Theme 7 Pre-Clinical Therapeutic Strategies

Background: ALS is a devastating neurodegenerative disorder characterized by the progressive degeneration of motor neurons in the brain and spinal cord, leading to a gradual loss of voluntary muscle control, paralysis, and fatal respiratory failure.Although familial forms of ALS exist, the multifactorial nature of sporadic ALS remains elusive.Recent animal studies have shown that Atxn2 deficiency disrupts the Ataxin-2-TDP43 interaction and ameliorates TDP43 proteinopathy, a causative factor in neurodegeneration in $97% of ALS and $50% of tau-negative frontotemporal dementia cases.Objective: To develop a miRNA gene silencing adeno-associated (AAV) gene therapy approach to modulate ATNX2 expression as a potential therapeutic approach for TDP43 proteinopathies.Methods: Using in vitro and in vivo model systems we screened ATXN2-targeting microRNA (miRNA) sequences.The most efficacious miRNA guide sequence was inserted into AviadoBio's proprietary vMiX TM platform to test efficacy in a dose-ranging study using human BAC-ATXN2-Q72 transgenic mice.Results: Fifty-one guides with optimal GC content and predicted binding were transfected in vitro, and 14 sequences were found to knockdown ATXN2 mRNA (up to 79%).The 14 most promising guides were cloned into our vMiX TM platform, AAV9 vectors produced, transduced in HEK 293T cells, and ATXN2 mRNA expression quantified.The vector with the greatest in vitro ATXN2 mRNA knockdown efficacy (AVB-205) was selected for in vivo evaluation in BAC-ATXN2-Q72 transgenic mice.Four doses of AVB-205, vMiX.CT (Control vector), or vehicle, were administered by intracerebroventricular (ICV) injection into neonatal heterozygous BAC-ATXN2-Q72 mice (n 7-10/group) and their wild-type littermates (n 5/ group).After eight weeks, all mice were euthanized.Molecular analyses of the cortex and spinal cord were conducted to establish ATXN2/Atxn2 mRNA knockdown, vector genomes per cell (VG/cell), and miRNA guide expression.AVB-205 knocked down human ATXN2 mRNA by 70% in the cortex and 29% in the spinal cord (p < 0.0001).Mouse Atxn2 mRNA was knocked down by 59% in the cortex and 24% in the spinal cord in wild-type littermates (p < 0.0001).In both genotypes and tissues, there was a significant dose-response relationship to knockdown (R 2 0.78, p < 0.001).In addition, a significant relationship between ATXN2/Atxn2 knockdown, VG/cell, and guide miRNA expression was found, such that an average of 1 VG/cell gave rise to up to 2.8x10 miRNA guides, which repressed ATXN2/Atxn2 expression by 50%.Discussion: Taken together, these data provide strong support for further testing the targeting of ATXN2 by AVB-205 in hTDP-43 transgenic mouse models and its translational potential as a therapy for TDP43 proteinopathies, including ALS and some forms of FTD.