This project is focused on the development of cell therapies, including stem cell therapies for the treatment of inherited primary immune deficiencies (PIDs) and primary immune regulatory disorders (PIRDs). The studies use primary human immune cells, hematopoietic stem cells, induced pluripotent stem cells (iPS cells), mouse and non-human primate models, gene editing, and clinical trials. We reported on side effects of granulocyte transfusions to manage severe infections in patients with chronic granulomatous disease, CGD (Ref #8), demonstrating these transfusions can cure infections but patients develop anti-HLA antibodies. To avoid anti-HLA antibody formation from granulocyte transfusions, we have developed a new method to correct the oxidase defect in patient granulocytes using mRNA. There is a new patent pending for this treatment (Ref #3 ). We used non-myeloablative allogeneic hematopoietic stem cell (HSC) transplant to treat chronic granulomatous disease (CGD) (Ref #13), demonstrating greater than 90% disease free survival even in presence of life-threatening infections. In the laboratory we contributed to preclinical development of effective lentivector gene therapy for artemis SCID (Ref #14). We developed a new CRISPR based gene editing method to efficiently and seamlessly repair in blood stem cells a specific X-CGD mutation causing X-linked CGD (Ref #2). We edited human patient iPS cells to treat X-CGD involving insertion of a corrective cDNA for CYBB at the CYBB gene exon 2, while showing that retaining intron 1 regulatory elements was critical to correction (Ref #18). We showed that editing correction of the constitutive deletion of the GT at Exon 2 of pseudogenes of NCF1 can correct p47phox CGD iPS cells (Ref #11). With John Parks lab, we contributed to developing methods to differentiated iPSC to functional neural microglia (Ref #12). With the Dunbar lab we contributed to achieving editing of iPSC from Rhesus macaque non-human primates (Ref #5). We developed a new mouse model of X-linked CGD NSG mice that can accept human HSC xenografts (Ref #17). For our work to develop adenosine receptor 2A agonists for prevention and treatment of graft versus host disease, we developed a new in vitro model for testing agonists of adenosine receptors (Ref #6). Also relating to work on treatments for GvHD, we worked with the Dvesker lab to assess pregnancy related placenta derived immune suppressants for inducing immune tolerance resulting in a new patent (Ref #4). A major new initiative in the laboratory has been to develop a novel method of achieving bone marrow conditioning using chimeric T cell receptor (CAR) incorporating antibody components that target cKit (CD117) a major marker of hematopoietic stem cells. This is the major scientific accomplishment reported for this annual report and detailed description of the method and its impact is included in that section of this report (Ref #1). We also continue to be major contributors to the clinical trials assessing the use of plerixafor (Mozobil; CXCR4 antagonist) to treat patients with the WHIM syndrome immunodeficiency defect in collaboration with the Philip Murphy laboratory conducting the studies (NIH Clinical Trial 14-I-0185; ClinicalTrials.gov NCT02231879; NIH Clinical Trial 09-I-0200; ClinicalTrials.gov NCT00967785). Other accomplishments related to this project are outlined in the brief comments below related to each reference listed. Accomplishments by publication and patent in 2017-2018 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 et al: Developed novel method of gene editing repair of X-CGD causing mutation in patient blood stem cells using the CRISPR/Cas 9 editing. 3. 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. 4. Dveksler and Malech: New patent from Dveksler lab using pregnancy specific glycoproteins to induce immune tolerance. 5. Hong et al: Collaboration with Dunbar lab to gene edit Macaque non-human primate iPS cells to target a safe harbor site to express therapeutic proteins. 6. Jones et al: Generation of novel cell model system to assess Agonists of the Adenosine 2a receptor for development of treatments to induce tolerance for prevention/treatment of graft versus host disease. 7. Keller, Notarangelo and Malech: Review of current management of chronic granulomatous disease and development of gene therapy. 8. Marciano et al: Experience in the use of donor granulocyte transfusions for infection management in CGD; providing tbackground rationale for development of patent indicated in Ref #3. 9. Marciano et al: Correlates percent oxidase positive neutrophils in x-linked carriers of CGD with infection susceptibility providing necessary information to inform gene therapy targets. 10. Margolis et al: Provides important information regarding management of CGD patient care in the transition from childhood to adulthood. 11. Merling et al: Shows that gene editing correction of the GT deletion in the pseudogenes (NCF1B and 1C) of p47phox gene (NCF1) can resurrect full function and p47phox protein production fix oxidase defect in the neutrophils derived from corrected iPS cells, providing rational to develop mutation specific gene editing treatment. 12. Pandya et al: 1st demonstration of microglial cells generated from iPS cells, providing model for this cell type that could include gene editing. 13. Parta et al: Largest single center cohort of approach to allogeneic transplant for CGD demonstrating most importantly that transplant can cure CGD patients who have persistent life-threatening fungal infections. 14. Punwani et al: 1st demonstration of an effective lentivector to treat ARTEMIS SCID; vector now been brought forward to a clinical trial in the Cowan lab. 15. Sowriraian et al: Demonstrates p47phox phagocyte oxidase component regulates dendritic cell differentiation from monocyte derived macrophages. 16. Straughan et al: Current update of management of liver abscesses occurring in CGD patients. 17. Sweeney et al: CRISPR/Cas9 generation of the CGD genotype/phenotype in the nsg mouse that can accept human marrow xenografts, as a new important model for testing gene therapy. 18. Sweeney et al: 1st demonstration in human CGD patient derived iPS cell model that retention of the first intron of CYBB is essential for efficient cDNA mediated expression for gene editing correction. 19. Van de Geer at al: Contributed to collaborators demonstrating range of phenotypes in a large cohort of patients with p40phox deficient CGD. 20. Wingfield et al: Report from our surgical collaborators of largest series of neck dissection treatment experience for severe neck infections in CGD.
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