Neuronal Silencing of ATXN3 Using Peripherally Administered Antibody/ASO Conjugates That Penetrate the Blood-Brain Barrier

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SCA3, also known as Machado–Joseph disease, is the most common dominantly inherited ataxia in the world. The mutant SCA3 disease protein, ATXN3, acts through a dominant toxic mechanism, and mice lacking ATXN3 are phenotypically normal. Thus, suppression of the disease gene, ATXN3, represents a promising approach to slow or block the neurodegenerative cascade in SCA3. Anti-sense oligonucleotides (ASOs) represent a nonviral gene suppression approach that has emerged as a compelling therapeutic strategy for treating SCA3 and other neurodegenerative disorders. However, this approach suffers from three main limitations: i) highly invasive (intrathecal or intracerebroventricular) route of administration; ii) poor deep brain penetration, and iii) lack of cell- specific targeting. The goal of this proposal is to address each of these limitations using bispecific antibodies conjugated to ASOs (bAb-ASOs) to enable: i) intravenous administration; ii) efficient transport across the intact blood-brain barrier (BBB) and deep and widespread brain penetration; and iii) selective targeting of neurons using antibodies that target cell-surface proteins that mediate neuron-specific internalization and intracellular release of ASOs. Our approach has three main components. First, we use a validated IgG (M6) specific for a neuronal membrane protein (Neuronal Glycoprotein M6a) that is highly conserved within mammals and highly expressed in the brain. Second, we genetically fuse a single-chain antibody to the C-terminus of the IgG M6 that recognizes an understudied BBB target for mediating efficient transport into the brain parenchyma, namely CD98hc. Third, we attach ASOs to the M6/CD98hc bispecific antibody that will be released after antibody internalization and mediate gene silencing. The overall objective of the current proposal is to establish the feasibility of using bAb-ASOs to silence disease-specific neuronal genes in transgenic mice and suppress disease phenotypes. Our central hypothesis is that the bispecific antibody will enable delivery of ASO across the BBB and into neurons, resulting in cell-specific gene silencing and improvement in disease severity. To test this hypothesis, we will first evaluate PK/PD of bAb-ASO conjugates and silencing of ATXN3 (Aim 1). We will conjugate our validated M6/CD98hc bispecific antibodies to an optimized ATXN3 ASO and test in cell culture. For in vivo analysis, we will use the YAC SCA3 transgenic mouse line, which harbors the full human SCA3 disease gene and recapitulates molecular and behavioral features of SCA3. We will perform PK/PD analysis to evaluate the silencing of ATXN3 protein and the duration of this effect with repeated dosing. Next, we will evaluate the efficacy of silencing ATXN3 using bAb-ASOs in mouse models of SCA3 (Aim 2). We will directly compare the efficacy of peripherally delivered bAb-ASO conjugate to that of intracerebroventricular injection of the naked ASO in aging SCA3 mice to determine whether peripherally delivered bAb-ASO conjugate prevents the development of disease-associated phenotypes and, if so, whether the efficacy equals or surpasses that of naked ASOs.