Basic Research on Hematopoietic Human Stem Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

This project is focused on the development of cell therapies, including stem cell therapies for the treatment of inherited primary immune deficiencies (PIDs). In the reporting period for this project we published manuscripts about our clinical studies relating to this project. To that end we reported on the efficacy and side effects of granulocyte transfusions in the clinic to manage severe infections in patients with chronic granulomatous disease (Annual Report reference: 8), demonstrating that granulocyte transfusions can assist to cure infections but are limited by the development of anti-HLA antibodies. We reported our experience in the use of non-myeloablative allogeneic hematopoietic stem cell (HSC) transplant to treat chronic granulomatous disease (CGD) (Ref. 11), demonstrating greater than 90% disease free survival even in the presence of life-threatening infections not responding to conventional therapy. We reported our outcome of clinical trial of lentivector gene targeting of autologous HSC to treat X-linked severe combined immune deficiency (XSCID) (Ref. 3), in which we report that the combined used of low dosing busulfan conditioining (6mg/kg total) together with lentivector transduction of autologous HSC can restore both cellular and humoral immunity. In the laboratory we contributed to our collaborators (Mort Cowan) use of HSC as the target cell for preclinical development of effective lentivector gene therapy for artemis SCID, and this will lead soon to a clinical trial for this disorder using the methods developed (Ref: 11). In previous years reporting we indicated our development of improved methods of developing induced pluripotent stem cells (iPSCs) from patients using peripheral blood, and employment of this model system to develop models of HSC and differentiated immune cells in culture, and the use of iPSC as the target for development of methods of gene editing to correct CGD. In the reporting period for this annual report we have used iPSC models to: Develop a new CRISPR based gene editing method to efficiently and seamlessly repair a specific mutation causing X-linked CGD (Ref. 1); Develop an efficient safe harbor gene insertion, gene editing approach to treat X-linked CGD (Ref. 2); Develop a gene editing approach to treat X-CGD involving insertion of a corrective cDNA for CYBB at the CYBB gene exon 2, which included work showing that retaining intron 1 regulatory elements was critical to the success of this approach (Ref: 14); Using the iPSC system in differentiation to HSC and then to mature neutrophils in culture to delinate the kinetics of key regulatory of hematopoiesis, including identifying key microRNAs that appear to regulate this process at different stages (Ref: 13); Demonstrating that gene editing correction of the constituitive deletion of the GT splice acceptor at Exon 2 of the pseudogenes of NCF1 can lead to resurrections of the function of these pseudogenes and correct the p47phox deficient autosomal recessive CGD oxidase activity (Ref: 8); with collaborators (John Park) developing for the first time efficient methods to differentiated iPSC to functional neural microglia (Ref: 9); with collaborators developing iPSC from Rhesus macaque non-human primates as safe harbor gene editing platform in this important animal model (Ref: 4). Also using gene editing of murine embryo at the mouse pluripotent stem cell level we developed a new animal model of X-linked CGD NSG mice that can accept human HSC xenografts and will be critical to development of new methods of gene therapy for CGD. (Ref: 12). We have been developing adenosine receptor 2A agonists for prevention and treatment of graft versus host disease because GvHD is a major undesired complication of allogeneic transplant for PIDs. We developed a new in vitro model system for testing the activity of new small molecule agonists of adenosine receptors (Ref: 6). We also demonstrated that the pregnancy related placenta derived immune suppressant PSG9 as a recombinant protein works by stimulating increase in FoxP3 positive regulatory T cells (Ref: 5), and in ongoing experiements in a in a mouse transplant GvHD system this agent can suppress GvHD through this mechanism.