Profiling of target molecules of human astrocytes for selective transduction by the Adeno-associated virus variant AAV9P1

Bauer, Amelie (2021): Profiling of target molecules of human astrocytes for selective transduction by the Adeno-associated virus variant AAV9P1. Dissertation, LMU München: Fakultät für Biologie

Abstract

Astrocytes are among the most abundant cell types in the human central nervous system (CNS). They have critical functions in the brain, including the maintenance of neuronal homeostasis and active contribution to the formation, regulation, and maintenance of synapses and synaptic transmission. Further, astrocytes react to CNS damage with proliferation and have been shown to adapt neuronal functions in mice after the artificial expression of neurogenic factors. Astrocytic dysregulation is associated with a variety of neuropathologies, including psychological and neurodegenerative diseases. Treatment strategies for these diseases that rely on modifying the astrocytic gene or protein expression are subject of current research. The main obstacle in this pursuit is the lack of efficient and specific vectors for targeted astrocyte transduction. Adeno-associated virus (AAV) vectors are considered the gold standard for gene therapy due to their favorable biological characteristic. The variant rAAV9P1 has been described to efficiently transduce astrocytes in vivo and discriminate between astrocytes and neurons. However, the molecular base of this transduction behavior is still elusive. In this work, we have investigated the underlying molecular profile that enables efficient and selective transduction of astrocytes by rAAV9P1. We could show that rAAV9P1 transduces astrocytic cell lines more efficiently than vectors derived from its parental serotype AAV9 and with higher selectivity than vectors carrying a capsid from the well-investigated serotype AAV2. It was found that rAAV9P1 follows a transduction mechanism that is distinctly different from the HSPG-dependent, ubiquitous transduction of rAAV2. On the molecular level, rAAV9P1 engages with αv-containing integrins, likely via the RGD-sequence in the inserted P1 peptide. These integrins include αvβ8 as a central receptor and αvβ3/αvβ5 as potential redundant auxiliary receptors. Besides, rAAV9P1 transduction is dependent on classical AAV9 receptors such as terminal galactose on N-linked cell surface glycans, the 37/67 kDa laminin receptor (LamR), and the essential AAV receptor KIA00319L (AAVR). Furthermore, a genome-wide CRISPR/Cas9 screening in a human glioblastoma cell line revealed that intra-cellular pathways with astrocyte-relevance might be involved in efficient and selective transduction of astrocytes by rAAV9P1. Taken together, this work presents the detailed receptor profile of rAAV9P1, which achieves high efficiency and cell-type selectivity by combining the binding to new receptors through capsid modifications with pre-existing receptors of the parental serotype. This multi-factorial binding might pave the road for the future development of more cell-type-selective rAAV vectors, but also refining of rAAV9P1 for future in vivo and gene therapy approaches.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Astrocytes, Adeno-associated virus, Integrins, Receptors, CRISPR/Cas9 screen
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 25. November 2021
• 1. Bericht­erstatter:in: Brack-Werner, Ruth