Molecular Signature of Astrocytes for Gene Delivery by the Synthetic Adeno‐Associated Viral Vector rAAV9P1

Abstract

Astrocytes have crucial functions in the central nervous system (CNS) and are major players in many CNS diseases. Research on astrocyte-centered diseases requires efficient and well-characterized gene transfer vectors. Vectors derived from the Adeno-associated virus serotype 9 (AAV9) target astrocytes in the brains of rodents and nonhuman primates. A recombinant (r) synthetic peptide-displaying AAV9 variant, rAAV9P1, that efficiently and selectively transduces cultured human astrocytes, has been described previously. Here, it is shown that rAAV9P1 retains astrocyte-targeting properties upon intravenous injection in mice. Detailed analysis of putative receptors on human astrocytes shows that rAAV9P1 utilizes integrin subunits 𝜶v, 𝜷8, and either 𝜷3 or 𝜷5 as well as the AAV receptor AAVR. This receptor pattern is distinct from that of vectors derived from wildtype AAV2 or AAV9. Furthermore, a CRISPR/Cas9 genome-wide knockout screening revealed the involvement of several astrocyte-associated intracellular signaling pathways in the transduction of human astrocytes by rAAV9P1. This study delineates the unique receptor and intracellular pathway signatures utilized by rAAV9P1 for targeting human astrocytes. These results enhance the understanding of the transduction biology of synthetic rAAV vectors for astrocytes and can promote the development of advanced astrocyte-selective gene delivery vehicles for research and clinical applications.

Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders

“…This also pertains to the two bioengineered capsids that we have introduced in this work, AAVMYO2 and AAVMYO3, as their biology remains enigmatic at this early point, including their unknown cellular receptor(s) and their activity in other species beyond the mouse. Notably, we have recently obtained several lines of experimental evidence supporting our prior notion (6) that the P1 peptide and other RGD-containing sequences interact with various types of integrins on the surface of target cells and harness these for transduction (56,57). This is further supported by the latest work from the Sabeti laboratory, which also identified integrins as cellular receptors for their own AAV9-RGD capsid variants (23).…”

Astrocyte-derived SerpinA3N promotes neuroinflammation and epileptic seizures by activating the NF-κB signaling pathway in mice with temporal lobe epilepsy

“…The expression of SerpinA3N was driven by the GFAP promoter. The serotype AAV9 was selected for investigation in this study because in previous studies they have been characterized to exhibit the greatest propensity astrocytes in various CNS regions including the spinal cord by various constitutive promoters or glial-specific promoters [ 38 – 42 ]. Detailed procedures are described in the supplementary materials and methods.…”

An integrin-targeting AAV developed using a novel computational rational design methodology presents improved targeting of the skeletal muscle and reduced liver tropism

“…Virus-host interaction is the foundation for improved viral vectors, yet skeletal muscle receptors that allow effective AAV transduction are poorly de ned. However, top hits from two independent studies with different screening schemes identi ed myotropic AAVs with a common RGD motif, 15,17,19 . In addition, it has previously been described that integrin functions as cellular receptor for natural AAV 23,24 .…”

“…Importantly, the myotropic AAVs [15][16][17] identi ed by muscle-directed high-throughput screening (HTS) were shown to share an Arg-Gly-Asp (RGD) motif, presumably targeting the integrin complex [17][18][19][20] . Integrins are a group of heterodimeric proteins composed of an α-and a β subunit that serve various cellular functions, including cell adhesion, cell migration, and cell signaling 21 .…”