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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.

Aging of the hematopoietic system is associated with a number of observations, including diminished regenerative potential, skewed lineage differentiation, increased incidence of anemia, and higher rates of neoplastic transformation. Despite advanced age being a strong poor prognostic factor, an increasing number of older patients are receiving hematopoietic stem and progenitor cell (HSPC) transplantation. Most previous investigations of the effects of aging on hematopoiesis have been obtained in murine models. The rhesus macaque is a powerful model to study human hematopoiesis and aging, based on a close phylogenetic relationship to humans, and similar telomere lengths, lifespans and aging phenotypes. To quantitatively elucidate the age-related changes that compromise hematopoietic function at a clonal level, we applied a genetic barcoding approach to quantitatively track the clonal behavior of HSPCs in young versus old macaques following autologous transplantation. We delivered high diversity barcodes via lentiviral transduction of CD34+ HSPC (detailed in our prior study: Wu et al Cell Stem Cell, 2014), allowing quantitative tracking of the output of thousands of individual HSPC clones labeled by unique barcodes, over time and in various lineages post-transplantation. We successfully transplanted barcoded HSPCs into 2 macaques aged 19 and 25 years, constituting "old" macaques based on an average lifespan in captivity of 20-30 years, and compared results to clonal patterns observed in 5 "young" macaques aged 3-5 years. Both old macaques engrafted promptly, and CD3+ T cells, CD3-CD20+ B cells, CD33+ Granulocytes (Gr), CD3-CD20-CD14+ Monocytes (Mo), and CD3-CD20-CD14-CD16+/or CD56+ NK cells were purified from the peripheral blood monthly following transplantation. In terms of overall polyclonality and diversity (Shannon index), analyzed through 4 months to date, there were no marked differences between the old and young recipients, with thousands of individual clones contributing to hematopoiesis in both sets of animals during the initial post-transplant time period studied. However, there were marked differences in the patterns of clonal lineage relationships between young and old animals, as assessed via pairwise Pearson correlations of all contributing clones as well as clustering algorithms allowing interrogation of patterns of clonal behavior. In both young and old, there was no correlation (i.e. no shared clones and thus no shared progenitors) between lineages at 1m, and clones contributing at 1m did not contribute to any lineage at 3m or later, indicating the existence of short-term, lineage-restricted progenitors in both age groups. By 3m in young animals, B and Gr/Mo became correlated, and by 3-6m, B/T/Mo/Gr multilineage clones appeared and constituted the majority of hematopoietic output. However, in old animals clones contributing to Gr/Mo versus B or T lineages remained almost completely distinct or markedly biased, up to 4m studied to date, without evidence for multi-lineage clones (Fig 1). In both young and old animals, the NK lineage remained clonally distinct, as previously reported for young animals. In summary, we transplanted 2 aged macaques with barcoded CD34+ HSPCs, and discovered a pattern of clonal reconstitution distinct from that in young animals, with persistent unilineage or highly-biased myeloid and B lymphoid progenitors in the aged animals. Longer follow-up will be required to determine if this biased pattern persists, and will be presented. This approach should improve our understanding of disorders of hematopoiesis in the elderly, and help improve transplantation and other therapies in this vulnerable patient population. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Dendritic cells (DCs) are effective in stimulating and controlling the outcome of T cell responses. Human immunodeficiency virus type 1-based lentiviral vectors can achieve sustained transduction of genes/antigens in dividing and nondividing cells, thus representing a candidate vector for stable expression of antigens in DCs. We previously established conditions for transduction of purified cytokine mobilized rhesus CD34(+) cells in vitro, and transplantation of the autologous transduced cells in a nonhuman primate model in vivo. In the present study, we transplanted DCs derived from EGFP-transduced CD34(+) cells into nonmyeloablated rhesus macaques. Transplantation of DCs stably expressing EGFP into autologous animals induces persistent, long-lived (up to 100 weeks) EGFP-specific T cell responses. Of note, no humoral responses against EGFP are detected in the transplanted animals. These studies provide, to our knowledge, the first demonstration that lentiviral transduction of CD34(+) progenitor cells subsequently differentiated to DCs is capable of priming a specific T cell response in a nonhuman primate in vivo. Taken together, our data provide formal in vivo evidence that lentivirus-transduced dendritic cells represent a potential approach in eliciting cellular immune responses in primates.

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.

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.

It has not been possible to evaluate immune recovery in tissues without invasive biopsy. Investigators have had to rely on peripheral blood lymphocytes (PBLs) for analysis. PBLs, however, represent only a very small percentage of cells in lymphoid tissues (LTs). Using a combination of SPECTanda radiotracer, rhesus IgG1 anti-CD4R1 Tc-99m(Fab')2,we sequentially imaged CD4+ cell recovery in rhesus macaques following varying doses of total body irradiation (TBI) and reinfusion of vector transduced, autologous CD34+ cells. Seven rhesus were used in this study. Two received a dose of 3Gy on two sequential days (3Gyx2days) of TBI (6Gy total), three received 4Gyx2days TBI, and two 5Gyx2days TBI. CD34+ cells were immunoselected from a leukapheresis product following G-CSF and SCF mobilization over 5 days. On the last day of TBI a mean (SD) of 5.9(1.7)x106/kg autologous CD34+ cells were reinfused after being transduced once (moi=50) with a SIV/HIV chimeric lentiviral vector expressing EGFP. SPECT/CT images were taken prior to transplant (Figure 1) and at approximately 6, 30, 90, 150, 260 and 380 days post-TBI. Development of immunogenicity to the radiotracer was monitored using size exclusion HPLC and an in vitro cell binding assay. Following 3Gyx2days TBI, both animals rejected their CD34+ cell graft despite achieving a PBL nadir of Unlike the 3Gyx2days TBI animals, no immunogenicity to the radiotracer was detected in the 4Gyx2days TBI rhesus monkeys until at least one year post transplant. After the 6 or 7th exposure, all three animals developed an immune response to the radiotracer. This suggests immune recovery remained incomplete in the animals receiving a dose of 4Gyx2days TBI or higher for at least one year following transplant. No immune response has been observed at 5Gyx2 at d.245. Our results present for the first time a non-invasive method in real time to evaluate CD4+ cell immune recovery in LTs following hematopoietic stem cell transplantation using SPECT/CT imaging. Despite myeloablation of circulating leukocytes following TBI, we did not observe total depletion of CD4+ cells in LTs such as the spleen. This is problematic if one is concerned about residual disease or endogenous contributions to recovery. The methodologies presented here have direct applications to the evaluation of immunosuppressive therapies and other immunosuppressive disorders, such as those associated with viral infections. Figure 1 Figure 1. Baseline SPECT image depicting lymphoid organs. Figure 2 Figure 2. SPECT images comparing 3Gyx2 (ZI12), 4Gyx2 (ZG21, ZH32, ZG41), and 5Gyx2 (ZG70 and ZI64) d.6 post-TBI and an animal chronically infected with SHIVDH12R(G43). Despite low circulating levels of CD4+ cells in all animals, residual CD4+ cells remain within LT such as the spleen following TBI, and more complete depletion associated with chronic SHIV infection. Figure 3 Figure 3. Control and d.6 post-TBI anti-CD3 IHC of spleen and axillary LN. Disclosures No relevant conflicts of interest to declare.

Leukapheresis procedures in very small volume subjects require procedure modifications related to anticoagulant, venous access, and volume. Historically, we accomplished this procedure in 4-5 kg rhesus macaques using a modified CS3000Plus cell separator, but this instrument is no longer available. The Spectra Optia apheresis system and its mononuclear cell (MNC) collection set were evaluated to develop leukapheresis procedures for such subjects. Five rhesus macaques, weight 7-9 kg, blood volume 60 mL/kg, were studied. They pre-donated 100-120 mL of autologous blood (25-30 mL/wk x 4 wks). Aspirin 81 mg was given daily for 3 days prior to 5 of 8 procedures. AMD3100 1 mg/kg SQ 2-3 hours prior to leukapheresis was given as a mobilizing agent. Autologous blood was pooled, diluted 1:1 with saline, and washed to remove citrate and plasma. The Optia was primed first with saline, and then with a customized albumin option in which 30-35 mL of the 40-50 mL of packed RBCs were pumped into the disposable apheresis kit at 10 mL/min. The albumin option shortens procedure ramp-up time. A closed circuit was created between the return line and the packed RBC bag and the flow rate increased to 40 mL/min until 400 mL was processed. To minimize citrate toxicity and avoid volume overload, animals received heparin 50 U/kg bolus IV followed by an ACDA solution containing heparin 5U/mL at an inlet:AC ratio of 25:1. A Collection Preference (CP) setting of 20 was selected using the automatic interface management mode. In the last procedure a semi-automatic mode was utilized so that collection deeper than CP of 20 was possible. Procedures were terminated at 120 min. Animals were anesthetized and catheters placed in the saphenous and cephalic as draw and return lines. Blood counts, chemistries, and ionized calcium (iCa) levels were monitored every 30 min. The elutriation chamber, which concentrates MNCs and allows platelets to return to the donor, was filled 1-2 times per procedure resulting in a product volume of 17-34 mL. A total of eight leukapheresis procedures were performed. Inlet flow rates were typically 10.5 mL/min. In one animal, an inlet rate of 12.0 mL/min was achieved; however, at 120 minutes severe citrate toxicity developed with bradycardia and an iCa level of 0.62 mmol/L, requiring IV calcium gluconate. Excluding this case, a mean(SD) baseline iCa value of 1.17(0.2) dropped to 0.84(0.03) mmol/L at120 min. Mean hematocrit (HCT) decreased from 40.4(1.7)% to a nadir of 36.8(2.6)% at 90 min. Mean platelets declined to a greater degree, from 342(42) x103/mcL to 109(33) x103/mcL at 60 min and 121(43) x103/mcL at 120 min (p MNC collections through the process of leukapheresis have proven to be effective in isolating cells for transplantation and cellular therapeutics. The use of leukapheresis in a pediatric setting is difficult due to small blood volumes and a high risk of adverse events. Here we have identified a novel means to perform leukapheresis procedures safely and effectively in small volume subjects. This methodology will have numerous therapeutic applications in both human and veterinary medicine. Disclosures Parker: Terumo BCT, Inc.: Employment. Lienesch:Terumo BCT, Inc.: Employment.

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 (γ c ), 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 γ c -transplanted animals are healthy. This system may prove useful for expression of therapeutic genes in human hematopoietic cells.

Background Historically lymphocyte (Lc) recovery following hematopoietic transplantation, immunosuppressive therapies, and infectious diseases has relied quite heavily on peripheral blood (PB) analysis. The PB, however, comprises less than 2% of total Lcs in the body, hence small changes in trafficking rates (e.g., lymphoid tissue (LT) homing) could have profound effects on PB Lc count. In this study we explored the use of a radiotracer rhesus IgG1 anti-CD4R1 Tc99mFab'2 and single photon emission computed tomography (SPECT/CT) to sequentially image CD4+ Lc recovery in the whole-body following total body irradiation (TBI) and autologous CD34+ cell transplant. Methods Three specific pathogen free rhesus macaques were used in this study. Each received 4Gy TBI on two sequential days (8Gy total). CD34+ cells were immunoselected from a leukapheresis product following cytokine mobilization over 5 days using G-CSF and Stem Cell Factor. 4-9x106/kg CD34+ cells were reinfused on the last day of irradiation after being transduced in culture once with a SIV/HIV chimeric lentiviral vector expressing EGFP at a multiplicity of infection of 50. SPECT/CT images were taken prior to transplant as a baseline; following mobilization with AMD3100 at least two months prior to transplant; and at approximately 6, 30, 90, 150 and 260 days post-TBI/transplant. Development of immunogenicity to the radiotracer was monitored using size exclusion high performance liquid chromatography (HPLC) and an in vitro cell binding assay. Results Dynamic SPECT acquisition for two hours following mobilization with 1mg/kg AMD3100 SQ did not reveal measurable changes in the whole-body CD4 pool, despite an ∼ 2-fold maximum increase in CD4+ PB cell counts. This is consistent with a small percentage of CD4+ cells being released from tissues to PB without significantly affecting total Lc counts in the whole-body. Following 4Gyx2 TBI, all 3 animals experienced dramatic depletion of PB CD4 counts (a 90-98% decrease from baseline) (Table 1). Variability in tissue CD4 depletion was observed between and within hosts, with more dramatic decreases observed in axillary lymph nodes (73-92%) and only mild decreases in the spleen (36-40%). Similarly, recovery of the CD4 pools in LTs following transplant varied between and within hosts and appeared overall slower than the dynamics of repopulation in the PB, suggesting changes in trafficking rates between LTs and PB has a role in transplant recovery. By month 9 post-TBI, one animal (ZG41) had full recovery of the PB CD4+ Lc count and the CD4 pool in axillary LNs quantified by the SPECT/CT, but minimal recovery of CD4 pools were observed in the spleen. In another animal (ZG21), full recovery of the PB CD4+ cell count was observed by month 5 concomitant with full recovery of the spleen CD4 pool, despite still greater than 70% depletion of CD4 pool in axillary LNs. In the last animal (ZH32), by month 3 the CD4+ Lc count in PB was fully recovered, however, minimal CD4 pool recoveries were observed in LT through month 9 post-TBI (Figure 1). EGFP expressing Lcs first appeared in the circulation on average(SD) 69(48) days following transplant and were CD8+ and CD20+. In all animals, CD4+ EGFP+ Lcs were not observed until after month 5, suggesting that endogenous recovery preceded graft recovery in the PB. Of importance is that no immunogenicity to the radiotracer was detected despite sequential imaging up to month 5 in one animal and up to month 9 in the other two animals. This is in contrast to animals that were imaged several years post-transplant. In these animals immune complex formation to the radiotracer was observed by HPLC after a single exposure to the rhesus IgG1 anti-CD4R1 Tc99mFab'2. This suggests either tolerance was induced in animals exposed to the radiotracer shortly following transplant or that immune recovery remains incomplete after 150 days in these animals. Conclusions These results demonstrate for the first time that immune reconstitution of lymphoid tissues can be effectively monitored following transplant of autologous CD34+ cells and TBI, that the reconstitution appears to be discordant between PB and LT, as well as identifying a potential approach to decreasing immunogenicity to complex antigens through lymphocyte mobilization during antigen exposure. Disclosures: No relevant conflicts of interest to declare.

Dendritic cells (DCs) play a central role in innate immunity and adaptive immunity. DCs are antigen presenting cells capable of inducing primary T cell responses or facilitating self-tolerance. Myeloid DCs (mDC) express CD11c. They are further divided into Type 1 myeloid DCs (mDC1) which are CD1c+CD141+ and Type 2 mDCs (mDC2) which are CD1c-CD141+, and plasmacytoid DC which are CD11c- and express CD303. Plasmacytoid DCs are the main source of type 1 interferon upon infection. CD34+ cells are a heterogeneous population of cells which contain both hematopoietic progenitors and stem cells. The microarray signature of CD34+CXCR4 (CD184)+ cells in both human and non-human primates suggest that this cell population plays a role in innate immunity. The signaling pathway for the Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) is the most up-regulated pathway in both human and non-human primate sorted CD34+CD184+ cells. The TREM family is involved in the amplification and regulation of inflammatory responses. Upon sorting both human and rhesus CD34+ cells into CD34+CD184+ and CD34+CD184-, we observe that CD34+CD184+ cells are both adherent and non-adherent in nature (Figure 1) and while maintained in Flt3-L, IL-3, and SCF form progeny which appear dendritic in nature (Figure 2). In addition, we have found the CD34+CD184+ subpopulation to be resistant to lentiviral vector transduction. Upon culturing in defined, serum free media supplemented with cytokines, the progeny of the CD34+CD184+ population using both flow cytometry and confocal microscopy are CD11c+ and contain both CD1c+ and CD1c- subpopulations (Figure 3) with a predominately CD80(B7-1)+, CD123(IL-3R)+, CD197 (CCR7)+, and HLA-Dr+ immunophenotype, having variability in CD86(B7-2)+/-, CD141(BDCA-3/Thrombomodulin)+/- , and CD144 (VE-cadherin)+/- expression, and being negative for CD303(BDCA-2)and CD309(VEGFR). Of special interest is that the CD34+CD184+ progeny contain both CD1c-CD16+ and CD1c+CD16- subpopulations of mDC. CD1c-CD16+ mDC have been shown to have strong pro-inflammatory activity, whereas CD1c+CD16- mDC are mainly inducers of chemotaxis. The progeny of the CD34+CD184+ cells can be stimulated by LPS and IFNγ to produce IL-12 based on an enzyme-linked immunosorbent assay. These results demonstrate the importance of the CD34+CXCR4+ progenitor in mDC development and allow one to speculate on how this mDC progenitor might prove of therapeutic benefit in vaccine development and cancer therapy. Download : Download high-res image (5MB) Download : Download full-size image Figure 1 . Human CD34+CD184+ Sorted Population Download : Download high-res image (5MB) Download : Download full-size image Figure 2 . Cultured Progeny CD34+CD184+ Download : Download high-res image (171KB) Download : Download full-size image Figure 3 . Non-Human Primate CD34+CD184+ Confocal Microscopy Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34(+) cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34(+) cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34(+) cells with granulocytes and lymphocytes in vivo. The transduced CD34(+) cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34(+) cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy.Molecular Therapy-Nucleic Acids (2013) 2, e122; doi:10.1038/mtna.2013.49; published online 17 September 2013.

Abstract 1007 CXCR4 is an α-chemokine receptor specific for the ligand stromal-derived-factor-1 (SDF-1). CXCR4 is expressed on several cell types, including CD34+ cells. In this study, we sought to evaluate the difference between subpopulations of human and rhesus CD34+ cells that express CXCR4. Rhesus CD34+ cells were mobilized using a five day course of G-CSF (10mcg/kg SQ) and a single dose (1mg/kg SQ) of the CXCR4 antagonist, AMD3100, on the 5th day. Leukapheresis was performed two hours following the last dose of G-CSF and AMD3100 and CD34+ cells were isolated by immunoselection from the product. Human CD34+ cells were collected by leukapheresis and immunoselection following a 5 day course of 480 mcg G-CSF SQ. CXCR4+ and CXCR4- fractions in the CD34+ cells were separated by cell sorting. Rhesus and human CD34+CXCR4+ and CD34+CXCR4- cells were used for microarray analysis as well as cultured and transduced with an EGFP-expressing lentiviral vector. On average (SD) 21.6 ± 4.4% of AMD3100+G-CSF mobilized rhesus (n=6) and 39.3 ± 4.3% of G-CSF mobilized human (n=3) CD34+ cells express CXCR4. Two days following in vitro culture, rhesus CD34+CXCR4+ cells showed greater numbers of adherent spindle-shaped cells (56.4 ± 7.4%) when compared to CXCR4- cells (1.3 ± 0.4%, p Disclosures: No relevant conflicts of interest to declare.

Abstract 3118 Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application. We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model. We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis. We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis. We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. Disclosures: No relevant conflicts of interest to declare.

Abstract 1301 CD34+ cells are a heterogeneous population of cells which contain the hematopoietic stem cell (HSC). Previously we determined that the microarray signature of CD34+ cells mobilized by G-CSF differed from those mobilized with either plerixafor (AMD3100) alone or in combination with G-CSF (Donahue RE, et al. Blood 2009; 114:2530–2541). Recently we also determined that a novel subpopulation of CD34+CD123+ cells was mobilized with plerixafor and not G-CSF in rhesus macaques (Uchida N, et al. Exp Hematol 2011; 39:795–805). To explore differences between CD34+CD123+ and CD34+CD123- cells, we evaluated their gene expression signatures. Following mobilization with plerixafor alone or in combination with G-CSF, we collected mononuclear cells by leukapheresis, isolated rhesus CD34+ cells by immunoselection, and further isolated CD34+CD123+ and CD34+CD123- by cell sorting. CD34+CD123+ cells were found not to express CXCR4 on their cell surface. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA were co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. Gene expression was analyzed by quantifying the fluorescence from the microarray. The raw data set was filtered according to a standard procedure to exclude spots with minimum intensity. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method. Statistical analysis was done using Array Class Comparison analysis. Pathway analysis was carried out using Ingenuity Pathway Analysis. Principal Component Analysis demonstrated that CD34+CD123+ and CD34+CD123- cells cluster separately and are not distinguishable on the basis of mobilization regimen. Using Supervised Hierarchical Cluster Analysis for genes which were significantly different (p Disclosures: No relevant conflicts of interest to declare.

Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p Disclosures: No relevant conflicts of interest to declare.

Abstract 350 Introduction: Leukapheresis is a widely utilized modality for collecting hematopoietic stem cells (HSCs). While collection of CD34+ cells with stem-cell activity is the primary goal of most mobilization and leukapheresis procedures, these cells only represent ∼1% of most leukapheresis products. The profile of the non-CD34+ cells is likely influenced by the choice of mobilization strategy, and has the potential to profoundly impact the post-transplant immune milieu of the transplant recipient. Two of the most critical of the CD34-negative cell populations that are collected during leukapheresis include effector and regulatory T cells. Thus, in evaluating mobilization regimens, the impact on these regimens on the mobilization of each of these T cell populations into the peripheral blood should be rigorously evaluated. Methods: We used a rhesus macaque model to determine the impact that mobilization with AMD3100 (a.k.a., Plerixafor or Mozobil®)+ G-CSF (“A+G”) had on peripheral blood CD4+ and CD8+ effector T cell populations as well as on FoxP3+/CD4+ T cells. Three rhesus macaques were mobilized with 10ug/kg SQ of G-CSF for five consecutive days prior to leukapheresis. AMD3100 was administered at 1mg/kg SQ in combination with the last dose of G-CSF two hours prior to leukapheresis. Leukapheresis procedures were performed for two hours using a modified CS3000 Plus cell separator. A peripheral blood sample was taken before cytokine therapy, just prior to leukapheresis following mobilization, one hour during leukapheresis, and at the end of the procedure. These samples were analyzed by multicolor flow cytometry using a BD LSRII flow cytometer. Results: Bulk, effector, and regulatory T cell subpopulations were analyzed flow cytometrically. The proportion of total CD3+ T cells remained stable during mobilization and apheresis: Thus, CD3+ T cells represented 77% of peripheral blood lymphocytes prior to mobilization, and 69% post-apheresis). The balance of CD4+ to CD8+ T cells was also relatively stable. Thus, for one of the three animals tested, the CD4+ and CD8+ proportions remained unchanged after apheresis. For two animals, the average CD4+ % decreased from 67% prior to mobilization to 52% post-apheresis. In these two animals, there was a reciprocal increase in the % of CD3+ T cells that were CD8+ (28% pre-G+A to 40% post-apheresis). The CD28+/CD95- naïve (Tn), CD28+/CD95+ central memory (Tcm) and CD28-/CD95+ effector memory (Tem) subpopulation balance of CD4+ and CD8+ T cells was also determined, by comparing the relative percentages of each subpopulation post-apheresis with their relative percentages prior to mobilization. Compared to their pre-G+A percentages, the post-apheresis CD4+ percentages of Tn, Tcm and Tem were 92%, 93% and 160%, respectively. Thus, the relative proportions of Tn and Tcm CD4+ cells decreased post-apheresis, while the relative proportion of CD4+ Tem increased compared to cytokine administration. For CD8+ T cell subpopulations, the post-apheresis proportions of Tn, Tcm, and Tem compared to their pre-G-CSF proportions were 99%, 70% and 130%, respectively–thus demonstrating the same direction of change as observed for CD4+ T cells. The most striking change in T cell subpopulations occurred in the CD4+/FoxP3+ compartment. The proportion of CD4+ T cells expressing FoxP3 increased by an average of 600% when post-apheresis samples were compared to pre-mobilization samples (FoxP3+ cells were 9.6% of CD4+ T cells post-apheresis versus 1.5% pre-GCSF). An average of 32% of these FoxP3+ CD4+ T cells expressed high levels of CXCR4. CXCR4 expression has been previously documented on human FoxP3+ T cells (Zou et al., Cancer Res, 2004), but this is the first observation of high level expression of CXCR4 on macaque FoxP3+ CD4 T cells, or of their ability to be efficiently mobilized with AMD3100. Discussion: These results suggest that treatment with AMD3100 and G-CSF may mobilize T cell subsets into the peripheral blood that could have beneficial effects during allo-transplantation. The combination of an increase in Tem cells, which have been observed to have decreased ability to cause GvHD (Zheng et al., Blood 2008), along with FoxP3+/CD4+ T cells, which may have regulatory functions, suggests that A+G mobilization could produce an apheresis product with a beneficial CD34-negative cell profile for allogeneic transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4798 Although CD34+ cells represent a small population of phenotypically defined cells within the bone marrow and peripheral blood circulation, they remain a very heterogeneous population of cells which contain hematopoietic stem/progenitor cells. CXC Chemokine Receptor 4 (CXCR4) is an α-chemokine receptor specific for stromal-derived-factor-1 (SDF-1 also called CXCL12) and is found on CD34+ cells. Plerixafor (also known as Mozobil® or AMD3100) is currently used as a mobilizing agent for CD34+ cells by interfering with the binding of CXCR4 to SDF-1. To explore differences between CD34+CXCR4+ and CD34+CXCR4- populations, gene and microRNA signatures were determined using microarray analysis. Three healthy human donors were cytokine mobilized with G-CSF, peripheral blood mononuclear cells were collected by apheresis, and CD34+ cells were immunoselected using the Miltenyi CliniMACS® system and cryopreserved. These cells were then thawed and allowed to sit overnight at 37°C in X-Vivo-10. The following day the cells were stained and sorted on a BD FACSAria III® cell sorter. All three individuals CD34+CXCR4+ and CD34+CXCR4- fell within two rather restrictive populations based on forward and side scatter gating with CD34+CXCR4+ cells routinely being smaller in size than the CD34+CXCR4- populations (Figure 1). Total RNA was isolated from the sorted cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye and co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. Using unsupervised and supervised Eisen's hierarchical clustering and Array Class Comparison analysis relationships were determined. Hierarchical cluster analysis demonstrated that the CD34+CXCR4+ and CD34+CXCR4- had different gene expression patterns (Figure 2). Ingenuity Pathway Analysis revealed that genes up-regulated in the CD34+CXCR4+ population were related to TREM-1 signaling (innate immunity), dendritic cell maturation, NFAT-regulation of the immune response, and T-cell receptor and NK cell signaling. In contrast, the CD34+CXCR4- population expressed more primitive genes such as those related to IGF-1, ERK/MAPK, IL-3, and Nitric Oxide signaling as well as transcription factors such as N-myc and HOX-A9. MicroRNA profiling of the CD34+CXCR4- population also express the miR-17-92 polycistron cluster (miR17, 18a, 19a, 19b, 20a, 92) (Figure 3). These results are consistent with the CD34+CXCR4- population being more primitive than the CD34+CXCR4+ population. It is interesting to speculate whether the CD34+CXCR4+ population includes a progenitor for T cell and innate immune responses while progenitors for hematopoietic stem cells and adaptive immune responses are within the CD34+CXCR4- populations. Disclosures: No relevant conflicts of interest to declare.

Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 3587 Poster Board III-524 The mechanism whereby neutrophils traffic from the circulation in response to G-CSF has remained unclear despite the observation of ourselves and others that there is a dramatic, yet transient, loss of circulating neutrophils shortly following the administration of G-CSF in humans, non-human primates, and mice (Gordon BC, et al. Exp Hematol. 35:872-8, 2007). To determine the role of the CD18 leukocyte integrin on neutrophils in the egress of neutrophils from the circulation, we used dogs with canine leukocyte adhesion deficiency (CLAD), a genetic disease in which a mutation in CD18 prevents CD18 surface expression. We selected CLAD dogs who had 5-10% CD18+ neutrophils following either matched littermate allogeneic transplant or autologous gene therapy for CLAD. Three CLAD dogs meeting these criteria were evaluated. Three carrier dogs served as controls. G-CSF was administered at 10μg/kg SQ to all six animals. Peripheral blood samples (EDTA) were taken immediately prior to G-CSF administration, and at 15, 30, 60, 120, 240 minutes, and 24 hours following G-CSF administration. Total white blood cell counts, neutrophil counts, and the number and percentage of CD18+ peripheral blood leukocytes were assessed. As anticipated, the control dogs had a 60% decrease in circulating neutrophils 30 minutes following G-CSF administration: the mean +/− standard of deviation (SD) absolute neutrophil baseline count decreased from 6806+/−1072/μL to 2727+/−767/μL. In five control animals the neutrophil nadir occurred at 30 minutes post-G-CSF, and in one control dogs it occurred 15 minutes following G-CSF administration. Experimental CLAD dogs had only a 35% decline in neutrophil numbers at 30 minutes, from a mean baseline of 6777+/− 672/μL to 4433+/−265/μL. In these dogs the neutrophil count returned to pre-G-CSF levels by 60 minutes post-G-CSF. By 24 hours after G-CSF, the neutrophil level was increased 3-fold from baseline. Immunophenotyping using an anti-CD18 and a canine specific anti-neutrophil PE conjugated antibody indicated that only the CD18+ neutrophils disappeared from the circulation following G-CSF administration. At baseline the transplanted CLAD dogs had a mean of 15.2+/−3.9% CD18+ peripheral blood leukocytes, of these 50.7+/−7.1% were CD18+ neutrophils. Thirty minutes following G-CSF administration the mean+/−SD percentage of CD18+ leukocytes declined to 13.7+/−3.7% with 33.4+/−5.8% being neutrophils. There was also a slight decline in CD14+CD18+ monocytes from 6.2 +/− 1.5% to 4.0 +/− 1.2%, which was not observed in the controls. There was no change in CD18- leukocyte numbers. The percentage of CD18+ neutrophils returned to baseline by 60 minutes and remained there at subsequent time points. These results demonstrate that the CD18 leukocyte integrin on circulating neutrophils mediates the transient neutropenia associated with G-CSF administration. Disclosures: No relevant conflicts of interest to declare.

Abstract 2523 Poster Board II-500 Prostaglandin E2 (PGE2), a member of the eicosanoid family, is a local signaling molecule released from mammalian blood vessel walls and other hematopoietic niches. PGE2 produces tissue specific effects by interacting with G protein receptors in cell membranes. It has been found to be an important regulator of hematopoietic stem cell (HSC) number during embryogenesis and is required for the development of HSCs (North et al., Nature 447:1007–1011, 2007). Additionally, PGE2 enhances HSC engraftment in competitive transplantation assays (North et al., Nature 447:1007–1011, 2007) through alterations in the survival, proliferation and homing of transplanted cells (Hoggatt et al., Blood 113:5444–5455, 2009). Immunoselected CD34+ cells were isolated from the leukapheresis products of both human and non-human primates following cytokine mobilization with G-CSF. CD34+ cells were incubated in X-Vivo 10™ with either 50μM or no PGE2 for one hour at 37°C on RetroNectin™ coated plates. After one hour incubation, media was replaced with X-Vivo 10™ supplemented with 10ng/mL SCF and IL-6 and cells were kept at 37°C and collected either prior to or at 2, 6, 12 and 24 hours following PGE2 exposure. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA was co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method and gene regulation was analyzed at each time point using an Array scatter plot comparison. Statistical analysis was done using Array Class Comparison analysis and pathway analysis was carried out using Ingenuity Pathway Analysis. Many of the significantly up regulated genes were associated with pathways activated by PGE2, including genes involved in the inflammatory response, signal transduction, and G proteins. Interestingly the human CD34+ cells showed at 2 hours a 21.8-fold increase of 3′,5′-cyclic AMP phosphodiesterase mRNA consistent with prior observations in zebrafish and murine cells (Goessling et al. Cell 136: 1136–1148). Pathways that increased at 2 hours include molecular mechanisms of cancer, and those that were down regulated include CCR5, CTLA4, and leukocyte extravasation signaling. At 6 hours the molecular mechanisms of cancer pathway predominated even more (diminishing by 12 hours) while oxidative phosphorylation was down regulated. Genes involved in cAMP and WNT signaling are observed to be up regulated at earlier time points but diminish with time. These results correlate with protein expression observations that have been made in the zebrafish and murine models following PGE2 treatment (Goessling et al. Cell 136: 1136–1148) and are consistent with the observation that PGE2 may influence the self renewal of HSCs. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties.

Although the successful application of gene transfer to hematopoietic stem cells using HIV based vectors for the hemoglobinopathies and thalassemias has recently been achieved in rodents, concerns regarding possible side effects, including insertional mutagenesis, mandate large animal preclinical testing. HIV vectors transduce rhesus blood cells poorly due to a species specific block by TRIM5a and APOBEC3G which target HIV capsid proteins (CAs) and viral infectivity factor (Vif), respectively. We developed a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV and SIV by including SIV CA and SIV Vif. Titers of HIV vectors prepared with SIV Vif (8.4±0.69x106 IU/ml, p=0.73) or SIV CA (delta-HIV 8.1±1.1x106 IU/ml, p=0.73) were comparable to that of a normal HIV vector (9.2±0.46x106 IU/ml), while titers of chimeric HIV vectors that included SIV gag-pol or rev-tat were dramatically decreased (0.18-4.5x105 IU/ml, p Figure Figure

Background: Anti-donor antibodies remain a significant problem in discordant xenotransplantation, and are believed to play a significant role in chronic allograft rejection. We have previously shown that genetic engineering of autologous bone marrow stem cells to induce molecular chimerism can be used to establish B cell tolerance in mice. In this study, we examined whether a similar approach could be used to induce B cell tolerance in Rhesus macaques, a preclinical animal model. To this end, we investigated whether induction of molecular chimerism could be used to affect production of antibodies specific for the carbohydrate ep|[iacute]|tope Gal|[alpha]|1-3Gal|[beta]|1-4GlcNAc-R (|[alpha]|Gal).