulation factors IX Padua and FVIII. These findings highlight the need to reevaluate the criteria used to differentiate between full and empty rAAV capsids in the purification process. Conclusions Our approach was instrumental in discovering that fractions containing low full/empty ratios exhibited high biopotency. This information enabled us to pinpoint the location of truncated forms in the collected samples, thereby allowing us to choose the most suitable fractions for pooling. In addition, we hypothesize that an amount of these truncated forms might reconstitute into complete genes in host cells and therefore we propose a reconstitution model, challenging traditional views on the necessity of only full rAAV capsids for gene therapy treatments.