Evaluation of <i>in vitro</i> functional activity of ANB4 drug for the treatment of spinal muscular atrophy

Abstract

BACKGROUND: Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disease. SMA is caused by a deficiency of the functional survival motor neuron protein (SMN) as a result of a mutation in the SMN1 gene. BIOCAD is developing a domestic gene therapy drug for the treatment of SMA based on recombinant adeno-associated virus serotype 9 (rAAV9) carrying the SMN1 gene (ANB4). In vitro evaluation of the functional activity of ANB4 will allow a more complete characterization of the drug. AIM: Development of an accurate and reproducible in vitro functional test that reflects the clinical mechanism of action of ANB4. METHODS: To model SMA, the SMN1 gene was knocked down by transfection with small interfering RNA. Amount of the SMN protein was measured by enzyme-linked immunosorbent assay. The functional activity of the drug was evaluated by analysis of Gemin2 protein level using the western blot analysis. RESULTS: Analytical method has been developed to assess the functional activity of the ANB4 drug for the treatment of SMA type 1 (rAAV9 carrying the SMN1 gene). The developed technique made it possible to obtain accurate and reproducible results. Production of Gemin2 after knockdown of SMN1 and the introduction of exogenous SMN1 gene was restored to control values, comparable with the restoration of the level of the SMN protein. CONCLUSION: The developed technique closely reflects the clinical mechanism of action of the ANB4 drug for the treatment of SMA. By evaluating the functional activity in vitro, accurate and reproducible results were obtained in accordance with the required standards, including the principles of 3R.