B5 - Neurologic Diseases (excluding Ophthalmic and Auditory Diseases)
273: AAV-Based Anti-RAN Antibody Therapy for C9orf72 ALS FTD
Type: Oral Abstract Session
Presentation Details
Session Title: Neurologic Diseases III
Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that causes paralysis and death ~3-5 years after diagnosis. The most frequent, known, genetic cause of familial and sALS is the C9orf72 (C9) G4C2 repeat expansion. Strong evidence indicates toxic proteins expressed by repeat associated non-AUG (RAN) translation contribute to disease. Our previous studies show that targeting GA RAN proteins with a human antibody, α-GA1, improved disease in a BAC transgenic mouse model of C9orf72 ALS/FTD (C9-BAC). Surprisingly, the α-GA1 antibody not only reduced GA aggregates, but also lowered GP and GR RAN proteins through improved proteasome and autophagy function. While passive immunotherapy is promising, high manufacturing costs, the need for frequent dosing, and limited blood-brain-barrier (BBB) penetration (~0.1%) highlight the need to develop improved immunotherapy strategies. Methods: Here, we report a second-generation immunotherapy strategy that targets GA-RAN proteins using an AAV-based approach. We engineered IgG and single-chain antibody variants with high affinity to GA-RAN proteins for AAV delivery. Cell overexpression, mouse, and organoid models were used to test target engagement and therapeutic efficacy of these AAV-IgG-GA variants. C9-BAC and non-transgenic (NT) littermates were injected intracerebroventricularly with AAV-IgG1-GA or AAV-IgG-GFP (isotype-control). Hanging-wire and Open-Field behavioral analyses were performed on 16 and 23-week-old animals. Immunohistochemistry (IHC) and IF were used to evaluate the effects of AAV-IgG1-GA in the brain and spinal cord. Results: Our data show these novel a-GA antibody variants (IgG1, IgG2, or single-chain scFv) reduce GA-RAN proteins (n>3, p<0.0001) and their toxicity (n>3, p<0.0001) in cells. A pilot study identified AAV-IgG1-GA as the most effective AAV-antibody for reducing GA-RAN proteins in C9-BAC mice (n>5/group, p=0.0499). In a larger study (n=30 mice/group), we found that animals injected with AAV-IgG1-GA improved performance in open-field (e.g., ambulatory distance, n>5/group, p<0.0001), hanging-wire (n>5/group, p=0.0301) and importantly increase survival (n>5/group, p=0.0044). Histopathological studies show that the AAV-delivered IgG1-GA antibody is broadly distributed throughout the hippocampus and motor cortex and that GA aggregates in the brains of IgG1-GA-treated mice are reduced. Conclusion and Discussion: These data show that AAV-anti-RAN immunotherapy reduces RAN protein load, improves behavior, and increases survival of C9-BAC mice. We are continuing to analyze the histopathological and molecular phenotypes of the mice to fully understand the molecular effects of AAV-anti-RAN immunotherapy. AAV delivery of high-affinity anti-GA antibodies provides a novel strategy to achieve broad and sustained CNS expression and biodistribution of therapeutic antibodies. These data open new possibilities for developing AAV-antibody therapies as a novel approach for C9orf72 ALS/FTD and for a broader group of disorders characterized by RAN protein pathology.
Lisa E. L. Romano1, Camille Preston1, Tala Ortiz1, Ramadan Ajeridini1, Hannah Golliher1, Ellie Carrell2, Megan Keiser2, Elaine Ames1, Beverly L. Davidson2,3, Laura P. W. Ranum1,4
1Department of Molecular Genetics and Microbiology, University of Florida, Center for NeuroGenetics and the Genetics Institute, Gainesville, FL,2The Children's Hospital of Philadelphia, Center for Cellular and Molecular Therapeutics, Colket Translational Research Building, Philadelphia, PA,3Perelman School of Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA,4McKnight Brain Institute and the Fixel Institute for Neurological Diseases, College of Medicine, University of Florida, Gainesville, FL"
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