Aylin C. Bonifacino

Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates

RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34+ hematopoietic stem/progenitor cell transplant. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1.

Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize different CD34+ cell populations based on global gene and microRNA expression signatures

Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize peripheral blood stem cells by different mechanisms. A rhesus macaque model was used to compare plerixafor and G-CSF-mobilized CD34(+) cells. Three peripheral blood stem cell concentrates were collected from 3 macaques treated with G-CSF, plerixafor, or plerixafor plus G-CSF. CD34(+) cells were isolated by immunoselection and were analyzed by global gene and microRNA (miR) expression microarrays. Unsupervised hierarchical clustering of the gene expression data separated the CD34(+) cells into 3 groups based on mobilization regimen. Plerixafor-mobilized cells were enriched for B cells, T cells, and mast cell genes, and G-CSF-mobilized cells were enriched for neutrophils and mononuclear phagocyte genes. Genes up-regulated in plerixafor plus G-CSF-mobilized CD34(+) cells included many that were not up-regulated by either agent alone. Two hematopoietic progenitor cell miR, miR-10 and miR-126, and a dendritic cell miR, miR-155, were up-regulated in G-CSF-mobilized CD34(+) cells. A pre-B-cell acute lymphocytic leukemia miR, miR-143-3p, and a T-cell miR, miR-143-5p, were up-regulated in plerixafor plus G-CSF-mobilized cells. The composition of CD34(+) cells is dependent on the mobilization protocol. Plerixafor-mobilized CD34(+) cells include more B-, T-, and mast cell precursors, whereas G-CSF-mobilized cells have more neutrophil and mononuclear phagocyte precursors.

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.

Significant mobilization of both conventional and regulatory T cells with AMD3100

In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4(+)/CD25(high)/CD127(low)/FoxP3(+) Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8(+) T cells were mobilized to a greater extent than CD4(+) T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8(+) effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.

Dynamics of HSPC Repopulation in Nonhuman Primates Revealed by a Decade-Long Clonal-Tracking Study

In mice, clonal tracking of hematopoietic stem cells (HSCs) has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3-10 years), arising in sequential groups and likely representing self-renewing HSCs. The remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid, or both myeloid and lymphoid lineages. Over time, the 4%-10% of clones with robust dual lineage contribution predominated in repopulation. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically relevant model over a long time frame and provides a substantial system-level data set that is a reference point for future work.

Development of a Human Immunodeficiency Virus Type 1-Based Lentiviral Vector That Allows Efficient Transduction of both Human and Rhesus Blood Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5α and APOBEC3G, which target HIV-1 capsid and viral infectivity factor (Vif), respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV), including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (χHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34+ hematopoietic stem cells (HSCs), the χHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34+ HSCs, the χHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the χHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus, 15 to 30% versus 1 to 5%; second rhesus, 7 to 15% versus 0.5 to 2%, respectively) 3 to 7 months postinfusion. In summary, we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application.

Intracoronary infusion of autologous mononuclear cells from bone marrow or granulocyte colony-stimulating factor-mobilized apheresis product may not improve remodelling, contractile function, perfusion, or infarct size in a swine model of large myocardial infarction

AIMS In a blinded, placebo-controlled study, we investigated whether intracoronary infusion of autologous mononuclear cells from granulocyte colony-stimulating factor (G-CSF)-mobilized apheresis product or bone marrow (BM) improved sensitive outcome measures in a swine model of large myocardial infarction (MI). METHODS AND RESULTS Four days after left anterior descending (LAD) occlusion and reperfusion, cells from BM or apheresis product of saline- (placebo) or G-CSF-injected animals were infused into the LAD. Large infarcts were created: baseline ejection fraction (EF) by magnetic resonance imaging (MRI) of 35.3 +/- 8.5%, no difference between the placebo, G-CSF, and BM groups (P = 0.16 by ANOVA). At 6 weeks, EF fell to a similar degree in the placebo, G-CSF, and BM groups (-7.9 +/- 6.0, -8.5 +/- 8.8, and -10.9 +/- 7.6%, P = 0.78 by ANOVA). Left ventricular volumes and infarct size by MRI deteriorated similarly in all three groups. Quantitative positron emission tomography (PET) demonstrated significant decline in fluorodeoxyglucose uptake rate in the LAD territory at follow-up, with no histological, angiographic, or PET perfusion evidence of functional neovascularization. Immunofluorescence failed to demonstrate transdifferentiation of infused cells. CONCLUSION Intracoronary infusion of mononuclear cells from either BM or G-CSF-mobilized apheresis product may not improve or limit deterioration in systolic function, adverse ventricular remodelling, infarct size, or perfusion in a swine model of large MI.

Accelerated lymphocyte reconstitution and long-term recovery after transplantation of lentiviral-transduced rhesus CD34+ cells mobilized by G-CSF and plerixafor.

OBJECTIVE Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor produces significant mobilization of CD34(+) cells in rhesus macaques. We sought to evaluate whether these CD34(+) cells can stably reconstitute blood cells with lentiviral gene marking. MATERIALS AND METHODS We performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34(+) cells transduced with a lentiviral vector, and these data were compared with those of G-CSF and stem cell factor mobilization. RESULTS G-CSF and plerixafor mobilization resulted in CD34(+) cell yields that were twofold higher than yields with G-CSF and stem cell factor. CD123 (interleukin-3 receptor) expression was greater in G-CSF and plerixafor-mobilized CD34(+) cells when compared to G-CSF alone. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages, similar to animals who received G-CSF and stem cell factor-mobilized grafts. Lymphocyte engraftment was accelerated in animals receiving the G-CSF and plerixafor-mobilized CD34(+) cells. One animal in the G-CSF and plerixafor group developed cold agglutinin-associated skin rash during the first 3 months of rapid lymphocyte recovery. One year after transplantation, all animals had 2% to 10% transgene expression in all blood cell lineages. CONCLUSIONS G-CSF and plerixafor-mobilized CD34(+) cells accelerate lymphocyte engraftment and contain hematopoietic stem cell capable of reconstituting multilineage blood cells. These findings indicate important differences to consider in plerixafor-based hematopoietic stem cell mobilization protocols in rhesus macaques.

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.

High-efficiency Transduction of Rhesus Hematopoietic Repopulating Cells by a Modified HIV1-based Lentiviral Vector

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.