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AAV Capsids for Non-Human Primates (NHPs)

AAV Cross-Species Issues

Animal models are fundamental for screening and optimizing AAV capsids targeting specific tissues. However, a critical insight from recent studies is that AAVs often display significant performance differences across species or even within different strains of the same species. For example, AAV-PHP.B, known for its ability to cross the blood-brain barrier (BBB), shows this property only in certain mouse strains and not in non-human primates (NHPs)【Hordeaux et al., 2018; Matsuzaki et al., 2019】. To improve the chances of translating AAV variants from animal models to human applications, it is essential to conduct variant screening across multiple animal models and human-based systems, focusing on those with translatable performance【Tabebordba et al., 2021; Gonzalez et al., 2022】.

Directed Evolution of AAV Variants for CNS Targeting

Directed evolution of a random peptide insertion library based on the AAV9 capsid's VR-VIII region led to the creation of the AAV-PHP family, including AAV-PHP.B, which significantly enhances BBB crossing in mice. Subsequent efforts to re-diversify this family produced AAV-PHP.eB, which maintains the peptide insertion of AAV-PHP.B but adds flanking substitutions. This modified variant demonstrated more efficient CNS transduction in mice, achieving a 55–76% transduction rate for neurons, a more than 2.5-fold increase over AAV-PHP.B.

Further engineering of AAV-PHP.eB by modifying the VR-IV region resulted in liver-detargeted variants like AAV.CAP-B10 and AAV.CAP-B22:

AAV.CAP-B10: This variant retains CNS targeting in mice but shows decreased transduction of peripheral organs and a significant reduction (50-fold) in liver tropism following IV delivery compared to AAV-PHP.eB. It exhibits robust CNS transgene expression in marmosets, achieving a 4-fold increase over AAV9, with 17-fold lower liver expression.

AAV.CAP-B22: Exhibits 12-fold higher CNS transduction in marmosets than AAV9, with increased astrocyte transduction. However, it does not translate effectively in newborn rhesus macaques, highlighting the challenge of cross-species variability.​

Note: All results are compared with AAV9.

​NHPs in AAV Research

Non-human primates (NHPs) are valuable models for AAV capsid evolution and evaluation due to their close similarities to humans in terms of tissue structure and cognitive function. However, one AAV variant selected in one species may not necessarily perform well in another. For instance, AAV.CAP-B22 showed efficacy in marmosets but not in rhesus or cynomolgus macaques (AAVnerGene’s Data). This highlights the importance of using multiple models to validate AAV performance before considering clinical applications.

Performance of AAV Variants Across Models

Non-human primates (NHPs) have been proposed as good models for AAV capsid evolution and evaluation due to close similarities to humans in terms of tissue structure and cognitive function.  However, one AAV selected from one species may not work in another species, such as AAV.Cap.B22, which is only work in marmoset, but not in rhesus macaques and cynomolgus macaques (AAVnerGene’s Data)

ig.1 B6C3 mice, 2 Weeks, IV injection of AAV Capsid Library(I), RNA expression ranking of common used AAV serotypes. 

Among the tested AAV variants, AAV9P31 showed the highest activity for crossing the BBB in B6C3 mice at both DNA and RNA levels, with significantly reduced liver expression. Other variants like AAV-F, AAV-MDV1A, AAV-PHP.C2, and PHP.eB also exhibited increased brain transduction and liver detargeting properties. However, the liver-detargeted variants AAV.CAP-B10 and AAV.CAP-B22 did not show improved brain transduction in the B6C3 mouse model. Furthermore, AAV variants selected from NHPs, such as MaCPNS1, MaCPNS2, AAV.Cap-Mac, AAV.cc47, and AAV9P81, also did not perform well in this mouse strain.

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