Regulation of viral overlapping genes and AAV vector engineering for antibody gene delivery and overcoming the immunological barrier

Abstract

Gene therapy is one of the next-generation biomedical interventions that could precisely edit or supplement human genes, providing great potential to the treatment of conventionally incurable diseases. Recombinant Adeno-associated virus (rAAV) is one of the leading platforms due to its extensively examined safety profiles. However, current applications are hampered by the suboptimal efficacy, the host anti-AAV immune responses and the low manufacturing capacity. I hypothesized that these challenges could be addressed by the AAV vector engineering and the regulation of AAV packaging genes. Therefore, my study focuses on three objectives: (i) development of a system to regulate the viral overlapping genes, (ii) optimization of the AAV vector genome for antibody gene delivery, and (iii) AAV capsid engineering for overcoming the immunological barrier. First, the poor scalability of rAAV production is largely attributed to the absence of a stable rAAV packaging cell line due to the toxicity of viral overlapping genes, and current regulation systems are not compatible with the overlapping gene structure. In this study, I tested multiple strategies and discovered that an intron-based regulation system was a potent approach to regulate viral overlapping genes. Particularly, I developed a synthetic intron containing a splicing trap flanked by loxP. This intron showed ultra-low leakiness in the expression of the intron-inserted gene. Meantime, the co-expression of Cre led to the increased expression of target gene by 116,700-fold. Importantly, conditional expression of AAV overlapping genes cap and rep were successfully achieved by the intron. Hence, the newly established platform opens an avenue to the establishment of rAAV packaging cell lines. Second, I elucidated that the inadequate antibody expressions after gene transfer were resulted from the improper peptide self-cleavage of Furin-2A (F2A), which was associated with the formation of incorrect proteins, protein fusions, protein aggregations and upregulated host immune response. To address these issues, I tested the internal ribosome entry site (IRES) for antibody chains’ coupling expression, resulting in remarkably enhanced antibody expressions by 20-49-fold in cell lines or mice. I also found that the incorporation of ubiquitous chromatin-opening element (UCOE) into the rAAV genome further improved the antibody expressions by 2-fold at 22-week post rAAV administration into mice. These results demonstrated that antibody chains’ coupling expression and the engineered rAAV genome improved antibody gene expression in vivo, which has important implications to the conventional gene transfer system based on F2A. Third, host immune response to viral vectors is the largest barrier to rAAV-based gene therapy. I validated that the ablation of rAAV cellular tropisms was a feasible method to evade the host immune response. Considering that hepatocyte was immune tolerant to rAAV due to the lack of type I interferon response, I engineered synthetic nanobodies (synbodies) specific to ASGR1, a specific marker expressed on hepatocytes. I found that capsid-engineered rAAV strains displaying 9 synbodies exhibited ASGR1-directed transduction, respectively. These results demonstrated that the manipulation of AAV cellular tropism is a promising approach to evade host immune response, providing a useful direction for addressing the issue of anti-AAV host immune responses during gene therapy.