Periodic Reporting for period 1 - TARGETNMDs (A novel and empowered TARGETed gene addition approach at a relevant microglia locus for the treatment of inherited NeuroMetabolic Diseases)
Período documentado: 2022-10-01 hasta 2025-03-31
Hematopoietic stem cell (HSC) gene therapy based on self-inactivating integrating vectors has proven unprecedented therapeutic potential in inherited neurometabolic diseases (NMDs). However, phenotypic effects are delayed after treatment due to the slow replacement of resident microglia by transplant-derived cells that hampers the broad application of this approach. Moreover, unregulated gene expression driven by the in-use promoters could in the long term cause unwanted effects. Finally, recent events suggest that the treated patients might be at risk of developing side effects related to vector integration. Therefore, novel strategies anticipating therapeutic benefit and reducing these potential safety concerns are desirable to address the still unmet medical need of NMD patients.
Our long-term goal is to develop a novel, broadly effective and safe treatment platform for NMDs based on a newly empowered HSC targeted gene addition approach at a newly identified microglia locus. Our central hypothesis is that correcting the gene defect by targeted addition at this locus in HSCs of patients affected by NMDs could generate in a timely manner a microglia-like progeny endowed with unprecedented therapeutic potential. Indeed, based on our recent findings, gene editing and targeted integration at this locus are expected to uniquely favor the timely engraftment and efficient, rapid myeloid/microglia differentiation of transplanted, edited HSCs in the recipients’ brain, and to induce robust and regulated expression of the integrated transcript in transplant-derived microglia-like cells. Based on this hypothesis, we aim at developing a targeted gene addition approach at the newly selected microglia locus for correcting the underlying genetic defect in HSCs and obtaining proof of concept of its therapeutic potential in NMDs animal models. Thus, the proposed work could generate the basis for a novel treatment platform for these devastating conditions.
Our long-term goal is to develop a novel, broadly effective and safe treatment platform for NMDs based on a newly empowered HSC targeted gene addition approach at a newly identified microglia locus. Our central hypothesis is that correcting the gene defect by targeted addition at this locus in HSCs of patients affected by NMDs could generate in a timely manner a microglia-like progeny endowed with unprecedented therapeutic potential. Indeed, based on our recent findings, gene editing and targeted integration at this locus are expected to uniquely favor the timely engraftment and efficient, rapid myeloid/microglia differentiation of transplanted, edited HSCs in the recipients’ brain, and to induce robust and regulated expression of the integrated transcript in transplant-derived microglia-like cells. Based on this hypothesis, we aim at developing a targeted gene addition approach at the newly selected microglia locus for correcting the underlying genetic defect in HSCs and obtaining proof of concept of its therapeutic potential in NMDs animal models. Thus, the proposed work could generate the basis for a novel treatment platform for these devastating conditions.
We showed that Cx3cr1 haplo-insufficient (Cx3cr1-/+) HSPCs are favored in generating microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1+/+) HSPCs upon transplantation in mice. Because of this evidence, we designed a CRISPR-based gene addition strategy at the human CX3CR1 locus that resulted in enhanced ability of the edited human HSPCs to engraft and repopulate the hematopoietic system and the central nervous system myeloid compartment of transplant recipients, in the latter with MLCs, and in lineage specific, regulated and robust transgene expression in their MLC progeny. We also demonstrated that this approach benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells.
By performing research as per project specific aims we demonstrated that i) haplo-insufficiency at the CX3CR1 locus of hematopoietic stem cells favors the appearance of microglia-like cells in the central nervous system of transplant recipients and ii) this phenotype could be reproduced by editing and targeted gene addition at the CX3CR1 locus of human HSPCs, while enabling a robust and regulated expression of the integrated cassette in transplant-derived cells. This evidence is of great translational relevance as it may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases, to patients who are already symptomatic at time of proposed treatment or are affected by rapidly progressive diseases. The findings and approach had been protected at an early stage by a dedicated patent application. At due time, plans for exploitation will be elaborated with appropriate stakeholders.