Isolation, engineering, and characterization of intracellular antibodies specific for the huntingtin protein

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of an N-terminal proteolytic htt fragment containing the glutamine-expanded region to misfold and adopt a conformation which is prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the htt fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene therapy approach to treatment of the disease. However, high expression levels of previously reported intrabodies have been required to obtain even limited reductions in htt aggregation. We have used yeast surface display (YSD) of antibodies combined with fluorescence activated cell sorting (FACS) to isolate novel single-chain antibody (scFv) clones against huntingtin from a non-immune human antibody library; these scFv's did not inhibit htt aggregation. Engineering analysis, including the derivation of equations that describe the probability that cells will form htt aggregates as a function of time and concentration, were used to estimate the intracellular expression level and binding affinity required for robust aggregation inhibition.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Colby, David W. (David Wesley)

Advisor dc:contributor.advisor

• K. Dane Wittrup and Vernon M. Ingram.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/30358

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/30358