Biomedical subjects

Atsushi Miyanohara

Publications and source records attributed to Atsushi Miyanohara.

3 recordsLinked to original sources

PubMed2026

Base editing rescues a hereditary motor neuron disease in mouse and patient-derived iPSC organoid models.

In hereditary motor neuron diseases (MNDs), including forms of amyotrophic lateral sclerosis (ALS) caused by single-nucleotide variants, effective therapeutic strategies need to address both gain- and loss-of-function mechanisms. Genome editing-based gene therapy represents a promising approach for simultaneously targeting these mechanisms. To establish proof-of-concept for base editing in a hereditary MND, we targeted the P285L variant in the TRK-fused gene (TFG), which causes hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P), a disorder that shares clinical and histopathological features with ALS. We identified the optimal adenine base editor by comparing candidate editors in HMSN-P patient-derived induced pluripotent stem cells (iPSCs). We then generated a transgenic mouse model expressing human TFG P285L and evaluated the selected editor by subpial delivery of adeno-associated virus (AAV) vectors to the spinal cord. AAV-mediated base editing prolonged survival, preserved motor neurons, and attenuated axon loss in ventral nerve roots. Treatment with the selected base editing vector reduced TFG aggregation and suppressed neuronal death in HMSN-P iPSC-derived neuromuscular organoids. Collectively, these findings support the therapeutic potential of base editing for hereditary MNDs.

AAV↗

PubMed2026

Treatment of spinal injury muscle spasticity by spinal subpial AAV9-GAD65/VGAT delivery: An efficacy and safety study in rat, pig, and NHP.

The loss in segmental inhibitory GABAergic tone plays the key role in the development of spinal injury-induced muscle spasticity. We use a subpial segment-targeted delivery of adeno-associated virus vector(s) expressing GAD65 (glutamic acid decarboxylase-65) and VGAT (vesicular GABA transporter) transgenes in rats with spinal transection-induced spasticity. In treated animals, a significant suppression in spasticity was seen at 5-8 weeks after treatment. Naive rats, pigs, and non-human primates (NHPs) injected with human equivalent dose of treatment vectors and surviving for 3 weeks to 4.5 years showed normal motor function and pinch-evoked response. A significant increase in the number of VGLUT2 terminals co-expressing GAD65 and VGAT protein in vector-injected segment was seen. This corresponded with the presence of transgene-specific rat Gad2 or human GAD2 and rat Slc32a1 or human SLC32A1 mRNA signal. No spinal toxicity was noted in NHPs at 4.5 years post vector delivery. Analysis of peripheral organs (liver, spleen, and skeletal muscle) showed minimal or no detectable transgenes in pigs and NHPs. These data demonstrate that a single-time-point spinal-segment-targeted subpial delivery of GAD65/VGAT transgenes is effective in suppressing spinal injury-induced spasticity and has a favorable long-term safety profile as defined by normal neurological function and histopathology in naive pigs and NHPs.

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PubMed2002

Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.

Animals↗