Huntington’s Disease (HD) is a genetically dominant trinucleotide repeat disorder resulting from CAG repeats within the Huntingtin (HTT) gene exceeding a normal range (> 36 CAGs). Symptoms of the disease manifest in middle age and include chorea, dystonia, and cognitive decline. Typical latency from diagnosis to death is 20 years. There are currently no disease-modifying therapies available to HD patients. RNAi is a potentially curative therapy for HD. A popular line of research employs siRNA or antisense oligonucleotides (ASO) to knock down mutant Huntingtin mRNA (mHTT). Unfortunately, this modality requires repeated dosing, commonly exhibit off target effects (OTEs), and exert renal and hepatic toxicity. In contrast, a single AAV-mediated short-hairpin RNA (shRNA) dose can last years with low toxicity. In addition, we highlight research indicating that shRNA elicits fewer OTEs than siRNA when tested head-to-head. Despite this promise, shRNA therapy has been held back by difficulties controlling expression (oversaturating cells with toxic levels of RNA construct). In this review, we compare RNAi modalities for HD and propose novel methods of optimizing shRNA expression and on-target fidelity.

Mitochondria-dependent apoptosis plays an important role in the embryonic development of the midbrain dopaminergic system as well as in Parkinson’s disease. Central to mitochondria-dependent apoptosis is the Bcl2 family of apoptosis-regulating proteins. However, it was unclear which Bcl2 proteins are important for the survival of dopaminergic neurons. Here, we identify Mcl1 as a critical Bcl2 pro-survival factor in midbrain dopaminergic neurons. Using a chemical biology approach to inhibit various components of the apoptotic machinery in the dopaminergic MN9D cell line or the control neuroblastoma N2A cell line, we find that functional inhibition of Mcl1 with the high affinity small molecule inhibitor UMI-77 results in a rapid and dose-dependent loss of viability, selectively in dopaminergic cells. In-depth analysis of the apoptotic signaling pathway reveals that chemical inhibition of Mcl1 results in the activation of Bax, activation of cleaved caspase-3 and finally cell death. The dependence of mouse dopaminergic midbrain neurons on Mcl1 was confirmed using ex vivo slice cultures from Pitx3GFP/+ and wildtype mice. In mouse dopaminergic midbrain neurons positive for the midbrain dopaminergic marker Pitx3, or tyrosine hydroxylase, UMI-77 treatment caused a dramatic increase in cleaved caspase 3, indicating that Mcl1 activity is required for basal neuronal survival. Overall, our results suggest that Mcl1 is of critical importance to dopaminergic neurons and is a weak link in the chain controlling cellular survival. Boosting the pro-survival function of Mcl1 should be pursued as a therapeutic approach to augment the resilience of midbrain dopaminergic neurons to apoptotic stress in Parkinson’s disease.

No abstract

Induced cell turnover (ICT) is a theoretical intervention in which the targeted ablation of damaged, diseased, and/or nonfunctional cells is coupled with replacement by partially differentiated induced pluripotent stem cells in a gradual and multiphasic manner. Tissue-specific ablation can be achieved using pro-apoptotic small molecule cocktails, peptide mimetics, and/or tissue-tropic adeno-associated virus-delivered suicide genes driven by cell type-specific promoters. Replenishment with new cells can be mediated by systemic administration of cells engineered for homing, robustness, and even enhanced function and disease resistance. Otherwise, the controlled release of cells can be achieved using implanted biodegradable scaffolds, hydrogels, and polymer matrixes. In theory, ICT would enable in situ tissue regeneration without the need for surgical transplantation of organs produced ex vivo, and addresses non-transplantable tissues (such as the vasculature, lymph nodes, and the nervous system). This article outlines several complimentary strategies for overcoming barriers to ICT in an effort to stimulate further research at this promising interface of cell therapy, tissue engineering, and regenerative medicine.

No abstract