Brian A. Agricola

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) shortens the period of neutropenia after autologous bone marrow transplantation in a primate model.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on hematopoietic reconstitution after autologous bone marrow transplantation was evaluated in a primate model. Animals were given a continuous intravenous infusion of recombinant human GM-CSF for several days both before and after transplantation or only after the transplant procedure. Marrow ablation was accomplished by total body irradiation. In both groups of animals, the neutrophil count reached 1,000/mm3 by 8-9 d posttransplant compared with an interval of 17 and 24 d for two concurrent controls. After withdrawal of GM-CSF, neutrophil counts fell to values comparable to those observed in untreated controls. Accelerated recovery of platelet production was also observed in four of the five animals. Two additional animals were initially given GM-CSF several weeks posttransplantation because of inadequate engraftment. Prompt and sustained increases in neutrophil and platelet counts were observed. We conclude that GM-CSF may be useful in accelerating bone marrow reconstitution.

Marking and Gene Expression by a Lentivirus Vector in Transplanted Human and Nonhuman Primate CD34+Cells

ABSTRACT Recently, gene delivery vectors based on human immunodeficiency virus (HIV) have been developed as an alternative mode of gene delivery. These vectors have a number of advantages, particularly in regard to the ability to infect cells which are not actively dividing. However, the use of vectors based on human immunodeficiency virus raises a number of issues, not the least of which is safety; therefore, further characterization of marking and gene expression in different hematopoietic lineages in primate animal model systems is desirable. We use two animal model systems for gene therapy to test the efficiency of transduction and marking, as well as the safety of these vectors. The first utilizes the rhesus animal model for cytokine-mobilized autologous peripheral blood CD34+ cell transplantation. The second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal model useful specifically for human T-lymphoid progenitor cell reconstitution. In the rhesus macaques, detectable levels of vector were observed in granulocytes, lymphocytes, monocytes, and, in one animal with the highest levels of marking, erythrocytes and platelets. In transplanted SCID-hu mice, we directly compared marking and gene expression of the lentivirus vector and a murine leukemia virus-derived vector in thymocytes. Marking was observed at comparable levels, but the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in either animal model system. Thus, these results indicate that while lentivirus vectors have no apparent deleterious effects and may have advantages over murine retroviral vectors, further study of the requirements for optimal use are warranted.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

Lentivirus Vector-Mediated Hematopoietic Stem Cell Gene Transfer of Common Gamma-Chain Cytokine Receptor in Rhesus Macaques

ABSTRACT Nonhuman primate model systems of autologous CD34+ cell transplant are the most effective means to assess the safety and capabilities of lentivirus vectors. Toward this end, we tested the efficiency of marking, gene expression, and transplant of bone marrow and peripheral blood CD34+ cells using a self-inactivating lentivirus vector (CS-Rh-MLV-E) bearing an internal murine leukemia virus long terminal repeat derived from a murine retrovirus adapted to replicate in rhesus macaques. In vitro cytokine stimulation was not required to achieve efficient transduction of CD34+ cells resulting in marking and gene expression of the reporter gene encoding enhanced green fluorescent protein (EGFP) following transplant of the CD34+ cells. Monkeys transplanted with mobilized peripheral blood CD34+ cells resulted in EGFP expression in 1 to 10% of multilineage peripheral blood cells, including red blood cells and platelets, stable for 15 months to date. The relative level of gene expression utilizing this vector is 2- to 10-fold greater than that utilizing a non-self-inactivating lentivirus vector bearing the cytomegalovirus immediate-early promoter. In contrast, in animals transplanted with autologous bone marrow CD34+ cells, multilineage EGFP expression was evident initially but diminished over time. We further tested our lentivirus vector system by demonstrating gene transfer of the human common gamma-chain cytokine receptor gene (γc), deficient in X-linked SCID patients and recently successfully used to treat disease. Marking was 0.42 and .001 HIV-1 vector DNA copy per 100 cells in two animals. To date, all EGFP- and γc-transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.

Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20-60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency.

Direct comparison of RD114-pseudotyped versus amphotropic-pseudotyped retroviral vectors for transduction of rhesus macaque long-term repopulating cells

Recently, RD114 (feline endogenous retrovirus envelope protein)-pseudotyped retroviral particles have been shown to transduce human NOD/SCID repopulating cells efficiently. In this study, we compared directly transduction of repopulating cells with RD114-pseudotyped vector to that with standard amphotropic vector in the rhesus macaque model. G-CSF/SCF-mobilized CD34(+) rhesus peripheral blood cells were cultured in the presence of SCF, Flt-3 ligand, and MGDF on Retronectin-coated flasks. To assess directly the ability of the two pseudotypes to transduce primitive cells, both vectors were added simultaneously to the target cells every 24 h, for a total of four exposures in 96 h. The cells were reinfused after the animals received 1000 cGy total body irradiation. At the end of transduction, gene marking efficiency of CFU was higher with amphotropic LNL6 vector (mean 88.4%) vs RD114-G1Na vector (mean 18.5%). After long-term engraftment in three animals, total neo gene marking levels were 4-5% in PBMNCs and 1.5-4% in granulocytes. The RD114-G1Na marking levels were consistently higher in granulocytes than in mononuclear cells, while amphotropic LNL6 marking levels were higher in PBMNCs than in granulocytes. The differential gene marking patterns suggest that RD114 and amphotropic vectors may target distinct progenitor or stem cell populations. There was no clear advantage for RD114-pseudotyped vectors in this predictive preclinical model in terms of overall long-term marking levels; however, optimization of transduction conditions by increasing m.o.i. or inducing the receptor could potentially improve results with this novel vector system.