Secreted therapeutics: monitoring durability of microRNA-based gene therapies in the central nervous system.

The preclinical development of microRNA-based gene therapies for inherited neurodegenerative diseases is accompanied by translational challenges. Due to the inaccessibility of the brain to periodically evaluate therapy effects, accessible and reliable biomarkers indicative of dosing, durability and therapeutic efficacy in the central nervous system are very much needed. This is particularly important for viral vector-based gene therapies, in which a one-time administration results in long-term expression of active therapeutic molecules in the brain. Recently, extracellular vesicles have been identified as carriers of RNA species, including microRNAs, and proteins in all biological fluids, whilst becoming potential sources of biomarkers for diagnosis. In this study, we investigated the secretion and potential use of circulating miRNAs associated with extracellular vesicles as suitable sources to monitor the expression and durability of gene therapies in the brain. Neuronal cells derived from induced pluripotent stem cells were treated with adeno-associated viral vector serotype 5 carrying an engineered microRNA targeting huntingtin or ataxin3 gene sequences, the diseases-causing genes of Huntington disease and spinocerebellar ataxia type 3, respectively. After treatment, the secretion of mature engineered microRNA molecules was confirmed, with extracellular microRNA levels correlating with viral dose and cellular microRNA expression in neurons. We further investigated the detection of engineered microRNAs over time in the CSF of non-human primates after a single intrastriatal injection of adeno-associated viral vector serotype 5 carrying a huntingtin-targeting engineered microRNA. Quantifiable engineered microRNA levels enriched in extracellular vesicles were detected in the CSF up to 2 years after brain infusion. Altogether, these results confirm the long-term expression of adeno-associated viral vector serotype 5-delivered microRNAs and support the use of extracellular vesicle-associated microRNAs as novel translational pharmacokinetic markers in ongoing clinical trials of gene therapies for neurodegenerative diseases.

Targeting RNA-Mediated Toxicity in C9orf72 ALS and/or FTD by RNAi-Based Gene Therapy.

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts.

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.

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

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

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

The single mutation underlying the fatal neuropathology of Huntington's disease (HD) is a CAG triplet expansion in exon 1 of the huntingtin (HTT) gene, which gives rise to a toxic mutant HTT protein. There have been a number of not yet successful therapeutic advances in the treatment of HD. The current excitement in the HD field is due to the recent development of therapies targeting the culprit of HD either at the DNA or RNA level to reduce the overall mutant HTT protein. In this review, we briefly describe short-term and long-term HTT-lowering strategies targeting HTT transcripts. One of the most advanced HTT-lowering strategies is a microRNA (miRNA)-based gene therapy delivered by a single administration of an adeno-associated viral (AAV) vector to the HD patient. We outline the outcome measures for the miRNA-based HTT-lowering therapy in the context of preclinical evaluation in HD animal and cell models. We highlight the strengths and ongoing queries of the HTT-lowering gene therapy as an HD intervention with a potential disease-modifying effect. This review provides a perspective on the fast-developing HTT-lowering therapies for HD and their translation to the clinic based on existing knowledge in preclinical models.

Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing.

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.

Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.