TS-004703 — For AAVs that harbor gene knockdown machinery, this IP is a novel potency assay development for release testing that creates a HEK293T stable cell line to integrate two genetic components.

TS-004598 — This system may help improve the safety of AAV vector products by decreasing cross-packaged bacterial sequences, increasing correctly packaged AAV payloads and blunting ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences.

TS-002303 — WHO: Scott Harper WHAT: RNAi therapy WHY: DNM1 Developmental and Epileptic Encephalopathy (DEE) HOW: (from IDF) The DNM1 gene encodes dynamin-1, a large GTPase involved in clathrin-mediated endocytosis of synaptic vesicles in neurons and in related processes. Dynamin monomers assemble into multimers that interact with each other and with various other proteins to form ring structures for GTPase-catalyzed membrane scission. Dominant mutations in one allele of DNM1 cause neurological disease in humans and mice. Children with DNM1 mutations suffer from intractable conditions manifesting as early-onset seizures, global developmental delay, profound intellectual disability, lack of speech, muscular hypotonia, dystonia, and spasticity. As is the case with many severe DEEs, affected individuals do not respond well to antiepileptic drugs, leaving >80 % of patients with intractable seizures and little to no improvement of the severely debilitating other neurological features. At least 20 de novo pathogenic DNM1 variants have been identified, and all are presumed to operate with dominant effect, most likely by interfering with the assembly or function of normal dynamin-1 monomers. Importantly, knockout studies in mice support that at least 50% normal DNM1 levels are required, as homozogous DNM1 null animals die by postnatal day 8. We propose that a successful gene therapy approach must eliminate or reduce the expression or translation of the mutant DNM1 variant mRNA or protein, while still enabling expression of the remaining wild-type allele. We are pursuing two different approaches to accomplish this: (1) isoform-specific silencing of mutant DNM1 transcripts; (2) a knockdown-and-replace strategy; expressing exogenous wildtype Dnm1 via AAV while eliminating endogenous Dnm1 mRNA entirely. We already filed an IDF and provisional patent application on approach 1. This IDF covers the knockdown-and-replace strategy. In brief, we will develop artificial microRNAs that non-selectively knockdown both mutant and wild-type DNM1 alleles using RNAi, while at the same time adding back an RNAi-resistant, wild-type DNM1 cDNA. We will test this in mouse models of DNM1-related DEE in the Frankel lab at Columbia University.