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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.

Neurological disorders - disorders of the brain, spine and associated nerves - are a leading contributor to global disease burden with a shockingly large associated economic cost. Various treatment approaches - pharmaceutical medication, device-based therapy, physiotherapy, surgical intervention, among others - have been explored to alleviate the resulting extent of human suffering. In recent years, gene therapy using viral vectors - encoding a therapeutic gene or inhibitory RNA into a "gutted" viral capsid and supplying it to the nervous system - has emerged as a clinically viable option for therapy of brain disorders. In this Review, we provide an overview of the current state and advances in the field of viral vector-mediated gene therapy for neurological disorders. Vector tools and delivery methods have evolved considerably over recent years, with the goal of providing greater and safer genetic access to the central nervous system. Better etiological understanding of brain disorders has concurrently led to identification of improved therapeutic targets. We focus on the vector technology, as well as preclinical and clinical progress made thus far for brain cancer and various neurodegenerative and neurometabolic disorders, and point out the challenges and limitations that accompany this new medical modality. Finally, we explore the directions that neurological gene therapy is likely to evolve towards in the future. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders".

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.

Alpha-one antitrypsin deficiency (AATD) is a genetic disorder that causes defective production of alpha-one antitrypsin (AAT), leading to decreased AAT activity in the blood and lungs, and deposition of excessive mutant Z-AAT protein in liver cells. There are several forms and degrees of deficiency, principally depending on whether the patient has one or two copies of the affected gene. Approximately 4% of the North American and Northern European populations possess at least one copy of a mutant allele.The current standard of care is protein replacement by frequent infusions, but gene replacement therapy could provide long-term correction of the serum levels after a one-time treatment only, which would offer a significant improvement in the quality of life of the patients. Towards this goal, a recent clinical trial tested injecting recombinant adeno-associated virus serotype 1 expressing M-AAT intramuscularly. However the therapeutic AAT serum threshold of 11μμM was not reached. Future efforts to achieve therapeutic levels will revolve around dose escalation and more efficient physical delivery.While the next trial may achieve therapeutic levels with the current AAV1-CB-AAT vector, it is worth further optimizing as it may result in a more efficient vector requiring a lower dose to achieve therapeutic levels. Achieving therapy with a lower dose has substantial benefits in the long run in terms of cost of production and more importantly potential immune responses to capsid. The current AAV1-CB-AAT gene therapy vector will be optimized through four independent strategies hypothetically leading to an increase of alpha-one antitrypsin serum levels in mice: 1) replacement of the secretion signal by secretion signals from other human secreted proteins, 2) modification of the AAV capsid either by triple tyrosine mutation or DNA shuffling, 3) optimization of the transgene to create self-complementary vectors with a codon-optimized AAT sequence, 4) addition of a hepatocyte-specific cis-regulatory module allowing recruitment of liver-specific transcription factors.In vivo data will be presented for each strategy. Future work will combine the validated candidates into a single vector for further evaluation; the use of this optimized vector may translate into a therapeutic effect for the AATD patients.

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.