Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.

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.

of 10e5 variants via DNA shuffling of eleven different parental AAV capsids. Our libraries selectively replicate in human cells when coadministered with wild-type adenovirus, making chimeric humanized liver mice an excellent tool to allow for selection of capsids with tropism for human liver. Screens were carried out for five rounds of selection and rather than waiting for the screen to go to completion, we ended the screen with some library diversity remaining for use in a subsequent screen for neutralizing antibody evasion. All variants from round five of the human liver screen were carried forward and screened for two additional rounds for their ability to resist binding to pooled human immunoglobulins in IgG immunocapture assays. The top 100 highly selected variants were sequenced and vectorized into CAG-driven GFP preparations in seven pools based on capsid relatedness at the amino acid level. Each pool was then resubjected to additional pooled human immunoglobulin screening and the best pool was chosen. The top seven candidates from this pool were tested alongside control serotypes that represent the extremes for humoral neutralization: highest neutralization (AAV-2), lowest neutralization (AAV-DJ), as well as LK03 that falls in between. One variant from this highly selected pool has neutralization profiles on par with AAV-DJ and further characterizations are underway in humanized liver mice in vivo and various human hepatoma lines in vitro to determine if both desired characteristics were robustly retained-human liver tropism and human immune evasion. 263.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 neuron...