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Searching the hairpin in the haystack: Engineering the replication origin of AAV vectors
Gene therapy using adeno-associated virus (AAV)-based vectors has advanced significantly in recent years, with multiple approved therapeutics now available on the market. Engineering of the capsid, transgene, and promoters has shown immense potential to improve the safety and efficacy of these drugs. The only vector component that has mostly remained in its original state is the viral replication origin, the inverted terminal repeats (ITRs). Several ITR variants have so far shown potential to improve vector efficacy and safety as well, implying that the parallel screening of large ITR libraries could improve the identification of superior variants. The reason this has never been attempted for variants of the central part of the ITR may be the strong secondary structure of the ITRs, severely impairing the cloning of novel variants. Additionally, the partial loss of the ITRs due to processing by the host’s DNA repair machinery prohibits tracing of the ITR variants upon transduction. To overcome these bottlenecks, a rarely used plasmid design was harnessed in the present work to facilitate the cloning of more than 110 newly designed ITR variants. This was complemented with a novel Sanger sequencing based method for fast and reliable ITR sequence verification in plasmid DNA, which proved effective for all conventional and most alternative ITR structures. To trace the ITR variants during production and transduction, they were tagged with a barcode sequence in the transgene of the vector genome. Intriguingly, after vector production with a pool of ITR variants, the barcoding also enabled the identification of a novel ITR repair pathway that relies on the presence of ITR sequences as repair templates in trans. This ITR trans-repair was detectable in all parts of the ITR and impedes the barcode-ITR association in pooled productions. Importantly, though, this repair could be circumvented by separate production of each ITR variant, which also facilitated the maintenance of highly diverse ITR hairpin sequences within vector genomes. Subsequently, after confirmation of the vector hairpin integrity in 90 ITR variants with high similarity to the wild-type ITR of AAV2 (wtITR2), the vectors were screened in vitro and in vivo for their effects on transduction. This revealed that the wtITR2 exhibited superior functionality in vitro but not in vivo, suggesting an immense potential for alternative ITR variants to improve transgene expression in human patients. As AAV vectors exhibit semi-random integration into the host genome, which could lead to adverse events in patients, a method was developed that enables interrogation of ITR-associated barcodes as well as the integration region. This allows the quantification of the integration propensity of different ITR variants, thereby complementing the comprehensive pipeline to screen for effects of ITR modifications on vector functionality. Long-term gene expression mediated by AAV vectors relies on vector genomes persisting in the cell as circular episomes or integrated into the host genome. To avoid the reliance on ITR sequences for vector genome circularization by the inefficient host cell machinery and to reduce the risks associated with host genome integration, the packaging of circular DNA genomes in AAV capsids may represent an alternative strategy to enhance the safety and efficacy of AAV-based gene therapy. Therefore, the possibility of generating circular vector genomes using a circovirus-inspired engineered AAV replication origin was examined. As hoped for, this generated circular replication intermediates using the AAV replication machinery, although packaging in AAV particles in a circular conformation was not detected. Collectively, this work has yielded a pipeline for (i) the generation of ITR variant plasmids, (ii) the validation of the ITR sequence in the plasmids, (iii) the confirmation of ITR integrity in the vector genomes, (iv) the analysis of transduction of ITR variants by barcodes, and (v) the interrogation of the effects of ITR variants on host genome integration. Taken together, this forms a comprehensive basis for larger-scale ITR variant screens. Concomitantly, the validation of the formation of circular replication intermediates could serve as a starting point to engineer AAV vectors with circular genomes. As a whole, the results of this work could therefore facilitate and accelerate the development of next-generation AAV gene therapy vectors.
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570 Biowissenschaften, Biologie
570 Life sciences