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Summary Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntington’s disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the cysteine protease families of caspases and calpains. Identifying critical protease families involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a high-throughput western blot-based screen to examine the generation of the smallest N-terminal polyglutamine-containing Htt fragment. Using this approach, we screened a set of 514 siRNAs targeting the repertoire of human protease genes. This screen identified 11 proteases that, when inhibited, reduced Htt fragment accumulation. Three of these belonged to the matrix metalloproteinase (MMP) family. One family member, MMP-10, directly cleaves Htt, and prevents cell death when knocked down in striatal Hdh111Q/111Q cells. Correspondingly, we found MMPs are activated in mouse models of HD, and loss of function of Drosophila homologs of MMPs and four other proteases suppress Htt-induced neuronal dysfunction in vivo.

In this report, we look at a range of classes of reaction involving microwave heating and show how different processing techniques can be used to address scale-up needs. We look at both batch and continuous-flow processing. We have shown that when using batch methodologies working using an open reaction vessel offers operational advantages while still giving good yields of desired products. In cases where open-vessel conditions are not amenable or where particularly volatile or toxic reagents are used, parallel sealed vessels can offer an alternative approach. For continuousflow processing, homogeneity of the reaction mixture is key. When the mixture is homogeneous, it is possible to move from smallscale sealed-vessel conditions to the continuous-flow apparatus without any modification of reaction conditions or loss in product yield. When either the starting materials or the product mixture contains particulate matter, continuous processing can prove a challenge, but reoptimization of reaction conditions as well as reduction of the concentration may allow these difficulties to be overcome.

Adeno-associated virus (AAV)-based vectors are widely used for gene therapy, but the effect of pre-existing antibodies resulting from exposure to wild-type AAV is unclear. In addition, other poorly defined plasma factors could inhibit AAV vector transduction where antibodies are not detected. To better define the relationship between various forms of pre-existing AAV immunity and gene transfer, we studied valoctocogene roxaparvovec (BMN 270) in cynomolgus monkeys with varying pre-dose levels of neutralizing anti-AAV antibodies and non-antibody transduction inhibitors. BMN 270 is an AAV5-based vector for treating hemophilia A that encodes human B domain-deleted factor VIII (FVIII-SQ). After infusion of BMN 270 (6.0 × 10 13 vg/kg) into animals with pre-existing anti-AAV5 antibodies, there was a mean decrease in maximal FVIII-SQ plasma concentration (C max ) and AUC of 74.8% and 66.9%, respectively, compared with non-immune control animals, and vector genomes in the liver were reduced. In contrast, animals with only non-antibody transduction inhibitors showed FVIII-SQ plasma concentrations and liver vector copies comparable with those of controls. These results demonstrate that animals without AAV5 antibodies are likely responders to AAV5 gene therapy, regardless of other inhibiting plasma factors. The biological threshold for tolerable AAV5 antibody levels varied between individual animals and should be evaluated further in clinical studies.

SUMMARY Huntington’s Disease (HD) is characterized by a mutation in the huntingtin gene encoding an expansion of glutamine repeats on the N-terminus of the huntingtin (Htt) protein. Numerous studies have identified Htt proteolysis as a critical pathological event in post mortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD targets. We report the use of the substrate activity screening method against caspases-3 and -6 to identify three novel, pan-caspase inhibitors that block proteolysis of Htt at caspase-3 and -6 cleavage sites. In HD models, these irreversible inhibitors suppressed Hdh111Q/111Q-mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study the identified nonpeptidic caspase inhibitors were used to confirm the role of caspase-mediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development.

SUMMARY Huntington’s disease (HD) is caused by a mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats at the N-terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knockout mouse (Casp6−/−) Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6−/− mice, regardless of HD genotype. We predicted the Casp6−/− mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6−/− mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6−/− mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 amino acid Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.

A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.

Background: Huntington disease is characterized by the generation of mutant huntingtin fragments, which correlate with disease progression. Results: The transgenic mice N171-Q148 and N552-Q148 display a significantly accelerated phenotype and shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. Conclusion: Some HTT proteolysis fragments have distinct neurotoxicity. Significance: Reducing proteolysis of huntingtin is a viable therapeutic treatment for Huntington disease.