# Identification of CD164 as an essential entry receptor for divergent adeno-associated viruses

## TL;DR

This study identifies CD164 as a key receptor for Clade G adeno-associated viruses, revealing a new mechanism for gene therapy vector entry into cells.

## Contribution

The discovery of CD164 as an essential entry receptor for Clade G AAVs introduces a novel AAVR-independent pathway for viral transduction.

## Key findings

- CD164 is essential for the entry and transduction of Clade G AAVs.
- AAV4 capsids bind to CD164 with high affinity and colocalize during cell entry.
- CD164 knockout mice show nearly complete loss of transgene expression from Clade G AAVs.

## Abstract

rAAVs are widely used vectors for human gene therapy, yet the mechanisms governing their entry into host cells remain incompletely understood. While multiple AAV serotypes depend on the broadly expressed AAV receptor (AAVR) for cellular entry, AAV4-related Clade G AAVs transduce cells via an AAVR-independent mechanism. In this study, we identify that CD164, a transmembrane sialomucin located on the cell surface and in endosomal compartments, is essential to the entry and transduction of Clade G AAVs. CD164 directly binds to AAV capsids in vitro and colocalizes with the capsids during cell entry and endosomal trafficking. These findings expand our understanding of AAV–host interaction and reveal a distinct AAVR-independent entry mechanism with implications for vector design and gene therapy.

Recombinant adeno-associated viruses (rAAVs) are widely used for in vivo gene delivery. While KIAA0319L, known as AAV receptor (AAVR), is essential for the transduction of multiserotype AAVs, it is dispensable for AAV4-related (Clade G) AAVs. We conducted a genome-wide CRISPR/Cas9 screen and identified CD164, a type I transmembrane sialomucin, as an essential entry receptor for Clade G AAVs. Ablation of CD164 expression substantially impaired both entry and transduction of Clade G AAVs. CD164-targeting antibodies and soluble CD164 ectodomain effectively blocked transduction. AAV4 capsids colocalized with CD164 at the plasma membrane and in endosomal compartments. In vitro, CD164 interacted with AAV4 or AAVrh32.33 capsids at high affinity. Importantly, systemic administration of rAAV4 or rAAVrh32.33 in CD164 knockout (KO) mice resulted in nearly complete loss of transgene expression. These findings establish CD164 as an essential entry receptor for Clade G AAV vectors and uncover a distinct AAVR-independent mechanism of AAV tropism.