FANCC mutation correction using homology-independent targeted integration for gene therapy of Fanconi Anemia group C

项目总结

Project summary Fanconi Anemia (FA) is a devastating inherited disease associated with progressive bone marrow failure (BFM), congenital abnormalities, and cancer predisposition. FA patients harbor biallelic mutations in any one gene member of the FA pathway consisting of 22 genes. Most mutations happen in the FA core complex including the FA Complementation Group C (FANCC) gene. FANCC-mediated FA (group C) patients show typical clinical symptoms of FA. Currently, the treatment focuses on mitigating BMF, the leading cause of early mortality in pediatric patients, and secondary malignancies. Allogenic stem cell transplantation is the preferred therapy to treat BMF in patients with matched donors. However, transplanted patients show enhanced risk of graft-versus-host disease (GVHD) and secondary cancer. An alternative approach that overcomes the limitations of allogenic stem cell transplantation involve the gene therapy to correct mutations in patient stem cells, and then transplant back corrected stem cells into the patient. CRISPR/Cas9 is the state-of-the-art technology that allows modifying the genome seamlessly. Scientists have used this technology to precisely correct mutations in blood stem cells that can be applied for the treatment of genetic blood diseases (hemoglobinopathies and immunodeficient disorders). This approach depends on a pathway called homology-directed repair (HDR) that is only active in dividing cells. However, blood stem cells from FA patients are defective in cell growth due to sustained DNA damage. Thus, the efficiency of HDR approach might reach the therapeutic threshold for FA gene therapy. Here, we propose an alternative approach called homology-independent targeted integration (HITI) to introduce an intact DNA sequence encoding for functional FANCC gene into the endogenous FANCC promoter (a regulatory DNA sequence that controls expression of the FANCC gene). This system can be applied to correct all mutations occurring in all FA group C patients. The HITI approach is dependent on the DNA repair pathway called non-homologous end joining (NHEJ). Unlike HDR, NHEJ is highly active in all cells including slow/non-dividing cells. Thus, we expect that HITI-mediated gene correction will be efficient in FA patient derived stem cells. We have developed all necessary systems to validate the gene editing efficiency and functions of the edited cells both in vivo and in vitro. We also generated a surrogated model of FA by knockout of FANCC in human CD34+ cells. These cells show typical phenotypes of FA-HSPCs, thus providing a powerful model for optimizing our gene editing system. Of note, albeit low efficiency, the function of HDR-corrected mouse HSPCs was partially rescued in vitro. Thus, high editing efficiency using HITI will fully rescue functions of corrected stem cells. Our proposal will provide an improved approach to precisely correct patient FANCC mutations with high efficacy. Our long-term goal is to develop a comprehensive pre-clinical model for gene therapy of FA group C. Outcomes from this proposal will create a strong foundation for developing gene therapy to treat FA group C disease as well as understanding the disease mechanism.