Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing

Abstract

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder resulting from a polyglutamine expansion in the huntingtin (HTT) protein. There is currently no cure for this disease, but recent studies suggest that RNAi to downregulate the expression of both normal and mutant HTT is a promising therapeutic approach. We previously developed a small hairpin RNA (shRNA), vectorized in an HIV-1-derived lentiviral vector (LV), that reduced pathology in an HD rodent model. Here, we modified this vector for preclinical development by using a tat-independent third-generation LV (pCCL) backbone and removing the original reporter genes. We demonstrate that this novel vector efficiently downregulated HTT expression in vitro in striatal neurons derived from induced pluripotent stem cells (iPSCs) of HD patients. It reduced two major pathological HD hallmarks while triggering a minimal inflammatory response, up to 6 weeks after injection, when administered by stereotaxic surgery in the striatum of an in vivo rodent HD model. Further assessment of this shRNA vector in vitro showed proper processing by the endogenous silencing machinery, and we analyzed gene expression changes to identify potential off-targets. These preclinical data suggest that this new shRNA vector fulfills primary biosafety and efficiency requirements for further development in the clinic as a cure for HD.

AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington’s Disease Minipig Model

“…Currently, several strategies are under development to lower huntingtin protein synthesis, and preclinical studies in HD rodent models have demonstrated that lowering mutant huntingtin protein reduces downstream deleterious effects 5, 6, 7. Most approaches of huntingtin lowering aim to decrease translation of huntingtin exploiting the endogenous RNAi mechanism by using synthetic small interfering RNA (siRNA) or by using modified single-stranded antisense oligonucleotides 7, 8, 9, 10, 11, 12, 13, 14. Both siRNAs and antisense oligonucleotides need to be delivered by continuous infusion or repetitive intrathecal injections into the cerebrospinal fluid (CSF), and their therapeutic effect is thought to be most potent in the brain areas adjacent to the ventricular system 7, 15.…”

“…Next to repetitive infusion of synthetic oligonucleotides, RNAi-based gene therapy approaches using short hairpin RNA (shRNA) and microRNA (miRNA) have been explored as well. RNAi-based gene therapy comprises a single administration of an adeno-associated virus (AAV) or lentiviral (LV) vector, resulting in continuous expression of shRNA or artificial miRNA precursors and subsequent long-lasting huntingtin lowering 5, 6, 12, 13, 17, 18, 19, 20. Intracranial parenchymal injections of artificial miRNA delivered by AAV vectors resulted in huntingtin reduction in HD animal models without signs of toxicity 21, 22, 23, 24…”

Translation of MicroRNA-Based Huntingtin-Lowering Therapies from Preclinical Studies to the Clinic

“…100 Because gene expression signatures are often species-specific, these human-cell-based models have become the preferred test system in parallel to animal studies to study therapy-induced neuronal protection as well as specific off-target silencing of other genes. 71,104 Preclinical Outcome Measures for AAV-miRNA Gene Therapies…”

“…et al, unpublished data). 39,64,72,104,144 Measurements of On-Target Lowering Efficacy miRNA-based lowering strategies are designed to lower HTT mRNA levels and thereby reduce the overall mutant HTT protein. Already in early preclinical development, it is essential to establish the on-target activity of therapeutics by measuring their effects on HTT mRNA and protein levels.…”

“…10 Off-target effects may be predicted by seeking for genomic sequences with partial homology to the artificial miRNA targets and addressing the potential gene expression changes in HD animal and cell models. 104,162 Long-Term Wild-Type HTT Lowering…”