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

Abstract

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 two 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.

A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease

“…17,49,50 In addition, a recent study confirmed that amiRNAs are present in vesicles 2 years post injection into the brains of NHPs. 51 These results support the choice of AAV5 as a delivery vehicle for amiR136-A2; however, the high variability in the silencing efficiency between individuals and the low silencing efficiency in the cortex suggest that the delivery and distribution of RNAi vectors in the brain should be improved. The uneven distribution of amiR136-A2 may be a cause of the weak improvements in motor deficits.…”

Functional Intercellular Transmission of miHTT via Extracellular Vesicles: An In Vitro Proof-of-Mechanism Study

“…Human iPS cells (ND42229*B) containing 71 CAG repeats were reprogrammed from HD patient-derived fibroblasts (Coriell Institute Stem Biobank, Camden, NJ, USA) [ 47 ]. These cells were differentiated and maintained in culture as previously described [ 47 ].…”

“…Isolation of EVs from HEK293-miHTT cell line conditioned culture media was performed as previously described [ 47 ]. Briefly, culture medium was collected and centrifuged at 4000× g for 15 min at room temperature to remove cells and cell debris.…”

“…How efficiently AAV vector transport occurs through neuronal connections in the context of HD-related neurodegeneration, or whether other mechanisms contribute to its biodistribution beyond target regions, remains unknown. Previously, we have shown that AAV5-delivered miRNA molecules were released from HD patient-derived neuronal cells in association with EVs [ 47 ]. In addition, EV-associated miHTT was documented in CSF of NHPs two years after a single intrastriatal infusion of AAV5-miHTT [ 47 ].…”

“…Previously, we have shown that AAV5-delivered miRNA molecules were released from HD patient-derived neuronal cells in association with EVs [ 47 ]. In addition, EV-associated miHTT was documented in CSF of NHPs two years after a single intrastriatal infusion of AAV5-miHTT [ 47 ].…”