The goal of this proposal is to optimize engineered intracellular antibodies (intrabodies) as novel clinical reagents and drug discovery tools for the treatment of Huntington's Disease (HD), with broad, long-term relevance to other neurodegenerative disorders caused by misfolded proteins. Intrabodies use the target specificity of antibodies to form complexes with intracellular proteins, and are already in clinical trials for treatment of cancers and AIDS. The research design starts with in vivo testing with a single-chain Fv anti-huntingtin (htt) intrabody (scFv C4) that has shown significant rescue of HD phenotypes in cell lines, organotypic slice cultures and a Drosophila HD model;plus a newer single domain intrabody (VL 12.3)that shows even stronger anti-htt aggregation properties in situ. Delivery of the intrabody genes will utilize a non-primate lentivirus, Equine Infectious Anemia Virus (EIAV), with either a VSVG or Rabies-g envelope, as one gene therapy vector, with some experiments to compare with delivery using AAV vectors provided by a collaborator. Quantitative assays of abnormal nuclear htt accumulation and aggregation, DARPP- 32 levels, and open field activity behavior will be used to assess the efficacy of the intrabodies delivered to the brains of Exon 1 transgenic (R6/1) and Hdh knock-in (Q111) mouse models on the same inbred genetic background. Simultaneously, screening and testing of a small pool of newer intrabodies will be done using anti-aggregation, protection, and toxicity assays in neuronal cell lines. The most successful of the new intrabodies will then be tested as above. If correction is incomplete with individual intrabodies, combination therapies will be tested in cells and in vivo. At the end of these studies, we will have established the optimal characteristics of intrabodies for eventual HD therapeutics and further drug discovery. These approaches should also be generally applicable for other neurodegenerative diseases that result from abnormal protein folding and accumulation, including Alzheimer's, Parkinson's, and prion diseases.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CDIN-D (01))
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Sutherland, Margaret L
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Wadsworth Center
United States
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Ramsingh, Arlene I; Manley, Kevin; Rong, Yinghui et al. (2015) Transcriptional dysregulation of inflammatory/immune pathways after active vaccination against Huntington's disease. Hum Mol Genet 24:6186-97
De Genst, Erwin; Messer, Anne; Dobson, Christopher M (2014) Antibodies and protein misfolding: From structural research tools to therapeutic strategies. Biochim Biophys Acta 1844:1907-1919
Butler, D C; Snyder-Keller, A; De Genst, E et al. (2014) Differential nuclear localization of complexes may underlie in vivo intrabody efficacy in Huntington's disease. Protein Eng Des Sel 27:359-63
Tome, Stephanie; Manley, Kevin; Simard, Jodie P et al. (2013) MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice. PLoS Genet 9:e1003280
Messer, Anne; Joshi, Shubhada N (2013) Intrabodies as neuroprotective therapeutics. Neurotherapeutics 10:447-58
Bhatt, Mansi A; Messer, Anne; Kordower, Jeffrey H (2013) Can intrabodies serve as neuroprotective therapies for Parkinson's disease? Beginning thoughts. J Parkinsons Dis 3:581-91
Joshi, Shubhada N; Butler, David C; Messer, Anne (2012) Fusion to a highly charged proteasomal retargeting sequence increases soluble cytoplasmic expression and efficacy of diverse anti-synuclein intrabodies. MAbs 4:686-93
Butler, David C; McLear, Julie A; Messer, Anne (2012) Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins. Prog Neurobiol 97:190-204
Hathorn, Tyisha; Snyder-Keller, Abigail; Messer, Anne (2011) Nicotinamide improves motor deficits and upregulates PGC-1α and BDNF gene expression in a mouse model of Huntington's disease. Neurobiol Dis 41:43-50
Butler, David C; Messer, Anne (2011) Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments. PLoS One 6:e29199

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