A3 - AAV Vectors - Capsid Engineering
489: Identification of AAV2-13 Hypervariable Regions Mediating Liver Transduction and Detargeting in Rodents and Non-Human Primates
Type: Poster Session
Poster Board Number: 489
Presentation Details
Session Title: Wednesday Posters: AAV Vectors - Capsid Engineering
Adeno-associated viral (AAV) vectors are widely used in gene replacement strategies in preclinical and clinical contexts. Along the years gene therapy with AAV vectors showed efficacy for the targeting of different organs and sustained expression over time (Mendell JR, 2017; Nathwani AC, 2014; D’Antiga L, 2023). The excellent safety profile and the proven efficacy resulted in the multiplication of clinical trials to address unmet medical needs that eventually led to achieve marketing authorization for some of the AAV-based products. Despite these enormous successes, limitations in their use still exist, and a better understanding of the mechanisms mediating liver targeting and detargeting is required.
In this work, we conducted a shuffling study of the hypervariable regions (HVRs) of AAV8 with homologous regions from AAV2-13 capsids isolated from human liver (La Bella T, 2020) to pinpoint regions involved in liver tropism. HVRs were shuffled in AAV8, a capsid with a robust liver tropism, by substituting them individually or as combinations. Shuffled capsids were combined with a ubiquitous or a hepato-specific promoter to evaluate the in vivo and in vitro transduction efficiency in mouse.
Our results highlighted the HVR5 as a region of the AAV capsid able to improve the specificity of liver targeting. Noteworthy, HVR5 shuffled capsids combined with ubiquitous promoter led to a modest improvement in liver transduction. On the contrary, when combined with the hepatic promoter, shuffled capsids outperformed AAV8 transduction efficiency, suggesting that modifications of the HVR5 may increase the selectivity of AAV vectors for hepatocytes. We then tried to combine other HVRs with the HVR5 to evaluate whether a synergic effect was observed. The combination of HVRs 1, 7, 8 and 10 with HVR5 led to a dramatic decrease of liver targeting suggesting that the simultaneous replacement of multiple HVRs of AAV8 with the corresponding regions of wild type AAV impaired the liver tropism regardless of the promoter used. On the contrary, individual HVRs 1, 5, 7, 8 and 10 substitutions did not independently impact liver transduction.
Validation of the association of certain HVR regions with liver targeting and detargeting was performed in non-human primates. Importantly, we found that shuffled AAV capsids containing regions that mediated liver detargeting in mice had a similar profile in primates pointing to an inter-species conservation of the detargeting characteristics associated to these regions.
To conclude, in this study, rational shuffling of regions derived from wild-type AAV2-13 capsids isolated in human livers into homologous regions of the AAV8 capsid, allowed to identify determinants of liver transduction and detargeting across species. This knowledge is fundamental to develop AAV capsids for diseases where concomitant muscle and liver gene transfer maybe required to reduce the impact of the immune response by achieving sufficient transgene expression to induce liver-mediated peripheral tolerance without liver overloading (Sellier P, 2023). When validated in appropriate preclinical models and with clinically relevant transgenes, the use of capsids derived from shuffling of HVRs may thus provide safer AAV vectors tailored to the specificities of the disease.
Plain Language Summary
Adeno-associated virus (AAV) are the vectors of choice for gene replacement in vivo. The excellent safety profile and the proven efficacy resulted in the multiplication of clinical trials to address unmet medical needs that eventually led to achieve marketing authorization for several AAV-based products. Despite these enormous successes, limitations in their use still exist, and a better understanding of the mechanisms mediating liver targeting and detargeting is required. The surface of the viral capsid plays a crucial role in driving the tissue targeting of AAV vectors by interacting with the host cell receptors and impacting the vector tropism. In this study we systematically exchanged the exposed regions of the AAV8 capsid, a capsid with a robust liver tropism, with homologous regions derived from a capsid with extensive liver detargeting to pinpoint the regions responsible for liver tropism. Liver targeting and detargeting properties were then validated in mouse and primates.
Justine Nozi1,2, Novella Tedesco1,2, Christian Leborgne1,2, Bérangère Bertin1,2, Louis Bergantz1,2, Ante Mihaljevic1,2, Coralie Pecquet1,2, Theoni-Maria Michalopolou1,2, Nathalie Daniele1, Laetitia Van Wittenberghe1, David A. Gross1,2, Tiziana La Bella1,2, Giuseppe Ronzitti1,2
1GENETHON, Evry, France,2Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, Evry, France"
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