Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins.

The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins.

Early or late-stage anti-N-terminal Huntingtin intrabody gene therapy reduces pathological features in B6.HDR6/1 mice.

Huntington disease (HD) is a progressive neurodegenerative disease caused by an expansion of a polyglutamine sequence in mutant huntingtin (mhtt) that produces abnormal folding and aggregation that results in the formation of nuclear and cytoplasmic neuronal inclusion bodies. Although the precise role of mhtt aggregates in the pathogenesis is unclear, attempts to reduce accumulated mhtt protein have ameliorated the phenotype in multiple cellular and in vivo HD models. Here, we provide critical results on intracranial delivery of a single-chain Fv intrabody, C4, which targets the first 17 amino acids of the htt protein, a region of httExon1 that is increasingly being recognized as pivotal. To assess long-term efficacy and safety issues, we used adenoassociated viral vectors (AAV2/1) to deliver intrabody genes to the striatum of inbred B6.HDR6/1 mice. Treatment initiation at various stages of the disease showed that early treatment preserved the largest number of cells without nuclear aggregates and that the accumulation of aggregated material could be delayed by several months. Even when intrabody treatment was not initiated until the clinical disease stage, significant, albeit smaller, effects were seen. These data indicate that neuronal intrabodies against critical N-terminal epitopes can be safely and effectively delivered using AAV2/1 to delay the aggregation phenotype during a sustained period in this HD model, even when delivery is initiated after disease onset.

In vivo inactivation of pRb, p107 and p130 in murine neuroprogenitor cells leads to major CNS developmental defects and high seizure rates.

Nestin-positive cells were targeted for pRb, p107 and p130 (pRb(f)) inactivation by expression of T(121), a truncated SV40 large T antigen that selectively binds to and inactivates pRb(f). Cre expression was initiated under GFAP control, resulting in T(121) expression restricted to neuroprogenitor cells beginning at embryonic day 11.5 (E11.5). Bi-transgenic embryos showed aberrant central nervous system (CNS) cell proliferation and apoptosis by E13.5. Defects in cortical development were evident with primary effects resulting in depletion of neural progenitors and aberrant cellular migration. Consequently, juvenile and adult brain morphology was reproducibly abnormal, including disorganization of neocortical, hippocampal and cerebellar regions. These aberrations resulted in behavioral phenotypes, including ataxia and seizures. The data indicate that inactivation of pRb(f) in radial glial cells, a population of neuroprogenitor cells, leads to specific disruptions in CNS patterning. The neuroprogenitor-restricted transgene expression provides a model in which to explore both developmental mechanisms and functional neurological outcomes.