This project is focused on developing curative gene therapies for primary immune deficiencies (PIDs) and primary immune regulatory disorders (PIRDs). The project also must include clinical studies to understand the basic physiology, genetic defect, clinical problems and management issues affecting the patient groups for which we are developing gene therapies. We use a variety of cell lines, primary patient cells, and animal models to develop these gene treatments; and a variety of tools including integrating and non-integrating gene transfer vectors, as well as gene editing reagents/approaches; as well as methods to transiently correct function in mature immune cells by transfection with mRNA. To these ends, we developed and studied integrating self-inactivating lentivectors and are using them in ongoing clinical trials that are open in our program at the clinical center to treat X-linked chronic granulomatous disease ( NIH Protocol #15-I-0008; ClinicalTrials.gov NCT02234934; 4 patients treated at NIH, 12 patients at all the centers including the United Kingdom- ClinicalTrials.gov NCT01855685; Also see Abstract report of these 9 patients: Kohn DB, et al. 2018 Mol Ther 26(Suppl 1):157) and older children and young adults with X-linked severe combined immune deficiency (NIH Protocol #11-I-0007; ClinicalTrials.gov NCT01306019; 13 patients treated at NIH with early report on the first 5 patients, De Ravin et al. Sci. Transl. Med. 2016 8:335ra57) as well as contributing scientifically to the conduct of an ongoing clinical trial of lentivector gene therapy for newborn infants treated at St. Jude Childrens Research Hospital (ClinicalTrials.gov NCT01512888; report of 8 patients.Ref #9). We developed improved methods of gene editing to correct PIDs in cellular models of gene correction/mutation repair of primary immune deficiencies (Refs #s 4,12,13,16); developed and studied stem cell models that include both primary CD34+ hematopoietic stem cells (HSCs) obtained from patients and healthy volunteers or iPSC developed from patients and healthy volunteers and from animal models that may be used as targets for gene therapy (Refs #s 2,12,13,16); and published reviews of gene therapy (Refs #: 7,14); developed a new method of marrow conditioning using CAR-T cells targeting the cKIT surface receptor on hematopoietic stem cells in a mouse model (Ref #: 1); developed a novel method to correct the oxidase activity in CGD neutrophils by electoporating oxidase subunit mRNA (See new patent in Ref #: 2). We developed a new methods of treating/preventing graft versus host disease based on an immunosuppressive pregnancy related protein and applied for a patent (See Refs #s: 3,6). A very important element of developing gene and cell therapies is to understand standard diagnostic, genetic and therapeutic features of the primary immune deficiencies for which we are developing gene therapies (See Refs #s: 5,8,10,11,15,17,18). Accomplishments by publication and patent in 2018-2019 period Reference #: 1.Arai et al: Developed novel method of bone marrow conditioning using anti-cKit Chimeric Antigen Receptor T cells introduced by retrovirus vector in a mouse model achieving high efficiency of donor engraftment. 2.De Ravin and Malech: New patent from Malech lab using mRNA to correct oxidase defect in CGD patent neutrophils that they may be used as treatment for infections. 3.Dveksler and Malech: New patent from Dveksler lab using pregnancy specific glycoproteins to induce immune tolerance to prevent graft versus host disease in a mouse tissue type mismatch transplant model. 4.Espinoza et al: Reported markedly abnormal dysplastic clonal hematopoiesis in a rhesus macaque transplanted with hematopoietic stem cells that were transduced with a lentivector containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the long terminal repeat, suggesting that that strong constitutive promoters should not be included in lentivectors. 5.Haddad et al: Prospective review of outcomes of transplant strategies for newly diagnoses patients with severe combined immune deficiency as part of the Primary Immune Deficiency Treatment Consortium projects. 6.Jones et al: Demonstration of a novel pregnancy related immunosuppressive protein used as a treatment to prevent graft versus host disease in a mouse model of transplant. 7.Keller et al: Provides a timely review of current management of chronic granulomatous disease, the potential of allogeneic hematopoietic stem cell transplant to cure CGD, and the emerging potential of gene therapy to provide clinically beneficial treatments to patients lacking suitable donors for transplant. 8.Kuhns et al: First pcr based genetic analysis of the NCF1 gene and its pseudogenes NCF1B and NCF1C quantifying the exon 2 GT deletion used as diagnostic tool to diagnose patients with the p47phox deficient autosomal recessive form of CGD; demonstration that there is extraordinarily high rates of recombination related loss or gain of pseudogene or gene alleles; and demonstration that carriers of p47phox deficient AR CGD have less than normal amounts of p47phox protein in their myeloid cells. 9.Mamcarz et al: Reported first in human restoration of both cellular and humoral immunity in 8 infants newly diagnosed with X-linked severe combined immune deficiency treated with lentivector transduced autologous stem cells following low dose targeted busulfan myeloid conditioning. 10.Marciano et al: Correlates percent oxidase positive neutrophils in x-linked carriers of CGD with infection susceptibility providing necessary information to inform gene therapy targeting indicating that there is significant CGD type infection risk at <10% oxidase normal neutrophils and normal resistance to infection at >20% oxidase normal neutrophils. 11.Marsh et al: First demonstration in a multicenter retrospective analysis of bone marrow transplant in patients with chronic granulomatous disease and significant CGD associated inflammatory disease that following successful transplant there is full resolution of inflammatory bowel disease by 1 year post transplant. 12.Pavel-Dinu et al: First demonstration of gene editing repair of function in situ at the IL2RG gene to achieve correction of the genetic defect causing X-linked severe combined immune deficiency in patient hematopoietic stem cells ex vivo (plus associated erratum correction). 13.Pavel-Dinu et al: Author added associated erratum correction for #13 above. 14.Salzman et al: Review and position white paper assessing the value of gene therapy to transform medicine in the form of one treatment for potentially life long clinical improvement or cure for both inherited and acquired disorders. 15.Straughan et al: Describes the pattern of presentation of liver abscess in patients with chronic granulomatous disease and improved methods of diagnosis and management of liver abscess. 16.Sweeney et al: A review of current methods of gene editing as applied to the correction of the genetic defects responsible for the various genetic forms of chronic granulomatous disease. 17.van de Geer et al: First report that the p40phox deficient form of autosomal recessive CGD affects may more patients and kindreds than initially appreciated and that the pattern of presentation is almost exclusively that of severe inflammatory bowel disease presenting early in childhood without significant history of CGD related infections in these patients. Cell functional diagnosis requires that oxidase activity be tested using a particulate stimulus such as opsonized zymosan as the standard test with PMA stimulation does not detect the functional defect in patient neutrophils. 18.Wingfield et al: First extensive description of outcomes of surgical neck dissection to treat severe cervical lymphadenitis in 9 patients with CGD.
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