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Slade, Peter

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Engineering Conferences International

 Poster Number 198 SIMPLIFYING MANUFACTURING AND CONTROL OF DUAL VECTOR AAV THERAPIES  Peter Slade, Decibel Therapeutics, USA pslade@decibeltx.com   Permanent hearing loss of all severities is reported in 1.7 per 1000. Congenital hearing loss is associated with impairments in cognitive development, language development, academic achievement, and reading.  Severe hearing losses are associated with poorer outcomes. More than half of all congenital hearing loss are caused by single gene mutations while biallelic mutations can cause severe to profound deafness. Decibel Therapeutics is focused on developing single and dual vector AAV gene therapy products to address the unmet therapeutic need for monogenic hearing loss.   Dual vector AAV therapies pose manufacturing challenges where formulation of the drug product (DP) necessitates controlling drug substance (DS) concentrations to target a specific ratio of the two vectors and final total vector concentration. Control of vector ratios relies on accurate titer measurements. Since each vector uses an identical serotype and the transgene mass only differs by a few kDa, ddPCR or qPCR are currently the only methods available which can distinguish the vectors in the final DP. Quantitative PCR methods are known to have significant assay variability and a biophysical method would be preferable, therefore analytical ultracentrifugation (AUC) was evaluated to directly measure the full capsid ratios of the two vectors in the final DP. Using AUC, the sedimentation values of the full capsids were modeled based on the known differences in the two transgene masses. The assay was successful at calculating a vector ratio that correlated well to the ddPCR measurements. This method can be used as an orthogonal means for targeting vector ratios and improving DP control.  To better understand the range in which vector ratios could vary without changing protein expression an ex-vivo assay was used by culturing utricles from the mouse inner ear. A dual vector DP was prepared over a wide range of ratios, utricles were infected, and mRNA expression of the therapeutic protein quantified. This assay identified a range of ratios and acceptance criteria that could be used during DP manufacturing where protein expression remained unchanged. The assay also demonstrated that the response of transgene expression was not equally dependent on each vector concentration, and one vector was more dominant or essential. In this case a dual vector DP functions more like a single vector system, with potential implications of simplifying manufacturing, reducing the overall total vector formulated and even the total amount of AAV administered. Studies are ongoing using a newly developed cell-based potency assay to further investigate vector ratios and protein expression as well as understand ratios and protein expression with respect to the cell specific promotor and recombination sites of the transgene.      

Simplifying manufacturing and control of dual vector AAV therapies

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1242&context=ccexviii&type=additional

Simplifying manufacturing and control of dual vector AAV therapies

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Engineering Conferences International