Abstract
Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ∼80–90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.
Highlights
Huntington’s disease (HD) is the most prevalent of nine known human neurodegenerative disorders linked to the expansion of polyglutamine tracts in specific disease-associated proteins [1]
We have previously shown that fusion of a nuclear localization sequence (NLS) derived from SV40 Large T antigen to Single-chain Fv antibodies (scFvs)-C4 was able to bind, transport, and sequester soluble httex1 protein fragments with 72 glutamine repeats fused to enhanced green florescent protein into the nuclei [18,19]
Because the targeted epitope of scFv-C4 is the N-terminal 17 amino acids of htt, scFv-C4 and scFv-C4-PEST are likely to bind a variety of N-terminal mhtt fragments produced from the cleavage of full-length mhtt during HD pathology
Summary
Huntington’s disease (HD) is the most prevalent of nine known human neurodegenerative disorders linked to the expansion of polyglutamine (polyQ) tracts in specific disease-associated proteins [1]. Intrabody-based therapies show significant potential for addressing the critical need to reduce the misfolded protein burden in HD [9]. These recombinant single-chain and single-domain variable fragments of full-length antibodies exhibit high specificity and affinity for targets, can be selected, engineered, and delivered as genes [10,11,12,13]. ScFv-C4 preferentially binds to soluble mhtt N-terminal fragments It is only weakly active against endogenous full-length mhtt and wild type htt, possibly due to epitope inaccessibility [20]. Additional optimization of scFv-C4 is required for this intrabody to be of future use in clinical applications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
