Rhesus CD34 Research Articles

CD117 Antibody-Drug Conjugate Conditioning Allows Efficient Engraftment of Gene-Modified CD34+ Cells with Fertility Preservation in a Rhesus Gene Therapy Model

Hematopoietic stem cell (HSC) gene therapy is curative for multiple genetic diseases; however, it is limited due to toxicities from chemotherapy-based conditioning. To target HSCs directly, we developed an anti-human CD117 ( c-KIT)-targeted antibody-drug conjugate (CD117-ADC) conjugated to a DNA-binding payload that cross-reacted with non-human primates. To investigate the activity of CD117-ADC in non-human primates, we administered a single injection of ADC without HSC infusion, resulting in a >99% depletion of CD34+CD90+ bone marrow cells in cynomolgus macaques (n=2 of 3 in 0.1 mg/kg and n=3 in 0.3 mg/kg); similar to the ablation observed with 4 doses of myeloablative busulfan (Bu) (n=3). With escalating ADC doses (0.2-0.6 mg/kg), the optimal timing of HSC infusion (6 days in 0.2 or 0.3 mg/kg and 10 days in 0.4mg/kg) was determined in rhesus macaques (n=1 each). The animal dosed 0.6 mg/kg required euthanasia on day 14 due to irreversible hematopoietic suppression and severe liver toxicity. In our preliminary transplantation study, dosing 0.2 mg/kg ADC to rhesus macaques (n=2) resulted in bone marrow depletion and modest-level engraftment of gene-modified cells (vector copy number per cell (VCN) 0.05±0.01) (ASH 2019). Therefore, we evaluated higher single doses of CD117-ADC in rhesus macaques (0.3 mg/kg for ZL13 and ZJ62, and 0.4 mg/kg for H635 and H96G, n=4) and directly compared to Bu conditioning (5.5 mg/kg x 4 days, n=2). Mobilized rhesus CD34+ cells (ADC 6.4±2.9e6/kg vs. Bu 4.1±0.1e6/kg, n.s.) were transduced with a lentiviral vector for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells ( in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals after ADC or Bu conditioning. The time course of blood cell count suppression and recovery was similar between ADC and Bu animals, except for a strong reduction of platelet counts observed only with ADC (ASH 2021). Two months post-transplant, substantial levels of vector-containing granulocytes were observed long-term in 1 of 2 animals receiving 0.3 mg/kg ADC (ZJ62, VCN 0.16) and in both animals receiving 0.4 mg/kg (H635, VCN 0.46 and H96G, VCN 0.21) at a similar range to the two Bu animals (0.44±0.17, n.s.). Robust HbF induction was detected in three ADC animals with substantial VCN as measured by both HbF-positive cells (F-cells 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC measurement of total HbF (8.0±2.9% vs. 11.1±5.2%, n.s.). In ZL13 (0.3 mg/kg ADC), lower gene marking (VCN 0.02) was observed, along with lower HbF induction (F-cells 1.0% and HbF amounts 0.9%). Importantly, ADC resulted in minimal off-target toxicity, unlike those associated with Bu (such as infertility, veno-occlusive disease, pulmonary dysfunction, mucositis, or seizures). High diversity of gene-modified cells (Simpson's diversity index >0.99) without any large clone (maximal integration site 0.3-3.7%) was observed by integration site analysis 1-year post-transplant with both ADC (0.3-0.4 mg/kg) and Bu. Infertility is a major complication of conventional myeloablative conditioning that limits the uptake of curative therapies; therefore, we investigated fertility in CD117-ADC animals. When monitored daily for menstrual bleeding, the menstrual cycles of all 4 female macaques (ZL13, ZJ62, H635, and H96G) were maintained post-ADC conditioning (0.92±0.11 per month) in contrast to our previously transplanted animals following myeloablative Bu (0.23±0.11, n=4, p<0.01) and total body irradiation (0.33±0.3, n=6, p<0.05). To confirm fertility, all transplanted animals post-ADC including females (n=4) and males (n=2) were housed for mating at a rhesus breeding colony. Two ADC females (ZL13 and H635) gave birth, and an ADC male (12U032) was paired with a pregnant female. Importantly, an ADC female (H635) was paired with an ADC male (13U047). In summary, a single-injection CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving similar levels as the current clinical standard of care, myeloablative Bu conditioning. Robust HbF induction was also confirmed at the protein level in our rhesus gene therapy model with ADC conditioning. The transplanted animals with ADC maintained their fertility. Overall, these data indicate that an ADC-based targeted approach offers safer conditioning and could improve the risk-benefit profile in HSC gene therapy.

HDAd6/35++ - A new helper-dependent adenovirus vector platform for in vivo transduction of hematopoietic stem cells

In previous studies, we achieved safe and efficient invivo hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6. We found a lower prevalence and titers of serum anti-HDAd6/35++ in human samples compared with HDAd5/35++. HDAd6/35++ vectors efficiently transduced human and rhesus CD34+ cells invitro. Intravenous injection of HDAd5/35++-GFP or HDAd6/35++-GFP vectors after G-CSF/AMD3100 mobilization of mice with established human hematopoiesis or human CD46 transgenic mice resulted in comparable GFP marking rates in HSCs in the bone marrow and spleen. In long-term invivo HSC transduction and selection studies with integrating vectors, stable GFP expression in >75% of PBMCs was show for both vectors. In contrast with HDAd5/35++, undesired transduction of hepatocytes was minimal with HDAd6/35++. Furthermore, HDAd6/35++ allowed for efficient invivo HSC transduction in Ad5-pre-immune mice. These features, together with the straightforward production of HDAd6/35++ vectors at high yield, make this new HDAd vector platform attractive for clinical translation of the invivo approach.

CD117 Antibody Drug Conjugate-Based Conditioning Allows for Efficient Engraftment of Gene-Modified CD34+ Cells in a Rhesus Gene Therapy Model

Hematopoietic stem cell (HSC) gene therapy is now curative for multiple genetic diseases; however, it is limited by morbidity and mortality from cytotoxic chemotherapy-based conditioning. To overcome these limitations, we developed an antibody drug conjugate (ADC) targeting CD117 (c-Kit) to specifically deplete both HSCs and progenitor cells. In our preliminary study, 0.2 mg/kg CD117-ADC conditioning resulted in >99% bone marrow depletion, detectable engraftment of gene-modified cells (vector copy number per cell (VCN) ~0.01), and minimal toxicities in a rhesus HSC gene therapy model (ASH 2019). In this study, we investigated escalating doses of CD117-ADC to determine the optimum conditioning dose to enable engraftment of gene-modified CD34+ HSCs in rhesus macaques.We evaluated autologous CD34+ cell transplantation with lentiviral gene marking following conditioning using a single injection of CD117-ADC at the 0.3 mg/kg dose for ZL13 and ZJ62, and the 0.4 mg/kg dose for H635 and H96G. The extent of gene marking was compared with myeloablative busulfan conditioning (5.5 mg/kg x 4 days) for 12U018 and 12U020. Mobilized rhesus CD34+ cells (ADC 3.8±1.9x10e7 vs. Busulfan 2.9±0.2x10e7, n.s.) were transduced with a lentiviral vector encoding BCL11A-targeting microRNA-adapted short hairpin RNA (shmiR-BCL11A) co-encoding a truncated human erythropoietin receptor (thEpoR) for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells (in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals 6 or 10 days after ADC conditioning (0.3 or 0.4 mg/kg, respectively) or 1 day after busulfan conditioning. Blood counts, gene-marking levels, and HbF induction were evaluated for 0.3-1.2 years post-transplant in ADC conditioning and for 1.5 years in busulfan conditioning. After a reduction of blood counts post-transplantation with ADC or busulfan conditioning, all lineages recovered. Granulocyte (>500/μl, day 6-9 vs. day 8-9), reticulocyte (>50,000/μl, day 10-14 vs. day 11), and platelet (>30,000/μl, day 2-8 vs. no reduction) recoveries were similar for ADC and busulfan conditioning, respectively. Only ADC conditioning resulted in a reduction of platelet counts as well as a novel transient rebound in all major lineages. Two months post-transplant, efficient gene marking (VCN in granulocytes 0.28±0.16 vs. 0.44±0.17, n.s.) was observed in 3 of 4 animals in ADC-conditioning (ZJ62 with 0.3 mg/kg ADC, and H635 and H96G with 0.4 mg/kg ADC). This marking level was similar to busulfan conditioning (Left panel in Figure). Robust and durable HbF induction was also detected by both HbF-positive percentages (F-cell 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC-quantitated HbF amounts (8.0±2.9% vs. 11.1±5.2%, n.s.) in these 3 animals, similar to busulfan conditioning (Right panel in Figure). In ZL13 (1 of 2 animals in 0.3 mg/kg ADC), lower gene marking (VCN in granulocytes 0.02) was obtained, along with low HbF induction (F-cell 1.0% and HbF amounts 0.9%), suggesting that 0.3 mg/kg ADC is marginal and 0.4 mg/kg ADC is sufficient for robust engraftment of gene-modified cells. Importantly, CD117-ADC conditioning resulted in minimal toxicities unlike busulfan conditioning.In summary, we demonstrated that a single dose of CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving a similar level as myeloablative busulfan conditioning. Robust HbF induction was also confirmed at the protein levels in this rhesus gene therapy model with ADC conditioning. This targeted approach for safer conditioning could improve the risk benefit profile in HSC gene therapy. [Display omitted] DisclosuresLatimer: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bhattarai: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Yoder: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Palchaudhuri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Li: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bertelsen: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Olson: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

Fetal Hemoglobin Induction with Forced Chromatin Looping in Gene-Modified Erythroid Cells Differentiated from Rhesus Hematopoietic Progenitor Cells

Substantial advances in genome engineering offer new therapies for hemoglobin (Hb) disorders including sickle cell disease. Fetal hemoglobin reactivation remains a viable strategy, as recently demonstrated in human erythroid cells with an artificial looping molecule ZF-Ldb1, tethering Ldb1 to the γ-globin promoter by specific zinc finger proteins (Deng et al., 2014). Large animal preclinical testing can improve the therapeutic potential of such approaches by optimizing safety and efficacy issues before launching clinical trials in humans. A robust in vitro erythroid evaluation system, however, does not exist to determine if fetal hemoglobin reactivation can be modeled in the rhesus macaque. Therefore, in the current work, we aimed to develop an efficient in vitro erythroid differentiation model from rhesus hematopoietic progenitor cells with Hb production at detectable protein levels, and to evaluate rhesus fetal hemoglobin reactivation with ZF-Ldb1.To adapt a human erythroid differentiation method to rhesus cells, we used C3H or OP9 feeder cells to culture rhesus peripheral blood mononuclear cells (PBMCs) with HEMA differentiation media containing 10ng/ml stem cell factor (SCF), 1ng/ml interleukin-3 (IL3), 2U/ml erythropoietin (Epo), and 20% fetal bovine serum (FBS) (Migliaccio et al., 2010). Two weeks after erythroid differentiation, robust Hb production evaluated by HB electrophoresis was obtained on both C3H and OP9 feeder cells but not in feeder-free conditions. Increased concentration of SCF (20x), IL3 (10x), and Epo (1.5x) allowed for Hb production even in feeder-free conditions; however, fetal Hb was predominant (due to 20x SCF). Both conditions provided similar levels of enucleation (39±6.3% vs 29±5.6%, ns), indicating efficient differentiation. We then compared HEMA media with serum-free StemSpan-based differentiation media to evaluate transduction with a GFP-encoding lentiviral vector at MOI 2.5 two days after initiating differentiation. Transduction was almost completely inhibited (0.4±0.5%) in HEMA media, while StemSpan-based transduction media followed by HEMA differentiation media resulted in significant increase of transduction efficiency (41±8.5%, p<0.01). These data demonstrate that feeder cells are required for rhesus erythroid differentiation in HEMA media, while FBS strongly interferes lentiviral transduction.Next, we sought to establish a reverse phase HPLC method to quantitatively analyze rhesus globins among differentiated erythroid cells. Peaks on the HPLC chromatogram were eluted and run in liquid chromatography-mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) for protein identification. We identified β-globin and γ-globin (HBG1 and HBG2) peaks using both rhesus peripheral blood (99±26% and 0.65±0.28%) and in vitro differentiated rhesus erythroid cells (63±13% and 37±16%), which is essential for Hb switching analysis. In addition, human β-globin and T87Q β-globin (with anti-sickling mutation) proteins were also separated from rhesus globins in the HPLC chromatogram and were detectable for quantitation.Finally, we evaluated whether fetal Hb expression can be induced by the ZF-Ldb1 molecule in rhesus erythroid cells. We transduced differentiating rhesus CD34+ cells with ZF-Ldb1-encoding lentiviral vectors that are driven by an erythroid specific ankyrin-1 promoter, and contain GFP along with a β-globin locus control region (LCR) enhancer or YFP without the LCR. After erythroid differentiation, we obtained both LCR/ZF-Ldb1/GFP+ (9.4%) and ZF-Ldb1/YFP+ (1.3%) rhesus erythroid cells, and higher γ-globin RNA expression was detected for GFP+ and YFP+ fractions (68.1±1.5% and 59.8±2.1%, respectively), as compared to GFP- and YFP- controls (28.1±3.1% and 31.8±1.9%, p<0.05). These data demonstrate successful γ-globin induction with ZF-Ldb1 vectors in the rhesus macaque.In conclusion, we developed an efficient rhesus erythroid cell differentiation protocol from hematopoietic progenitor cells with robust Hb production detectable at protein levels, analyzed in a newly established HPLC method. The ZF-Ldb1 vector transduction allows for induction of γ-globin expression in rhesus erythroid cells. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation for Engraftment of Gene-Edited CD34+ Cells with a Sickle Cell Disease Mutation in a Rhesus Transplantation Model

Sickle cell disease (SCD) is caused by a 20A&amp;gt;T mutation in the β-globin gene. State-of-the-art genome editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. We have demonstrated efficient gene correction in SCD CD34+ cells with SCD mutation-specific guide RNA, Cas9 mRNA/protein, and single strand donor DNA, resulting in ~30% gene correction and ~50% indels at the DNA level, and ~60% normal β-globin production at the protein level in in vitro erythroid differentiation (ASH 2018). Gene correction by homology directed repair is thought to be enhanced by cell proliferation; however, cell proliferation might reduce stemness of HSCs. To investigate this hypothesis, we sought to evaluate engraftment of gene-edited CD34+ HSCs in a non-human primate model. To model SCD gene correction, a β-to-βs globin conversion was designed in rhesus macaques. Mobilized rhesus CD34+ cells (n=2) were electroporated using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver rhesus β-globin-targeting guide RNA (the same target site as the SCD mutation-specific guide RNA), SpCas9 protein, and single strand donor DNA including a SCD mutation (20A&amp;gt;T). We also added an adjuvant to improve gene conversion efficiencies. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. We observed high-efficiency genome editing without the adjuvant (20-30% gene conversion and 61-64% indels), and further enhanced genome editing with the adjuvant (51-59% gene conversion and 36-39% indels). After erythroid differentiation, we observed production of βs-globin protein (~100%) but not normal β-globin in gene-edited cells. We then evaluated engraftment of gene-edited rhesus CD34+ cells with β-to-βs globin conversion (n=2, 13U005 and 12U011). Mobilized rhesus CD34+ cells (3.4-3.8e7) were pre-stimulated for 2 days, and edited cells were cryopreserved after electroporation with editing tools. Small aliquots of edited cells (before and after cryopreservation) were differentiated into erythroid cells in vitro, resulting in 17-26% of gene conversion and 57-71% of indels at the DNA level and 50-100% of β-globin production at the protein level, with no difference observed between aliquots taken before and after cryopreservation. Following 9.5 Gy total body irradiation, the frozen edited CD34+ cells (1.6-2.2e7) were injected into autologous macaques. We observed robust recovery of blood counts in 13U005, while peripheral blood recovery was delayed in 12U011, who was supported by serial whole blood transfusion. We observed 7-11% of gene conversion and 44-54% of indels in both granulates and lymphocytes in 13U005 1 month post-transplant. Around 15% sickle Hb production in red blood cells was detected by Hb electrophoresis in 13U005 three months post-transplant and ~7% in 12U011 two months post-transplant. Interestingly, ~10% of fetal Hb production was observed in 12U001, likely due to stress hematopoiesis. In summary, we developed a rhesus β-to-βs globin conversion model with HSC-targeted genome editing strategies. The gene-edited rhesus CD34+ cells are engraftable for at least 3 months post-transplant. Although further follow-up is necessary for transplanted animals, these findings are helpful in designing HSC-targeted gene correction trials. Figure Disclosures Li: MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Peshwa:MaxCyte, Inc: Employment.

Durable and Robust Fetal Globin Induction without Anemia in Rhesus Monkeys Following Autologous Hematopoietic Stem Cell Transplant with BCL11A Erythroid Enhancer Editing

Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p&lt;0.01) was observed in BCL11A edited cells (81-85% indels) as compared to 19-25% and 15-24% for non-electroporated and AAVS1 edited cells, respectively, with no significant difference in red blood cell (RBC) enucleation efficiency (44-47%) among groups. We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation. For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×106 CD34+ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs. For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×106 CD34+ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p&lt;0.01). In both edited and unedited animals γ-globin levels peaked in the first two months after transplantation and subsequently declined and plateaued. In ZM17 (~70% BCL11A enhancer indels at ~24 weeks), ~12% γ-globin was observed in peripheral blood (PB) at last measurement (compared to 0.5% γ-globin in RBC prior to transplant). In the same animal, editing ranged from 78-81% across all PB and bone marrow (BM) lineages (excluding CD3+ T-cells with 63% indels), including B-lymphoid, myeloid, erythroid, and HSPCs (in particular including 78% indels in CD71+ CD45- erythroblasts). Hemoglobin, hematocrit, and reticulocyte counts and peripheral smear appearance were all normal, suggesting no erythroid toxicity. Colony-forming ability of BM-derived mononuclear cells was similar in edited and control animals. In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction. Disclosures Weiss: GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy.

Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (&gt;20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p&lt;0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p&lt;0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Busulfan Combined with Immunosuppression Allows Efficient Engraftment of Gene-Modified Cells in a Rhesus Macaque Model.

Busulfan conditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene-therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppression is required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene-therapy model. We transduced half of rhesus CD34+ cells with an enhanced green fluorescent protein (GFP)-encoding vector (immunogenic) and the other half with a γ-globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfan conditioning (5.5mg/kg/day for 4days), we observed immunological rejection of GFP-transduced cells up to 3months post-transplant and stable engraftment of γ-globin-transduced cells in two animals, demonstrating that ablative busulfan conditioning is sufficient for engraftment of gene-modified cells producing non-immunogenic proteins but insufficient to permit engraftment of immunogenic proteins. We then added immunosuppression with abatacept and sirolimus to busulfan conditioning and observed engraftment of both GFP- and γ-globin-transduced cells in two animals, demonstrating that additional immunosuppression allows for engraftment of gene-modified cells expressing immunogenic proteins. In conclusion, myeloablative busulfan conditioning should permit engraftment of gene-modified cells producing non-immunogenic proteins, while additional immunosuppression is required to prevent immunological rejection of a neoantigen.

Robust erythroid differentiation system for rhesus hematopoietic progenitor cells allowing preclinical screening of genetic treatment strategies for the hemoglobinopathies

γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups (P < 0.01). In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.

Development of a New Generation, Forward-Oriented Therapeutic Vector for Hemoglobin Disorders

Ameliorating hemoglobin disorders such as sickle cell disease (SCD) using hematopoietic stem cell (HSC) gene therapy is under development. Unlike in other diseases, therapeutic globin vectors have demanding requirements including high-level β-globin expression, tissue specificity among erythroid cells, long-term persistence, and high-level modification at the HSC level. These demanding requirements necessitate the inclusion of complex genetic elements including the locus control region (LCR), β-globin promoter, β-globin gene, and the 3' untranslated region (3'UTR), all now feasible using lentiviral vectors. The additional requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent loss by RNA splicing during viral preparation. This reverse-orientation is in contrast to all other therapeutic vectors under clinical development. Current reverse-oriented globin vectors can drive phenotypic correction in mouse models for both b-thalassemia and SCD, while they are limited by lower viral titers and lower transduction efficiency in primary human HSCs, limiting their prospects, especially in SCD. We hypothesized that the reverse-orientation impedes both viral preparation and vector transduction, as despite deletion of cryptic polyadenylation (polyA) signals to optimize a conventional reverse-oriented globin vector, titers were still 10-fold lower than a standard GFP-vector.We thus designed a forward-oriented globin-expressing vector, which was further optimized by minimizing the size of the LCR, inclusion of a large segment of the β-globin promoter and an enhancer region of the 3’UTR lacking the polyA signal. Viral titers of the forward-oriented vectors (1.0±0.2x10e9 IU/mL) were 6-fold higher than the optimized vector in the reverse orientation (1.6±0.2x10e8 IU/mL, p<0.01), and comparable to a standard GFP-marking vector (1.9±0.2x10e9 IU/mL, p<0.01). The forward-oriented vector demonstrated 3-4 fold higher transduction efficiency among human erythroid cells derived from transduced CD34+ cells in in vitro culture (34±0% vs 13±1%, p<0.01) and in xenografted mice (31±9% vs 7±5%, p<0.05).To evaluate transduction efficiency for long-term HSCs, we transduced rhesus CD34+ cells with our optimized reverse-oriented vector and our forward-oriented vector including GFP or YFP genes (instead of β-globin gene) in a competitive repopulation assay following 10 Gy total body irradiation. In two animals, GFP and YFP signals from both vectors were detected exclusively in red blood cells, documenting tissue specificity. Gene marking levels were 10-fold higher out to 4 years with the forward-oriented vector, compared to the reverse-oriented vector, and were comparable to standard GFP or YFP-marking vectors in 2 other animals.We then replaced the GFP gene with the β-globin gene containing intron 2 in the forward-oriented vector construct. To positively select intron-2-containing β-globin vectors, essential viral components (packaging signal, rev response element (RRE), or central polypurine tract (cPPT)) were deleted in the backbone of the forward-oriented vector, and the deleted viral components were inserted into intron 2 of the β-globin gene. We observed that half of the forward-oriented vectors lost intron 2 during vector preparation when no elements were included into intron 2. Insertion of the RRE resulted in positive selection of intron-2-containing β-globin vectors. We confirmed β-globin expression from the forward-oriented vector by hemoglobin A production in human erythroid cells derived from transduced peripheral blood mononuclear cells from SCD patients. Finally, human β-globin expression was detected in rhesus erythroid cells following transplantation of transduced CD34+ cells in 2 animals.In summary, we have developed a clinically relevant forward-oriented globin-expressing vector, which has 6 fold higher viral titers and 4-10 fold higher transduction efficiency for hematopoietic repopulating cells, as compared to the optimized reverse-oriented vector. RRE insertion allowed positive selection of intron-2-containing β-globin vectors, and human β-globin production was observed in transplanted rhesus macaques with the forward-oriented β-globin vector transduction. These findings bring us closer to a curative gene therapy for hemoglobin disorders. DisclosuresNo relevant conflicts of interest to declare.

523. Human Umbilical Vein Endothelial Cell, HUVEC, Co-Culture Promotes Robust Expansion and Maintains Phenotypic Integrity of Rhesus Hematopoietic Stem and Progenitor Cells, HSPC, Prior to Autologous Tansplantation

The development of ex-vivo HSPC expansion techniques is particulary relevant for improving cord blood transplantation and gene therapy. Despite successful long-term, multilineage reconstitution of expanded human cord blood HSPC in immunodeficient mice, early phase clinical trials have failed to demonstrate improved outcomes. Thus, it is critical to develop a robust pre-clinical model to study ex-vivo expansion strategies, particularly function of long term HSPC and engrafment of all lineages, difficult in xenograft models. We have previously used retroviral insertion site retrieval and autologous competitive transplantation in rhesus macaques to track hematopoietic engraftment and ontogeny at a single HSPC level, in addition to comparing ex-vivo expanded and unexpanded HSPC (Gomes et al, Mol Ther). More recently we have developed retrieval of 31bp diverse barcodes as a more robust and quantitative in vivo HSPC tracking approach (Wu et al, Cell Stem Cell). Modified human endothelial cells (HUVEC) with the ability to be maintained in serum free media for prolonged periods have shown to support and expand human HSPC in murine Butler et al, Blood). We have developed a barcoded rhesus autologous transplant model to evaluate expansion of HPSC on HUVEC versus unexpanded or cytokine expanded rhesus HSPC. We first tested the feasibility of rhesus CD34+ cell expansion on HUVEC versus cytokines/fibronectin for 8 days. We also evaluated for the diversity of expanded HSPC by retrieving transduced viral barcode DNA tags from individual cells by low cycle PCR followed by Illumina sequencing. On average, rhesus CD34+ cells expanded 76 fold (± 53) on HUVEC versus 13 fold (±7) in cytokines (n=4; p=0.03). By morphologic assessment, the HUVEC expanded cell fraction contained higher numbers of progenitors and differentiating cells, with up to 97% CD34+CD45+ cells in the HUVEC expanded fraction and 65% in the cytokine expanded fraction. The HUVEC expanded rhesus CD34+ cells were capable of multi-lineage colony formation after 8 days of expansion. Within the HUVEC expanded cell fractions, fold expansion was similar whether CD34+ cells were frozen after (C1) or before (C2) transduction-expansion. However, viability and percentages of CD34+CD45+ cells were higher in C2 compared to C1 (55% versus 26%). GFP transduction efficiency was also higher in C2 compared to C1 (43% vs. 28%). We demonstrated a slightly lower percentage but higher absolute number of CD34+CD38-CD45RA-CD90+CD49f+Rho-low putative long-term HSC after expansion on HUVEC compared to in cytokines over 26 days in culture (0.018% vs. 0.016% pre and post expansion, respectively). Retrieved barcode analysis and quantitation revealed that in the HUVEC expanded fraction, the top 20 clones constituted up 15% of the total valid reads by day 8, in contrast to only 3.9% of total clones in the cytokine expanded fraction. Cells have been transplanted into autologous macaques, in a competitive model comparing non-expanded, cytokine-expanded and HUVEC-expanded conditions, and in vivo expansion and barcode analysis will be presented. Our data thus far show that peripheral blood CD34+ cells from rhesus macaques expand more robustly in the presence of HUVEC and cytokines as compared to expansion in cytokines alone, and barcode analysis suggests that the HUVEC fraction selectively expanded a subset of highly proliferative cells, which may represent true HSCs within the CD34+ cell fraction.

Total body irradiation must be delivered at high dose for efficient engraftment and tolerance in a rhesus stem cell gene therapy model

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) gene therapy applications. However, low gene marking was previously observed in gene therapy trials, suggesting that RIC might be insufficient for (i) opening niches for efficient engraftment and/or (ii) inducing immunological tolerance for transgene-encoded proteins. Therefore, we evaluated both engraftment and tolerance for gene-modified cells using our rhesus HSC gene therapy model following RIC. We investigated a dose de-escalation of total body irradiation (TBI) from our standard dose of 10Gy (10, 8, 6, and 4Gy), in which rhesus CD34+ cells were transduced with a VSVG-pseudotyped chimeric HIV-1 vector encoding enhanced green fluorescent protein (GFP) (or enhanced yellow fluorescent protein (YFP)). At ~6 months after transplantation, higher-dose TBI resulted in higher gene marking with logarithmic regression in peripheral blood cells. We then evaluated immunological tolerance for gene-modified cells, and found that lower-dose TBI allowed vigorous anti-GFP antibody production with logarithmic regression, while no significant anti-VSVG antibody formation was observed among all TBI groups. These data suggest that higher-dose TBI improves both engraftment and immunological tolerance for gene-modified cells. Additional immunosuppression might be required in RIC to induce tolerance for transgene products. Our findings should be valuable for developing conditioning regimens for HSC gene therapy applications.

294. Myeloablative Conditioning Is Required for Efficient Engraftment of Gene-Modified Cells and Prevention of Antibody Production Against Transgene Products in a Rhesus Stem Cell Gene Therapy Model

Reduced intensity conditioning (RIC) regimens are desirable for hematopoietic stem cell (HSC)-targeted gene therapy. However, in previous gene therapy trials, low gene marking was reported in peripheral blood cells, raising the question that RIC might be insufficient for (1) opening niches for efficient engraftment and (2) inducing immunological tolerance to transgenes. Therefore, we sought to evaluate both engraftment and tolerance for genetically modified cells using our rhesus HSC gene therapy model following RIC.We performed a dose de-escalation of total body irradiation (TBI) (10, 8, 6, and 4Gy) as RIC transplantation (total 19 animals), in which mobilized rhesus CD34+ cells were transduced with a VSV-G-pseudotyped chimeric HIV-1 vector encoding GFP (or YFP) and these cells were transplanted into autologous animals following TBI. We evaluated GFP-positive rates (%GFP), average vector copy number per cell (VCN), and antibody titers against GFP and VSV-G in peripheral blood 6 months after transplantation.When we evaluated in vitro %GFP in transduced rhesus CD34+ cells, efficient transduction (22-71%) was observed among all TBI groups (without correlation). After transplantation of the transduced CD34+ cells, GFP-positive cells were detected in peripheral blood cells in all animals. Increasing doses of TBI resulted in higher %GFP with logarithmic regression in both granulocytes (R2=0.64, p<0.01) and lymphocytes (R2=0.67, p<0.01)(Figure). Similar results were observed between TBI dose and VCNs in both granulocytes (R2=0.54, p<0.01) and lymphocytes (R2=0.52, p<0.01). These data suggest that higher doses of TBI improve engraftment of gene-modified hematopoietic repopulating cells.To evaluate immunological tolerance for gene-modified cells, we measured both anti-GFP and anti-VSV-G antibody titers using serum samples from transplanted animals. Lower dose TBI allowed anti-GFP antibody production with logarithmic regression (R2=0.84, p<0.01)(Figure), while no significant effect of anti-VSV-G antibody was observed among all TBI groups (R2=0.003, p=0.87). These data demonstrate that higher dose TBI is important to induce immunological tolerance for gene-modified cells in a rhesus transplantation model.In summary, higher dose of TBI increased gene marking levels in transplanted rhesus macaques, while lower dose of TBI resulted in anti-GFP antibody production. Additional immunosuppressive therapy might be required in RIC to induce immunological tolerance for transgene products. Our findings should be valuable to consider conditioning regimens for clinical gene therapy. View Large Image | Download PowerPoint Slide

TRIM5α Variations Influence Transduction Efficiency With Lentiviral Vectors in Both Human and Rhesus CD34+ Cells In Vitro and In Vivo

Human immunodeficiency virus type 1 (HIV-1) vectors can transduce human hematopoietic stem cells (HSC), but transduction efficiency varies among individuals. The innate immune factor tripartite motif-containing protein 5α (TRIM5α) plays an important role for restriction of retroviral infection. In this study, we examined whether TRIM5α could account for variations in transduction efficiency using both an established rhesus gene therapy model and human CD34(+) cell culture. Evaluation of TRIM5α genotypes (Mamu-1, -2, -3, -4, -5, and TrimCyp) in 16 rhesus macaques that were transplanted with transduced CD34(+) cells showed a significant correlation between TRIM5α Mamu-4 and high gene marking in both lymphocytes and granulocytes 6 months after transplantation. Since significant human TRIM5α coding polymorphisms were not known, we evaluated TRIM5α expression levels in human CD34(+) cells from 14 donors. Three days after HIV-1 vector transduction, measured transduction efficiency varied significantly among donors and was negatively correlated with TRIM5α expression levels. In summary, transduction efficiency in both rhesus and human CD34(+) cells was influenced by TRIM5α variations (genotypes and expression levels). Our findings are important for both understanding and mitigating the variability of transduction efficiency for rhesus and human CD34(+) cells.

CD34+CXCR4(CD184)+ Cells Differentiate Into Myeloid Dendritic Cell Progenitors

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. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34+ Cells Correlates With In Vivo Vector Copy Number

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.

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.

Evaluation of Human and Rhesus CD34+CXCR4+ and CD34+CXCR4- Subpopulations

Abstract 1007CXCR4 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<0.01) and bulk CD34+ cells (21.0 ± 2.0%, p<0.01). Following transduction, the rhesus CD34+CXCR4+ derived cells also had significantly lower EGFP expression (7.0 ± 0.3%) when compared to their CD34+CXCR4- counterpart (25.4 ± 1.2%, p<0.01) and bulk CD34+ cells (22.6 ± 2.2%, p<0.01). In CFU assay, human CD34+CXCR4+ cells showed fewer colony numbers (Erythroid (E): 6.2 ± 1.7/1000 cells, Myeloid (M): 7.0 ± 0.0/1000 cells) when compared to CD34+CXCR4- derived cells (E: 21.5 ± 7.9/1000 cells, ns, M: 24.7 ± 4.8/1000 cells, p<0.05) and bulk CD34+ cells without lentiviral transduction (E: 37.3 ± 1.2/1000 cells, p<0.05, M: 24.7 ± 4.8/1000 cells, p<0.01). To determine whether the CD34+CXCR4+ or CD34+CXCR4- subpopulation contained hematopoietic repopulating cells, human CD34+CXCR4+ cells (4×10e4 cells/mouse) and CD34+CXCR4- cells (4×10e4 cells/mouse or 2×10e6 cells/mouse) with lentiviral gene-marking were transplanted into NOD/SCID/IL2Rγ null mice. Human cell chimerism and %EGFP in the peripheral blood cells were analyzed every 4 weeks for over 6 months (Figure). CD34+CXCR4+ cell-transplanted mice showed no engraftment of human cells (less than 1% of human CD45+ cells) at all time points, while human cells were detected in the peripheral blood of the CD34+CXCR4- cell-transplanted mice (4×10e4 cells/mouse) until 12 weeks after transplantation. The CD34+CXCR4- cell-transplanted mice (2×10e6 cells/mouse) showed human cell engraftment for over 6 months. These data suggest that the CD34+CXCR4- cell population contain hematopoietic repopulating cells. To evaluate the nature of the CD34+CXCR4+ cell population, global microarray gene expression analysis was performed both on human and non-human primate CD34+CXCR4+ cells. Microarray signatures showed different gene profiles between CD34+CXCR4+ and CD34+CXCR4- populations in both human and rhesus CD34+ cells. Both human and rhesus CD34+CXCR4+ populations demonstrated strong monocyte- and neutrophil-mediated inflammatory response gene expression profiles, suggesting that the CD34+CXCR4+ subpopulation in both human and non-human primates contain progenitors instrumental in the innate immune response. These results indicate that while the addition of AMD3100 to G-CSF mobilization protocols may increase the number of CD34+ cells mobilized, some of these cells may not be as potent for gene therapy and transplantation applications and support the design of immunoselection protocols which target specific subpopulations of CD34+ cells for therapeutic applications in the future. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High Transgene Expression Rates After Extended Follow up Among Rhesus Macaque Recipients of Autologous Hematopoietic Stem Cells Transduced with a Modified HIV1-Based Lentiviral Vector

Abstract 3118Hematopoietic 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<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors.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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Microarray Signature of Rhesus Macaque CD34+CD123 (IL-3 Receptor)+ Hematopoietic Stem/Progenitor Cells Isolated Following Mobilization with Plerixafor Alone or in Combination with Granulocyte Colony-Stimulating Factor (G-CSF)

Abstract 1301CD34+ 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<0.05) there was further confirmation of clustering of the CD34+CD123+ cells versus the CD34+CD123- cells. Microarray analysis revealed that pathways including retinoic acid receptor activation and TGF-β and MAPK signaling were up-regulated in CD34+CD123+ cells, whereas glucocorticoid receptor and TREM-1 signaling, and genes involved in dendritic cell maturation and production of nitric oxide and reactive oxygen by macrophages were down regulated. The up regulated pathways and the down regulated pathways for the CD34+CD123+ population suggest that CD34+CD123+ cells are lymphoid progenitors. Upon comparing pathways between those previously reported to be unique to CD34+ cells mobilized with plerixafor alone or in combination with G-CSF, the majority are those that have been identified also to be in the CD34+CD123+ subpopulation. It appears as if the CD34+CD123+ population accounts for many of the differences in CD34+ cells mobilized with plerixafor observed in the earlier study. In conclusion, the microarray signature of the CD34+CD123+ cells subpopulation suggests that these cells are lymphoid progenitors which can be effectively mobilized with plerixafor. This may account for the more rapid lymphoid recovery we observed for rhesus macaques transplanted with CD34+ cells mobilized with plerixafor and suggests a therapeutic approach through immunoselection to selectively target CD34+CD123+ progenitor populations. Disclosures:No relevant conflicts of interest to declare.