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A - Viral Vector Development -> AAV Vectors – Preclinical and Proof-of-Concept In vivo Studies (excluding Non-Human Primates)

3: Novel AAV-Capsid-Mediated Delivery of an RNAi Targeting Atxn2 Extends Survival and Improves Strength and Neuroinflammation in a Mouse Model of Sporadic ALS

Type: General Session

Presentation Details
Session Title: Presidential Symposium and Presentation of Top Abstracts
Location: West Hall B
Start Time: 5/17/2023 15:00
End Time: 5/17/2023 15:15

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by death of motor neurons. A key pathologic feature is the cytoplasmic mislocalization of a nuclear transcription and splice regulator, Tar-DNA binding protein of 43kDa (TDP-43). TDP-43 aggregates in cytoplasmic stress granules (SGs) and leads to toxicity through both cytoplasmic gain- and nuclear loss-of-function. Inhibiting formation of SGs is therefore a promising strategy, and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASOs) prolongs survival by 35% in a mouse model of sporadic ALS (Becker et al. Nature 2017), a strategy that is now in human clinical trials. However, this strategy requires frequent CNS readministration of the Atxn2-targeting ASO and may not effectively reach the brain after intrathecal injections, limiting efficacy and safety. An alternative approach is to provide lasting knockdown throughout the brain and spinal cord after one treatment using AAV-mediated RNAi delivery. If successful, this strategy could be used to treat the vast majority of ALS.
We designed miRNAs targeting Atxn2 and tested their efficacy in N2A cells, packaging the most effective candidate into a novel AAV9 capsid variant, AAV1999, engineered in our lab for superior CNS targeting in both mice (Figure, A) and nonhuman primates. A dosing study demonstrated 55% knockdown of Atxn2 in the frontal cortex and 25% knockdown throughout the brainstem and cervical and lumbar spinal cord after a one-time intracerebroventricular injection, with GFP-tagging demonstrating selective localization within the spinal cord to anterior horn cells. We then conducted an efficacy study in the same ALS mouse model used in the prior ASO study, in which wildtype human TDP-43 is overexpressed in neurons starting at P7 and mice exhibit a rapid decline in strength, succumbing around P22. After treatment, mean survival was increased by 54% and median survival by 45% (p<0.002; Figure, B). Mice showed marked improvement across several strength-related measures, including rotarod (2X duration, p<0.02); gait (30% improvement, p<0.001); kyphosis (66% improvement, p<0.001); tremor (34% improvement, p<0.005); and foot angling (48% improvement, p<0.01), with a trend towards improvement in abdominal droop and limping. Interestingly, mice showed an increase in vertical activity above that seen in wildtype mice, perhaps suggesting an unmasking of an FTD phenotype in the setting of improved strength. Histologically, treated mice showed normalization of the astrogliosis seen in mutant mice to wildtype levels (p<0.05). AAV-mediated RNAi targeting Atxn2 is therefore a promising strategy for treatment of the 97% of ALS characterized by TDP-43 pathology.

Defne A. Amado1,2, Alejandro Mas Monteys2, Katherine Whiteman2, Alicia R. Smith2, Guillem Chillon Bosch2, Aleksandar Izda2, Beverly L. Davidson2

1Neurology, University of Pennsylvania, Philadelphia, PA,2Children's Hospital of Philadelphia, Philadelphia, PA
 D.A. Amado: None.

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