A4 - AAV Vectors - Preclinical and Proof-of-Concept In-Vivo Studies (Excluding Non-Human Primates)
252: Transgene Protein Evolution as a Novel Strategy for Next-Gen Gene Therapy in Canavan Disease
Type: Oral Abstract Session
Presentation Details
Session Title: AAV Vectors - Preclinical and Proof-of-Concept: Technology Focus
Canavan Disease (CD) is characterised as a progressive neurodegenerative disease caused by the accumulation of N-acetylaspartate (NAA) as a result of the deficiency of aspartoacylase (ASPA) enzyme associated with mutations in the ASPA gene. ASPA is responsible for the supply of acetate for the oligodendrocytes and is essential for normal myelination. Recombinant adeno-associated (rAAV) gene therapy (rAAV9-ASPA) has been shown to rescue the CD knock-out mouse model from premature death. This has been supported by the normalisation of NAA levels and growth, improved motor function, and ASPA gene expression detected in the CNS of the treated CD mice.
The advancements in the design of rAAV as a gene therapy delivery vehicle have provided significant potential for numerous rare diseases with unmet therapeutic options. One of the remaining challenges for rAAV gene therapies for CNS disorders is the high dose required to achieve sufficient transgene protein levels. The high cost associated with rAAV production and the risk of systemic toxicity is further exacerbated by the vector dose required. To offer solutions to the dosage-associated toxicity and costs, we have hypothesised that the amino acid sequence translated from the rAAV-delivered transgene can be evolved and optimised for enhanced transgene protein activity.
To explore if specific mutation(s) in the wild-type ASPA (WT-ASPA) amino acid sequence could achieve increased ASPA enzyme activity, we constructed a random mutant ASPA library (~103) using an optimised directed evolution approach to maximise mutation distribution and diversity. The use of a prokaryotic expression system allowed for ease of microscale protein expression and high throughput functional screening by a spectroscopic method benchmarked against the recombinant human WT-ASPA enzyme. This led to the identification of several evolved ASPA (ev.ASPA) enzyme candidates with increased enzymatic activity. Subsequently the ev.ASPA candidates underwent enzyme purification and secondary screening using a high-sensitivity chromatography method to confirm enhanced performance. To determine the in vivo performance of ev.ASPA, the leading candidate nucleotide sequences were cloned into rAAV9 (rAAV9-ev.ASPA) and delivered to CD mice on postnatal day 1 (P1) intravenously at a 3-fold lower dose than our clinical full dose vector expression WT-ASPA. Initial findings show disease rescue in the CD mouse model, supported by improvements in growth and motor function. In addition, none of the treated animals have succumbed to the disease. This contrasts with untreated animal’s overall survival of around 24 days of age. Studies to directly compare ev.ASPA to our clinical vector is underway to determine the side-by-side in vivo performance of our transgene evolution approach.
This approach could be a critical contribution to rAAV genome design for therapeutic applications. In terms of rAAV delivery to the CNS, this would allow for potential viral vector dose reduction, all while maintaining the therapeutic efficacy - providing a novel solution to CD and other gene therapy applications and benefiting the wider gene therapy research community.
Plain Language Summary
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Sarah Foley1, Morgan Mooney2, NhatAnh B. Nguyen2, Anoushka Lotun2, Guangping Gao2,3,4, Dominic J. Gessler2,4, Lee Coffey1,5
1Pharmaceutical and Molecular Biotechnology Research Centre, South East Technological University, Waterford, Ireland,2Horae Gene Therapy Centre, UMass Chan Medical School, Worcester, MA,3Li Weibo Institute for Rare Diseases Research, UMass Chan Medical School, Worcester, MA,4Co-corresponding author, Worcester, MA,5Co-corresponding author, Waterford, Ireland"
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