RNA Interference Targeting Small Heat Shock Protein B8 Failed to Improve Distal Hereditary Motor Neuropathy in the...

Missense mutations in the HSPB8 gene, encoding the small heat shock protein B8, cause distal hereditary motor neuropathy (dHMN) or an axonal form of Charcot-Marie-Tooth disease (CMT subtype 2L). Mice expressing mutant Hspb8 (Lys141Asn) mimic the human disease, whereas mice lacking Hspb8 show no overt phenotype. We aimed to design an RNA interference treatment strategy that rescues the mutant HSPB8 neuronal and muscle phenotype in patient-derived motor neurons and in a knock-in mouse model of CMT2L/dHMN.

We optimized RNA interference sequences targeting both human HSPB8 and mouse HspB8 transcripts with the aim to alleviate disease symptoms. We used human induced pluripotent stem cells and the Hspb8 knock-in mouse model. We designed lenti- and adeno-associated viral vectors that contained the short-hairpin RNA constructs. We performed expression and microscopy studies, magnetic resonance imaging, behaviour analysis and electrophysiology.

In CMT2L patient-derived induced pluripotent stem cells differentiated towards motor neurons, reducing the HSPB8 expression with a short-hairpin RNA (shRNA), directed towards the 3' untranslated region (3'UTR), ameliorated the morphology and fragmentation of mitochondria. The AAV9-mediated treatment of the 3'UTR shRNA construct, under neuron-specific regulation, in Hspb8 knock-in mice showed inconclusive results towards functional improvement upon expression studies, magnetic resonance imaging and neuropathological findings.

Given the limited beneficial effect of the treatment, the RNA interference-mediated reduction of HSPB8/Hspb8 expression might not be the best therapeutic strategy to treat dHMN/CMT2L, unless a higher viral load and earlier treatment can be applied to the mouse model.