Biology subjects

Oustelandt, J.

Publications and source records attributed to Oustelandt, J..

2 recordsLinked to original sources

bioRxiv2026

CRISPR-Mediated Linearization of IDLV Donor Enables Precise Targeted Integration in Human Hematopoietic Stem Cells

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.

molecular biology↗

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↗