Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina

Abstract

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across species. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

The infectivity of AAV9 is influenced by the specific location and extent of chemically modified capsid residues

“…These capsids may contain AAV genomes, which, if present, would be inactive as they require access to the cellular nucleus to become transcriptionally active. In contrast, the attachment of Mannose or GalNac to AAV2 and AAV8 capsids enhances transgene expression, normalizing or even improving transgene expression in the liver and retina, despite a significant reduction in AAV genomes [23,34]. We need to investigate whether attaching these glycans to the AAV9 surface at any of the four studied residues can enhance transgene expression in the target organs, similar to what has been observed in AAV2 and AAV8.…”

“…Most of these studies have not provided a detailed characterization of the molecule density on the AAV capsid. Interestingly, distinct outcomes are achieved when GalNac or Mannose is covalently linked to the AAV2 surface through the formation of a thiourea bond with the accessible amino groups or tyrosine bioconjugation of the rAAV capsid [23,34,35]. While there is no discernible effect on AAV2 transduction efficiency when ten times more Mannose is attached, an equivalent increase in GalNac leads to a notable reduction in AAV2's infectivity in the target organ.…”

“…Ultimately, chemical modifications of the AAV capsid have unveiled novel pathways for enhancing the properties of recombinant AAVs. Introducing GalNac or Mannose to exposed tyrosines has notably enhanced transgene expression in the liver and retina [23]. Moreover, the impact can be serotype-dependent; for instance, binding N-ethyl Maleimide to the AAV9 capsid has augmented its affinity for murine bone marrow while diminishing transduction in liver tissue, unlike AAV2 and AAV8 which did not exhibit this effect [24].…”

The infectivity of AAV9 is influenced by the specific location and extent of chemically modified capsid residues

“…Ultimately, chemical modi cations of the AAV capsid have unveiled novel pathways for enhancing the properties of recombinant AAVs. Introducing GalNac or Mannose to exposed tyrosines has notably enhanced transgene expression in the liver and retina (19). Moreover, the impact can be serotypedependent; for instance, binding N-ethyl Maleimide to the AAV9 capsid has augmented its a nity for murine bone marrow while diminishing transduction in liver tissue, unlike AAV2 and AAV8 which did not exhibit this effect (20).…”

“…Most of these studies have not provided a detailed characterization of the molecule density on the AAV capsid. Interestingly, distinct outcomes are achieved when GalNac or Mannose is covalently linked to the AAV2 surface through the formation of a thiourea bond with the accessible amino groups or tyrosine bioconjugation of the rAAV capsid (19,30,31). While there is no discernible effect on AAV2 transduction e ciency when ten times more Mannose is attached, an equivalent increase in GalNac leads to a notable reduction in AAV2's infectivity in the target organ.…”