DNM1 disease and new approaches to
gene therapy for DEE
The DNM1 gene encodes dynamin-1, a large GTPase that catalyzes endocytosis and synaptic vesicle recycling. Dynamin-1 is expressed in the CNS, localizing to the neuron presynaptic terminal. Pathogenic mutations reside in the GTPase and middle domains of the protein, driving severe developmental and epileptic encephalopathy (DEE). While DNM1 DEE is ultra-rare, with around 70 patients identified so far, clinical features are relatively homogeneous and significant, with affected children exhibiting unremitting seizures starting within the first year of life, severe to profound intellectual disability, developmental delay, muscular hypotonia and neurosensory deficits. Given that these children usually have intractable disease with limited, if any, efficacy of antiepileptic medications, we were compelled to explore genetic therapies in mouse models.
RNAi knockdown therapy for
isoform-specific DNM1 variants
In 2010 during his tenure at The Jackson Laboratory, Wayne Frankel’s group discovered an atypical mutation in Dnm1 - “fitful” - that conferred DEE-like features. This mutation was atypical because it arose on a mutually exclusive isoform (DNM1-1A), leaving the 1B isoform intact - unlike most human variants known at the time which were on a common isoform. The lab had also determined that DNM1-1A isoform-specific knockout mice were viable and relatively normal, suggesting a ready, early test of gene therapy whereby the DNM1-1A isoform could be selectively targeted, allowing DNM1-1B to provide sufficient function. Thus, in collaboration with Scott Harper’s lab (Nationwide Children’s Hospital, Columbus, OH) using a single treatment of neonatal mutant mice they developed an AAV9 vector designed to eliminate all DNM1-1A mRNA (mutant and wildtype) from transduced cells. As illustrated in the graphical abstract from the 2020 Molecular Therapy publication, they succeeded in converting a very severe disease, culminating in lethality before the 4th week of mouse life, to a mild disease with no early lethality, only mild seizures, and no obvious ataxia or neuropathology.
Knockdown-replace gene therapy for any DNM1 variant
While the gene therapy approach tested in fitful mice was promising, since pan-Dnm1 knockdown would result in early lethality in mice this therapy would not be expected to be effective on most DNM1 pathogenic variants since they reside on all DNM1 isoforms. Preparing for a new approach, we developed a second DNM1 DEE model, conditionally encoding the Gly-359-Ala (G359A) variant. This mutation maps to the same protein domain as fitful, but unlike fitful, G359A resides on an exon common to all DNM1 isoforms. As described in our recent Molecular Therapy publication (Jones, Soundararajan et al., 2024), we then tested several cre recombinase driver strains and identified one – Gad2-Cre – that expressed the mutation only in GAD2+ inhibitory neurons and resulted in fully-penetrant and severe growth delay and tonic-clonic seizures. In continued collaboration with Scott Harper, we developed an AAV9-based “knockdown-replace” vector which in a single viral genome harbored a pan-Dnm1 RNAi (“knockdown”) driven by the U6 promoter, and a wildtype Dnm1 mRNA (“replace”) engineered to be resistant to knockdown, driven by the human SYN1 promoter.
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Although neither the knockdown-only or the replace-only component separately improved the phenotypes, knockdown-replace together prevented the lethal seizures in these mice, improved overall growth and our collaborator Matthew Weston (Virginia Tech) demonstrated that synaptic transmission abnormalities were corrected. The treatment also rectified abnormal gene expression signatures observed in the transcriptome of mutant mice.
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As noted in the paper, while the treated mice are cured of severe disease they are not entirely normal and show behavioral and developmental symptoms that are either residual (uncorrected disease) or related to the therapy itself - we cannot distinguish between these two possibilities. However, we also note that this was a fairly basic first effort at a knockdown-replace vector to use for this purpose. We think that the expression of the RNAi and mRNA replacement components can be further optimized and possibly synchronized with alternative promoter-enhancers and delivery parameters, in work that lies ahead. It is also important to realize that because of the significantly more accelerated developmental periods and lifespans of mouse compared to human (mouse "childhood" is only 2 weeks long, and the healthiest normal mice only live for 2 years), the window of opportunity for treating affected DNM1 DEE children is comparatively much broader. Nevertheless, we view the conversion of a very severe (lethal in mice) form of DNM1 disease to a manageable state, using a one-time treatment that would potentially work for any pathogenic variant of DNM1, as strong indication of future promise for a disease that otherwise has very limited therapeutic potential.