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Julia C Morris

Publications and source records attributed to Julia C Morris.

8 recordsLinked to original sources
PubMed2006

Efficient transduction and engraftment of G-CSF-mobilized peripheral blood CD34+ cells in nonhuman primates using GALV-pseudotyped gammaretroviral vectors.

The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large-animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF); however, SCF is unavailable for clinical use in the United States and the European Union. A recent study in a competitive repopulation assay in the rhesus macaque showed very inefficient marking of G-CSF-mobilized (G/only) peripheral blood (G-PBSC) CD34(+) cells relative to G-CSF and SCF-mobilized cells using vectors with an amphotropic pseudotype. Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient transduction and engraftment of G-PBSC using Phoenix-GALV-pseudotyped vectors. We transplanted three baboons with G/only mobilized CD34(+) cells transduced with GALV-pseudotyped retroviral vectors. We observed high-level, persistent engraftment of gene-modified G-PBSC in all animals with gene marking levels in granulocytes up to 60%. We analyzed amphotropic (PIT2) and GALV (PIT1) receptor expression in G/only cells and found preferential expression of PIT1 after G/only, which may explain the inferior results with amphotropic pseudotypes. These findings demonstrate that high stem cell gene transfer levels can be achieved using G-CSF-mobilized PBSC with Phoenix-GALV-pseudotyped vectors.

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PubMed2004

Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia.

There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1-7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

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PubMed2003

Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning.

Lentiviral vectors are increasingly being used for transferring genes into hematopoietic stem cells (HSCs) due to their ability to transduce nondividing cells. Whereas results in in vitro studies and the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) model have been highly encouraging, studies in large animals have not confirmed the superior transduction of HSCs using lentiviral vectors versus oncoretroviral vectors. In contrast to the stable gene marking we have consistently achieved with oncoretroviral vectors in animals that received myeloablative conditioning, we observed the complete disappearance of genetically modified enhanced green or yellow fluorescent protein-expressing cells in 5 baboons that received transplants of HSCs transduced with lentiviral vectors alone or in combination with oncoretroviral vectors. Immune responses to transgene products have been found to be involved in the disappearance of gene-modified cells after nonmyeloablative conditioning. Thus, we examined whether the disappearance of gene-modified cells after ablative conditioning may be due to an immune response. In 4 of 5 animals, cytotoxic T lymphocytes specific for the transgene protein were readily detected, demonstrating that immune reactions were responsible for the disappearance of the gene-marked cells in the animals. In summary, we report the induction of transgene-specific immune responses after transplantation of lentivirally transduced repopulating cells in a myeloablative setting.

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PubMed2003

Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates.

The nonobese diabetic/severe combined immune-deficient (NOD/SCID) mouse xenotransplantation assay is the most commonly used surrogate assay for the study of human candidate stem cells. In contrast to large animal and human studies, however, it is limited by the short life span of the recipients, the limited proliferative demand placed on the transplanted cells, and the inability to support differentiation into all hematopoietic lineages. In the present study, we directly compared hematopoietic repopulation in NOD/SCID mice with autologous reconstitution in the baboon, a well-established preclinical large animal model for stem cell transplantation. Baboon CD34-enriched marrow cells were retrovirally marked and infused into the irradiated baboon and the NOD/SCID mice. Although the percentage of gene-marked cells was high and remained stable in NOD/SCID mice up to 12 weeks and in those that underwent secondary transplantation, we observed a considerable decline and overall a significantly (10-fold) lower percentage of gene-marked cells in the baboons. In addition, clonal integration site analysis revealed common proviral vector integrants in NOD/SCID repopulating cells and in the baboon at 6 weeks but not at 6 months after transplantation. These results suggest that distinct hematopoietic stem/progenitor cells are responsible for hematopoietic reconstitution in NOD/SCID mice compared with nonhuman primates.

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PubMed2002

Pharmacologically regulated in vivo selection in a large animal.

The inefficiency of gene transfer has greatly hindered gene therapy. In vivo selection may increase the frequency of genetically modified cells, thereby circumventing this critical limitation. Here we demonstrate regulated in vivo selection in a large animal. CD34(+) cells from 2 dogs were engineered to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Activation of the receptor through administration of a dimerizing drug, AP20187, produced reversible, drug-dependent rises in genetically modified red cells, white cells, and platelets in both animals, with minimal side effects. Cell growth switches could greatly enhance the efficacy and applicability of gene and cell therapy.

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PubMed2002

Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells.

Vector-containing medium harvested from murine packaging cell lines has been shown to contain factors that can negatively influence the transduction and maintenance of hematopoietic stem cells. Thus, we generated a human packaging cell line with a gibbon ape leukemia virus pseudotype (Phoenix-GALV), and we evaluated vectors produced by Phoenix-GALV for their ability to transduce hematopoietic progenitor/stem cells. In 3 baboons, we used a competitive repopulation assay to directly compare GALV-pseudotype retrovirus vectors produced by either Phoenix-GALV or by the NIH 3T3-derived packaging cell line, PG13. In 3 additional baboons we compared Phoenix-GALV-derived vectors to more recently developed lentiviral vectors. Gene transfer efficiency into hematopoietic repopulating cells was assessed by evaluating the number of genetically modified peripheral blood and marrow cells using flow cytometry and real-time polymerase chain reaction. Transduction efficiency of hematopoietic repopulating cells was significantly higher using the Phoenix-GALV-derived vector as compared with the PG13-derived vectors or lentiviral vectors, with stable transduction levels up to 25%. We followed 2 animals for more than one year. Flow cytometric analysis of hematopoietic subpopulations in these animals revealed transgene expression in CD13(+) granulocytes, CD20(+) B lymphocytes, CD3(+) T lymphocytes, CD61(+) platelets, as well as red blood cells, indicating multilineage engraftment of cells transduced by Phoenix-GALV-pseudotype vectors. In addition, transduction of human CD34(+) cells was significantly more efficient than transduction of baboon CD34(+) cells, suggesting that Phoenix-GALV-derived oncoretroviral vectors may be even more efficient in human stem cell gene therapy applications.

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