A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations.

Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

Effective knockdown-replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.

Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wild-type replacement. We evaluated such a knockdown-replace strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons-which resulted in growth delay and lethal seizures evident by postnatal week three-and delivered to newborn pups an AAV9-based vector encoding a ubiquitously expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated pups, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNA sequencing and functional annotation clustering. Mutants displayed abnormal expression of more than 1,000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating DNM1 and related genetic neurodevelopmental disease.

Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes.

Facioscapulohumeral muscular dystrophy (FSHD) arises from epigenetic changes that de-repress the DUX4 gene in muscle. The full-length DUX4 protein causes cell death and muscle toxicity, and therefore we hypothesize that FSHD therapies should center on inhibiting full-length DUX4 expression. In this study, we developed a strategy to accomplish DUX4 inhibition using U7-small nuclear RNA (snRNA) antisense expression cassettes (called U7-asDUX4). These non-coding RNAs were designed to inhibit production or maturation of the full-length DUX4 pre-mRNA by masking the DUX4 start codon, splice sites, or polyadenylation signal. In so doing, U7-asDUX4 snRNAs operate similarly to antisense oligonucleotides. However, in contrast to oligonucleotides, which are limited by poor uptake in muscle and a requirement for lifelong repeated dosing, U7-asDUX4 snRNAs can be packaged within myotropic gene therapy vectors and may require only a single administration when delivered to post-mitotic cells in vivo. We tested several U7-asDUX4s that reduced DUX4 expression in vitro and improved DUX4-associated outcomes. Inhibition of DUX4 expression via U7-snRNAs could be a new prospective gene therapy approach for FSHD or be used in combination with other strategies, like RNAi therapy, to maximize DUX4 silencing in individuals with FSHD.