FANCC mutation correction using homology-independent targeted integration for gene therapy of Fanconi Anemia group C
FANCC mutation correction using homology-independent targeted integration for gene therapy of Fanconi Anemia group C
批准号:
10653342
负责人:
Ngoc Tung Tran
金额:
$19.81万
依托单位国家:
美国
项目类别:
财政年份:
2023
资助国家:
美国
项目状态:
未结题
起止时间:
2023-07-15 至 2025-06-30
关键词:
AllelesAllogenicApoptosisAutologousBackBiological AssayBone marrow failureCD34 geneCRISPR correctionCRISPR/Cas technologyCell CycleCellsClinicalCodeComplementComplementary DNAComplexCongenital AbnormalityDNA DamageDNA IntegrationDNA RepairDNA Repair PathwayDNA SequenceDNA biosynthesisDataDefectDevelopmentDiseaseEngraftmentFaceFanconi Anemia pathwayFanconi&aposs AnemiaFoundationsFutureGenesGeneticGenomeGoalsHematological DiseaseHematopoietic Stem Cell TransplantationHematopoietic stem cellsHemoglobinopathiesHereditary DiseaseHumanHuman Cell LineImmunofluorescence ImmunologicImmunologic Deficiency SyndromesImpairmentIn VitroInterphase CellKnock-outLentivirusMediatingMendelian disorderMitomycin CModelingMusMutateMutationNonhomologous DNA End JoiningOutcomeOutcome StudyPathway interactionsPatientsPhenotypePre-Clinical ModelProliferatingProteinsProtocols documentationRegimenRiskSafetyScientistSecond Primary CancersStainsStem cell transplantSubgroupSymptomsSystemSystems IntegrationTechnologyTestingTherapeuticTimeTransplant RecipientsTransplantationVirus IntegrationWestern BlottingXenograft Modelarmcancer predispositioncell growthcell typeclinical applicationconditioningcongenital bone marrow failuredesignfunctional restorationgene correctiongene functiongene therapygraft vs host diseasehumanized mouseimprovedin vivoinsertion/deletion mutationinterestmembermortalitymutation correctionpediatric patientspromoterrepairedstem cellssuccesstransgene expressiontumor
中文摘要
项目总结
英文摘要
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.
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