This project is focused on developing curative gene therapies for primary immune deficiencies (PIDs). We also must understand the basic defects and clinical problems affecting the patient groups for which we are developing gene therapy. To that end we developed and studied integrating and non-integrating lentivectors (Annual report references: 3,18); developed and studied gene editing tools such as CRISP/Cas9 (CRISPR), Zinc Finger Nucleases (ZFNs), and TALENs used to correct PIDs in cellular models of gene correction/mutation repair of primary immune deficiencies (Refs: 1,2,5,15,20,22); 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 as targets for gene therapy (Refs: 1,2,5,15,16,18,20,21,22); studied immune system function and regulators (Refs: 6,7,8,12,16,19,23,); assessed the outcomes of allogeneic transplants and other cellular therapies given to patients with PIDs that serve as important guides to understand the target goals of gene therapy (Refs: 13,17); conducted studies of carrier females of X-linked PIDs, where such studies may also inform the target goals for gene therapy (Ref: 14); and conducted observational and standard of care studies of patients with PIDs of special interest, which for this project, in particular involves patients with the various genetic forms of chronic granulomatous disease (CGD) (Refs: 4,6,9,24) and the older children and young adults with X-linked severe combined immune deficiency (XSCID) (Ref: 3). We also conducted observational studies with our collaborators of patients with Wiskott-Aldrich Syndrome, patients with adenosine deaminase deficiency who have previously been treated with gene therapy, patients with WHIM syndrome (Warts, hypogammaglobulinemia, infections and myelokathexis) (Ref: 11) and other patients with rare forms of PIDs potentially treatable with gene therapy (Refs: 10,23). We list 24 publications from 2016 and 2017 in the reference section of this Annual Report that relate to aspects of this project outlined above. The importance and impact of the some of the most important of these particularly related to development and clinical application of gene therapy/gene editing are outlined in more detail below: 1. Reference #3: In this publication we report that in 5 children and young adults with XSCID that gene therapy with lentivector transduced autologous CD34 HSC following non-ablative busulfan conditioning can restore T cell, NK cell and humoral (B cell) immunity, leading to significant improvement of clinical status. We have treated 3 additional patients with similar encouraging clinically beneficial outcomes from this gene therapy. 2. Reference #2: We have developed a novel method of gene editing using zinc finger nuclease mRNA targeting the AAVS1 safe harbor genomic site to achieve >15% correction of the X-linked form of CGD in patient CD34+ hematopoietic stem cells by electroporation (MaxCyte instrument) together with delivery of a donor plasmid in an AAV6 vector. This demonstrates important proof of principle for safe harbor approaches to gene editing correction of monogenic disorders. 3. Reference #1: We demonstrate for the first time a CRISPR/Cas9 mutation repair approach for a mutation of the CYBB gene at the start of exon 7 responsible for 6% of cases of X-CGD, achieving unprecedented levels of correction (>20%) of human patient CD34+ HSC engrafting as human zenograft in the NSG mouse model. This leads the way to possible clinical application of this gene editing technology for mutation repair gene therapy of a monogenic disorder. 4. Reference #5: We provided major contribution to studies demonstrating the utility of using iPSC developed from rhesus macaque (non-human primate) as a model system gene editing platform. This system will help advance gene editing technologies by providing a non-human primate model to assess safety and efficacy. 5. Reference #15: We show for the first time using ZFN gene editing of iPSC from patients with the p47phox autosomal recessive form of CGD that correction of the GT deletion at the start of exon 2 of NCF1 (responsible for >90% of mutant alleles causing this form of CGD), can be functionally corrected by repair of not only the NCF1 gene, but by repair of this same mutation found in the two pseudogenes (NCF1B and NCF1C). This is a first demonstration of correction of a monogenic disorder by gene editing resurrection of function in a non-functional pseudogene. 6. Reference #18: We provided major contribution to studies that developed a lentivector capable of efficient correction of ARTEMIS deficient SCID. These studies provide the pre-clinical data needed for our collaborators to prepare an FDA IND and clinical protocol to use this vector to treat infants and children with ARTEMIS deficient SCID. 7. Reference #20: We used CRISPR/Cas9 to knock out the CYBB gene function (X-CGD) in the NSG mouse model (capable of hosting human bone marrow xenograft). This added the X-CGD genotype to this genetically complex model that will allow improved evaluation of gene therapy/gene editing of human X-CGD patient CD34+ HSC in the mouse xenograft model. 8. Reference 22: Using human X-CGD patient iPSCs to assess gene editing cDNA insertion into the CYBB gene to correct CGD, we found that addition of the gp91phox cDNA to the 1st exon worked very poorly with respect to expression of gp91phox, while addition to exon 2 provided excellent corrective production of gp91phox, demonstrating that there are critical control elements in intron 1 necessary to efficient production of gp91phox. This indicates the importance of knowing about intron control elements when planning a gene editing strategy for correction of monogenic disorders. 9. Not yet published is that we have opened here at the NIH a new clinical trial of lentivector gene therapy for X-chronic granulomatous disease as part of a multicenter study, Three patients have now been treated by us and two by our collaborators at Boston Childrens Hospital and at UCLA Childrens Hospital. Early results are very promising showing 20-45% of circulating neutrophils having restoration of normal oxidase activity, and this has been associated with clinical benefit in the control of ongoing infection present at the time of gene therapy.
Showing the most recent 10 out of 74 publications
