Investigation of adenoviral immune neutralization and tropism for improved targeted gene delivery

Human adenovirus serotype 5 (HAdV-5)-based gene delivery vectors are an attractive option for gene therapy applications because they can deliver large transgenes to a broad range of tissues, allow high level transgene expression, have negligible risk of insertional mutagenesis and are easily produced at high titers. Unfortunately, their high immunogenicity and liver and spleen-associated toxicity when intravascularly administered and high prevalence of neutralizing antibodies in patients remain challenges to be overcome for the generation of safe HAdV-5-based vectors for systemic gene therapy. The discovery that coagulation factor X (FX), a zymogen of a vitamin K-dependent serine protease that circulates in the bloodstream, simultaneously binds HAdV-5 hexon and heparan sulphate proteoglycans (HSPGs) to mediate hepatic transduction enabled the generation of HAdV-5 vectors with substantially reduced liver transduction via the manipulation of key amino acid residues in the adenoviral hexon protein. However, FX was also recently shown to protect HAdV-5 from neutralization by preventing binding of natural IgM antibodies to HAdV-5 capsids and it was reported that, in the absence of FX-binding and neutralization, HAdV-5 vectors can use alternative FX-independent transduction pathways for hepatocyte transduction. These findings highlight the complex interactions between adenoviruses and host blood factors and cells as well as the need for further research on these processes. Here, the interactions that mediate hepatic and splenic tropism of HAdV-5-based vectors and activation of the anti-viral immune response following intravascular delivery were investigated and targeted HAdV-5 delivery to the kidney was assessed. Liver and spleen transduction was assessed in immunocompetent C57BL/6 and immunocompromised Rag 2-/- or NSG mice lacking different components of the immune response, following intravascular administration of wild type or FX-binding deficient HAdV-5 vectors. HAdV-5 virions were neutralized in C57BL/6 mice in the absence of FX-binding, confirming a role for FX in protecting HAdV-5 from in vivo neutralization. However, NSG mice, which lack both innate and adaptive immunity, failed to neutralize FX-binding deficient HAdV-5 virions. Interestingly, administration of FX-binding deficient HAdV-5 vectors to IgM antibody-deficient Rag 2-/- mice revealed that IgM antibodies might not be required for in vivo neutralization of HAdV-5, indicating that innate immunity alone might be sufficient. In agreement with previous reports, exposure of FX-binding deficient HAdV-5 vectors to C57BL/6 serum or wild type HAdV-5 vectors to C57BL/6 serum pre-incubated with the FX inhibitor X-bp led to HAdV-5 neutralization in vitro. In contrast, Rag 2-/- and NSG serum failed to neutralize HAdV-5 in vitro in the absence of FX-binding, indicating that IgM antibodies are essential for in vitro HAdV-5 neutralization. This suggests in vitro and in vivo adenovirus neutralization is mediated by different mechanisms. Importantly, administration of FX-binding deficient HAdV-5 vectors to NSG mice, which were unable to neutralize HAdV-5, confirmed the existence of alternative FX-independent pathways for liver and spleen transduction in the absence of neutralization. CAR and αvβ3,5 integrins were assessed as possible host cell receptors for HAdV-5 transduction of these tissues in immunocompetent and immunocompromised mice. The use of CAR or αvβ3,5 integrin-binding ablated HAdV-5 vectors revealed that CAR and αvβ3,5 integrins might serve as receptors for HAdV-5 liver transduction in immunocompetent C57BL/6 mice in contrast to immunocompromised Rag 2-/- mice. Neither CAR nor αvβ3,5 integrins mediated HAdV-5 spleen transduction in either mouse strain. Furthermore, administration of HAdV-5 vectors simultaneously ablated for FX and αvβ3,5 integrin-binding to NSG mice showed that αvβ3,5 integrins play no role in liver or spleen transduction in the absence of neutralization. With the aim to define novel FX-independent pathways of HAdV-5 transduction in vitro that might be relevant in vivo, cell transduction was assessed for wild type or FX-binding deficient HAdV-5 vectors in the presence of immunocompromised Rag 2-/- serum or serum that had been pre-incubated with X-bp. These studies confirmed the existence of FX-independent mechanisms able to enhance HAdV-5 cell transduction in vitro in the presence of mouse serum and absence of neutralization. To identify the receptor(s) involved in in vitro HAdV-5 transduction in the presence of mouse serum, soluble recombinant HAdV-5 fiber knob was used to block access of virions to CAR and wild type or FX-binding deficient HAdV-5 cell transduction was assessed in high and low CAR-expressing cell lines in the presence of C57BL/6 or Rag 2-/- serum with or without X-bp. HAdV-5 predominantly used a FX-independent pathway for cell transduction of high CAR-expressing cell lines in the presence of Rag 2-/- serum and soluble fiber knob substantially reduced both C57BL/6 and Rag 2-/- serum-enhanced transduction in such cell lines, suggesting a role for CAR, Conversely, HAdV-5 used the FX-mediated pathway or other FX and CAR-independent pathways for low CAR-expressing cell line transduction. Importantly, the use of CAR-binding deficient HAdV-5 vectors demonstrated that CAR usage in this setting does not rely on direct interactions of HAdV-5 with CAR, thus implicating a role for a mouse serum protein(s) in this process. To investigate these different pathways further, virions were fluorescently labelled with Alexa Fluor 488 dye and HAdV-5 cell binding, uptake and endosomal membrane penetration were characterized in the presence of FX by microscopic assessment of individual virions at the single cell level. FX substantially enhanced virion cell binding but had minimal effect on virion uptake and it was suggested to decrease efficiency of endosomal membrane penetration, limiting escape of virions from endosomes into the cytosol. Finally, FX and CAR-binding deficient HAdV-5 vectors were engineered to incorporate renal-targeting peptides found by in vitro phage display into the HI loop of the fiber knob domain for kidney-specific gene therapy. The resultant mutant HAdV-5 vectors were tested for their specificity in mediating gene delivery to ligand-expressing cells in vitro but failed to achieve specific targeting. Together, these findings contribute to a deeper understanding of the interactions between HAdV-5 vectors and host blood components and cell receptors, and their implications for liver and spleen transduction and neutralization of virions. The studies presented in this thesis highlight the limitations of current re-targeting strategies and the need for further research to successfully develop efficient HAdV-5-based vectors for systemic gene therapy.