Biology subjects

Ronzitti, G.

Publications and source records attributed to Ronzitti, G..

3 recordsLinked to original sources

bioRxiv2026

Single-vector AAV delivery of En-ISPmu1 and donor DNA for in vivo targeted genome integration

TnpBs are compact RNA-guided nucleases ideally suited for in vivo delivery. By screening novel TnpBs identified from large metagenomic datasets, ISPmu1 was discovered and its protein and {omega}RNA were engineered to generate En-ISPmu1, which achieved editing efficiencies exceeding 65% across 12 human loci. Owing to its compact size, En-ISPmu1 was delivered together with a donor DNA in a single AAV vector, yielding up to 12.7% targeted integration in mouse liver. These findings establish En-ISPmu1 as a promising versatile platform for in vivo therapeutic genome editing applications.

bioengineering↗

bioRxiv2024

Rescue of lysosomal acid lipase deficiency in mice by rAAV8 liver gene transfer.

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive disorder caused by mutations in the LIPA gene, which results in lipid accumulation leading to multi-organ failure. If left untreated, the severe form of LAL-D results in premature death within the first year of life due to failure to thrive and hepatic insufficiency. Enzyme replacement therapy is the only available supportive treatment consisting in weekly systemic injections of recombinant LAL protein. Here, we characterized a novel Lipa-/- mouse model and developed a curative gene therapy treatment based on the in vivo administration of recombinant (r)AAV8 vector encoding the human LIPA transgene under the control of a hepatocyte-specific promoter. We defined the minimal rAAV8 dose required to rescue disease lethality and to correct cholesterol and triglyceride accumulation in multiple organs and blood. Finally, using liver transcriptomic and biochemical analysis, we showed mitochondrial impairment in Lipa-/- mice and its recovery by gene therapy. Overall, our in vivo gene therapy strategy achieves a stable long-term LAL expression sufficient to correct the disease phenotype in the Lipa-/-mouse model and offers a new therapeutic option for LAL-D patients. One Sentence SummaryWeve developed a liver-targeted gene therapy using recombinant AAV8 to effectively cure Lysosomal acid lipase deficiency by correcting lipid accumulation and by normalizing gene expression pattern and mitochondrial function in Lipa-/- mouse model.

molecular biology↗

bioRxiv2023

CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulation of utrophin (UTRN), a dystrophin paralogue, is a promising therapeutic avenue. Here, we present a CRISPR-Cas9-mediated strategy to increase utrophin expression by disrupting microRNA (miR) binding sites (BS). Using a Cas9/gRNA ribonucleoprotein (RNP) complex we disrupted several miR BS in DMD myoblasts and selected the Let-7c BS has crucial for UTRN repression. Interestingly, Cas9/gRNA indels were as efficient as the complete removal of Let-7c BS in upregulating UTRN expression, without any major off-targets. In three-dimensional human DMD cultures, Cas9/gRNA-mediated editing resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, Let-7c BS disruption in mdx animals by systemic rAAVs mediated delivery of Cas9 and gRNA resulted in utrophin upregulation and amelioration of the muscle histopathological phenotype. These findings provide the foundations for a universal (mutation-independent) gene editing therapeutic strategy for DMD. One Sentence SummaryCRISPR-Cas9 has the potential to upregulate utrophin to treat all DMD patients.

molecular biology↗