AAV Immunotoxicity: Implications in Anti-HBV Gene Therapy

Abstract

Hepatitis B virus (HBV) has afflicted humankind for decades and there is still no treatment that can clear the infection. The development of recombinant adeno-associated virus (rAAV)-based gene therapy for HBV infection has become important in recent years and research has made exciting leaps. Initial studies, mainly using mouse models, showed that rAAVs are non-toxic and induce minimal immune responses. However, several later studies demonstrated rAAV toxicity, which is inextricably associated with immunogenicity. This is a major setback for the progression of rAAV-based therapies toward clinical application. Research aimed at understanding the mechanisms behind rAAV immunity and toxicity has contributed significantly to the inception of approaches to overcoming these challenges. The target tissue, the features of the vector, and the vector dose are some of the determinants of AAV toxicity, with the latter being associated with the most severe adverse events. This review discusses our current understanding of rAAV immunogenicity, toxicity, and approaches to overcoming these hurdles. How this information and current knowledge about HBV biology and immunity can be harnessed in the efforts to design safe and effective anti-HBV rAAVs is discussed.

Keywords: adeno-associated viral vectors; gene therapy; hepatitis B virus; immunotolerance; immunotoxicity.

Figure 1

Commonly used tools in HBV gene therapy development. To silence HBV gene expression, the RNAi pathway is activated exogenously by artificial microRNAs (miRNAs), for example. A microRNA duplex is incorporated into an RNA silencing complex (RISC) before the selection of one strand that will guide the RISC to the target HBV messenger RNAs. This results in HBV RNA degradation or translation suppression. Transcription activator-like endonucleases (TALENs) and zinc finger nucleases (ZFNs) work in pairs and require a right TALEN or right ZFN and left TALEN or left ZFN, conjugated to a cleavage domain, for double-strand cleavage to occur. TALENs consist of tandem repeats comprising 33–35 amino acids, whereas ZFNs consist of only 3–6 ZFs. The single guide RNA (sgRNA) associate and directs Cas 9 to the DNA sequence of interest. The Cas 9 enzyme creates double-stranded DNA breaks. Once cleavage has occurred, a double-strand DNA break occurs and recruits host machinery for repair. The error-prone non-homologous end joining (NHEJ) repair pathway is favored, which leads to insertions and deletions (indels) within the cccDNA/rcDNA (created with Biorender.com, accessed on 29 November 2023).

Figure 2

Development of AAVs for anti-HBV gene delivery. The wild-type AAV rep and cap ORFs are replaced with the anti-HBV sequence, most preferably driven by a liver-specific promoter (LSP). For primary microRNA (pri-miRNA) or short hairpin RNA (shRNA) and zinc finger nucleases (ZFNs), the self-complementary AAVs (scAAVs) are commonly used, whereas single-stranded AAVs (ssAAVs) are used for transcription activator-like endonucleases (TALENs) and RNA-guided clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR associated (Cas) sequences. sgRNA expression is commonly driven by a polymerase III non-liver-specific promoter (NLSP). The AAV-mediated delivery of RNAi activators targeting Surface and X ORFs has been well characterized. AAVs carrying ZNFs targeting Surface, Polymerase, and Core ORFs have been reported. Although dual ssAAVs are promising for TALEN delivery, no anti-HBV TALEN expressing AAV has been reported. AAVs have been used to deliver CRISPR/Cas sequences against all HBV ORFs. The colored arrows on the HBV genome indicate the four open HBV reading frames, with all seven start codons and encoded proteins indicated (created with Biorender.com, accessed on 29 November 2023).

Figure 3

Treg response modulation by AAV and HBV in the liver. Liver infection with AAV increases IL-10 production by KCs and enhances Foxp3⁺CD4⁺ Tregs differentiation and expansion. Secreted IL-10 can also mediate the conversion of antigen-specific CD4⁺ T cells into Foxp3⁺CD4⁺ Tregs and inhibit CD4⁺ T cell activity. Hepatitis, caused by HBV infection, stimulates MHC-II expression by hepatocytes. This enhances antigen presentation to naïve CD4⁺ T cells and results in their activation and differentiation to form Foxp3⁺CD4⁺ Tregs (created with BioRender.com accessed on 21 November 2023).