Huntington's disease (HD) is a fatal neurodegenerative disorder caused by expansion of the polyQ domain in the huntingtin (Htt) protein. Mutant Htt protein forms intracellular aggregates and is cytotoxic to specific neurons in the striatum and cortex. We generated monoclonal antibodies (mAbs MW1-8) that bind the epitopes polyQ, polyP or the C-terminus of exon 1 in Htt. To use these mAbs as intracellular perturbation agents, we cloned their antigen-binding domains as recombinant, single chain Abs (intrabodies). In 293 cells co-transfected with expanded polyQ Htt, MW7 intrabody, recognizing the polyP domains of Htt, significantly inhibits aggregation as well as the cell death induced by mutant Htt. In contrast, MW1 and 2 intrabodies, recognizing the polyQ domain, stimulate Htt cell death. These results suggest MW7 as a candidate for gene therapy in HD. We propose to explore the therapeutic potential of MW7 and several new anti-Htt intrabodies in several models, and to produce new, more efficacious intrabodies. (i) Preliminary results indicate that MW7 is effective in an in vivo-like system: co-transfection of MW7 with mutant Htt in acute striatal slices inhibits Htt neurotoxicity. Further experiments are required to support this result, (ii) To engineer more potent intrabodies, we screened a naive human scFv phage library for binding to a proline-enriched peptide from Htt, followed by a screen for preferential binding to mutant Htt. Six new intrabodies were cloned and two of these appear very promising in blocking mutant Htt aggregation and toxicity in cell culture, (iii) Three animal models are being investigated in therapeutic experiments. First, a lentiviral vector encoding mutant Htt is injected in the adult striatum, causing neuropathology. Co-injection of vectors encoding the intrabodies will be tested for blocking this pathology. Second, AAV vectors encoding the best candidate intrabodies are injected in neonatal the R6/2 HD mouse striatum. These mice will be examined as adults for alterations in Htt aggregation, neuropathology and HD-like motor symptoms. Third, the AAV-intrabodies are injected into the adult striatum of pre- and post-symptomatic R6/2 and N171 HD model mice. These mice will be examined as above, (iv) To identify novel Abs with therapeutic potential we are developing a new screen for intrabodies that enhance the survival of PC12 cells induced to express mutant Htt. This non-biased approach could uncover new pathways involved in HD pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS055298-03
Application #
7576852
Study Section
Cell Death in Neurodegeneration Study Section (CDIN)
Program Officer
Sutherland, Margaret L
Project Start
2007-09-30
Project End
2012-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
3
Fiscal Year
2009
Total Cost
$375,296
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Khoshnan, Ali; Patterson, Paul H (2011) The role of IýýB kinase complex in the neurobiology of Huntington's disease. Neurobiol Dis 43:305-11
Southwell, Amber L; Patterson, Paul H (2011) Gene therapy in mouse models of huntington disease. Neuroscientist 17:153-62
Khoshnan, Ali; Ko, Jan; Tescu, Simona et al. (2009) IKKalpha and IKKbeta regulation of DNA damage-induced cleavage of huntingtin. PLoS One 4:e5768
Legleiter, Justin; Lotz, Gregor P; Miller, Jason et al. (2009) Monoclonal antibodies recognize distinct conformational epitopes formed by polyglutamine in a mutant huntingtin fragment. J Biol Chem 284:21647-58
Southwell, Amber L; Ko, Jan; Patterson, Paul H (2009) Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease. J Neurosci 29:13589-602
Thompson, Leslie Michels; Aiken, Charity T; Kaltenbach, Linda S et al. (2009) IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome. J Cell Biol 187:1083-99
Southwell, Amber L; Khoshnan, Ali; Dunn, Denise E et al. (2008) Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. J Neurosci 28:9013-20