Search

Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33–50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

Effective gene transfer to CNS is key for development of new therapies for neurological diseases. Relatively few natural AAV capsids cross the blood-brain barrier (BBB) upon systemic infusion and even then, transduction in adult brain after systemic delivery is limited to mainly glia and endothelium. Here we report the CNS tropism of a new AAV vector generated by genetic grafting of a small peptide into the capsid of AAV9.47, a liver-detargeted AAV9 mutant. The peptide insertion was tolerated well with no loss of packaging efficiency compared to AAV9.47 or AAV9. The scAAV-CBA-GFP vectors were infused via the tail vein in 6-8 week-old C57BL/6 mice at 5E11 vg. The new AAV vector transduced cortical and striatal neurons in the brain at high efficiency, as well as motor neurons and interneurons in the spinal cord. Significantly more vector genomes were found in the cerebrum (15-fold) and spinal cord (6-fold) compared to AAV9, while in peripheral tissues there were no significant differences. Western blot analysis of GFP expression levels confirmed the vector biodistribution results. Binding studies in parental and sialic acid-deficient CHO cells showed the interaction of the new AAV vector with terminal galactose to be unchanged compared to AAV9. The CNS tropism of the new AAV vector was evaluated in a 4 week-old cat infused with 1.26E13 vg via the carotid artery. Similar to the results in adult mice, the new AAV vector transduced cortical and striatal neurons in feline brain, and motor neurons in the feline spinal cord. These results suggest the remarkable CNS transduction properties of the new AAV may translate to higher organisms. Finally, we used the new AAV vector to knock down expression of human SOD1 in SOD1G93A mouse model of familial ALS. For these studies we used a self-complementary AAV vector encoding an artificial microRNA against human SOD1 in the 3’UTR of a CBA-GFP expression cassette. The scAAV-CBA-GFP-miR vector was infused via the tail vein in 55-day old SOD1G93A mice at 5E11 vg. At 4 weeks post-injection, there was 20% reduction in hSOD1 mRNA in the thoracic spinal cord of mice infused with the new AAV vector. The enhanced CNS targeting properties of the new AAV vector make it a promising candidate for gene therapy of neurodegenerative disorders.