This project studies development of gene therapy vectors tested in their targeting of a variety of cultured cells and in animal models, with the overall goal of better targeting gene transfer into human peripheral blood hematopoietic progenitors (PBHP) as a target for clinical gene therapy to treat inherited immune deficiencies. The specific goals relating to gene therapy were to develop the pre-clinical systems of gene therapy that could then be applied to correct the genetic defect in the X-linked genetic form of chronic granulomatous disease (CGD), the p47phox deficient autosomal recessive form of CGD and the X-linked form of severe combined immune deficiency (XSCID). Also in collaboration with Dennis Hickstein of the NCI we are also collaborating on studies to develop gene therapy for leukocyte adhesion deficiency (LAD). Specifically, we developed a retrovirus vector producer cell line that secretes high titers of the MFGS vectors containing the gp91phox cDNA and recently developed new methods to concentrate that virus to high titer that we plan in the future to use in the clinic. In collaboration with our collaborators at St. Jude Children's Research Hospitalwe have developed high titer Human Immunodeficiency Virus (HIV) self-inactivating mammalian internal promotor insulated lentivectors to correct the X-linked form of CGD and similar vectors to correct X-SCID. We are also in the process of developing similar lentivector to treat p47phox AR-CGD. Our collaborators at Indiana Universtiy are also testing our new lentivector for treatment of X-CGD. We also prior to that developed novel lentivirus vectors to correct X-CGD or X-SCID based on the Simian Immunodeficiency Virus of the macaque type (SIV-mac) that very efficiently targets human, non-human primate and dog hematopoietic stem cells. We have conducted and are in the process of conducting large animal studies with these gamma retrovirus and lentivirus vectors. The NOD/SCID immunodeficient mouse will accept grafts of human hematopoietic stem cells. Using the NOD/SCID mouse/human stem cell chimera we demonstrate the full functional correction of 10-20% of human neutrophils arising in this model from the mobilized peripheral blood stem cells of CGD patients transduced with SIV-gp91phox vector. This unprecedented level of gene correction in this model provides the basis for considering lentivector (probably the new higher titer HIV X-CGD lentivector) in a future clinical gene therapy trials for CGD. This X-CGD lentivirus vector has been shown capable of achieving full oxidase correction of human neutrophils that arise from gene transduced X-CGD patient CD34 hematopoietic stem cells both in vitro and in the NOD/SCID mouse xenograft system. We have also developed RD114 pseudotyped SIV-common gamma chain (gc) vectors to treat XSCID. We also collaborated with Dr. Felsburg from the University of Pennsylvania who has a dog model of XSCID in which we have tested the ability of both our MFGS-gc, SIV-gc and the clinical candidate HIV lentivector vector to cure this disorder with in vivo gene therapy in this animal model. The clinical lentivirus vector is based on the self-inactivating CL20 construction backbone devised by the St. Jude group, and into which we inserted the short version of the human Elongation factor-1 alpha (EF-1a) as the internal promotor plus the 400 bp version of the chicken beta globin insulator to further increase safety of the vector by reducing vector insertion mediated activation of nearby genes. In vitro we have achieved levels of over 80% marking of dog stem cells using these vector. Specifically, we have used a novel approach for in vivo gene therapy in the XSCID dogs where 3 day old dogs are injected intravenously with corrected gene therapy vector. Using this method, a number of dog treated with either the gamma retrovirus-gc, SIV-gc vectors or the clinical candidate HIV lentivector-cg have safely achieved long term high level restoration of the immune system. This is a novel and unprecedented demonstration of the feasibility of in vivo gene correction using direct injection of gene therapy vector, and validates the efficacy of the clinical lentivector we plan to bring to clinical trial within the year. In other studies we are examining the role of different growth factors in stimulating CD34+ stem cells to divide and to determine the relationship between entry into the cell cycle, ability to transduce with retrovirus vectors, and the maintenance or loss of long term engrafting potential. These studies are essential to achieving our goal of high levels of gene transfer into long term engrafting stem cell. In other studies we have studied the effects of low dose radiation or chemotherapy on the engraftment of stem cells in animal models. We have demonstrated high levels of engraftment of gene marked cells in mouse and non-human primate animal models using low intensity conditioning regimens consisting of non-ablative levels of total body radiation. Follow up studies are in progress looking at non-ablative chemotherapy regimens instead of using radiation, and preliminary studies suggested that the non-ablative combination of cyclophosphamide and fludarabine can achieve low level (0.3%) prolonged (greater than 1 year)gene transfer marking of blood cells in the primate model. Evidence from human and animal studies of gene therapy suggest that providing an in vivo growth or survival advantage to genetically corrected blood cells can improve the outcome of gene therapy by increasing the percent of corrected cells in the body. One approach to this is to co-express the therapeutic gene (such as the corrective gene for X linked CGD) with a gene that allows for selective enrichment. In studies with collaborators we have explored the use of the methyguanine methyl transferase (MGMT) which protects against alykating agents such as BCNU in a non-human primate model achieving marking rates of up to 20%. Finally, in a separate project we have established induced pleuripotent stem cells (iPS cells) from X-CGD patients. These iPS cells have primitive embryonic stem cell properties and will be used to explore gene repair methods of correcting the CGD gene defect in these cells.
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