48. In Vivo and In Situ Delivery of Huntingtin-Specific Single-Chain Fv Intrabody Genes as Therapeutic Agents for Huntington's Disease

Huntington's disease (HD) is one of several neurodegenerative disorders caused by expansion of a CAG repeat. The resulting expanded polyglutamine tract causes the huntingtin protein to form insoluble aggregates and participate in aberrant protein-protein interactions. Therapeutic intervention t...

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Veröffentlicht in:Molecular therapy 2004-05, Vol.9 (S1), p.S20-S21
Hauptverfasser: Miller, Todd W, Wolfgang, William J, Manley, Kevin, Zhou, Yangsheng, Zhou, Chun, Webster, Jack M, Mazarakis, Nicholas D, Messer, Anne
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Sprache:eng
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Zusammenfassung:Huntington's disease (HD) is one of several neurodegenerative disorders caused by expansion of a CAG repeat. The resulting expanded polyglutamine tract causes the huntingtin protein to form insoluble aggregates and participate in aberrant protein-protein interactions. Therapeutic intervention to prevent aggregation, reduce abnormal interactions, and allow proper proteolytic processing may reduce HD pathogenesis. Intrabodies (single-chain Fv antibody fragments) can bind with high specificity and affinity to intracellular targets, potentially altering their folding and interactions, and serving as curative agents and/or tools for drug discovery in an array of neurological disorders associated with altered protein structures. We have previously demonstrated that an intrabody targeting the N-terminus of huntingtin (upstream of the polyglutamine tract) dramatically reduces aggregate formation in HEK-293, BHK-21, and COS-7 cell lines, and provides functional protection against expanded polyglutamine toxicity in organotypic brain slice cultures. Transient dual expression of both intrabody and huntingtin exon 1 (httex1)-GFP proteins from a single vector in a rat striatal cell model (ST14A) shows that the intrabody enhances protein turnover, measured as a decrease in the amount of soluble httex1-GFP; effects were even more dramatic in the HEK293T cell line. We are now using recombinant equine infectious anemia virus (EIAV; pONY8.7), pseudotyped with either VSV-G or Rabies-G, encoding intrabodies alone or in bicistronic vectors to stably transduce neuronal cell lines and brain cells. Viral titers are 10 8-9 IU/ml for all constructs.We have generated stably transfected striatal cell lines previously derived from wild-type, heterozygous, and homozygous htt Q111 knock-in mice (Trettel, 2000). Coexpression of the anti-htt intrabody (or control intrabody) and a GFP marker from a bicistronic cassette allows flow-sorting of positive cells expressing different levels of intrabody. There is no sign of toxicity, even at high levels of the anti-htt intrabody, which was selected against an epitope common to both mutant and wild-type htt. These lines will allow further analysis of intrabody effects in the context of full-length, endogenous htt, which may better simulate aspects of the human disease.Intrastriatal injection of HD R6/1 transgenic mice with Rabies-G-pseudotyped EIAV encoding the anti-htt intrabody shows widespread, long-term (>3 months) neuronal expression in the st
ISSN:1525-0016
1525-0024
DOI:10.1016/j.ymthe.2004.05.071