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Ex vivo hematopoietic stem and progenitor cell (HSPC) expansion platforms are under active development, designed to increase HSPC numbers and thus engraftment ability of allogeneic cord blood grafts or autologous HSPCs for gene therapies. Murine and in vitro models have not correlated well with clinical outcomes of HSPC expansion, emphasizing the need for relevant pre-clinical models. Our rhesus macaque HSPC competitive autologous transplantation model utilizing genetically barcoded HSPC allows direct analysis of the relative short and long-term engraftment ability of lentivirally transduced HSPCs, along with additional critical characteristics such as HSPC clonal diversity and lineage bias. We investigated the impact of ex vivo expansion of macaque HSPCs on the engineered endothelial cell line (E-HUVECs) platform regarding safety, engraftment of transduced and E-HUVEC-expanded HSPC over time compared to non-expanded HSPC for up to 51 months post-transplantation, and both clonal diversity and lineage distribution of output from each engrafted cell source. Short and long-term engraftment were comparable for E-HUVEC expanded and the non-expanded HSPCs in both animals, despite extensive proliferation of CD34+ cells during 8 days of ex vivo culture for the E-HUVEC HSPCs, and optimization of harvesting and infusion of HSPCs co-cultured on E-HUVEC in the second animal. Long-term hematopoietic output from both E-HUVEC expanded and unexpanded HSPCs was highly polyclonal and multilineage. Overall, the comparable HSPC kinetics of macaques to humans, the ability to study post-transplant clonal patterns, and simultaneous multi-arm comparisons of grafts without the complication of interpreting allogeneic effects makes our model ideal to test ex vivo HSPC expansion platforms, particularly for gene therapy applications., Graphical Abstract, The rhesus macaque barcoded autologous competitive repopulation preclinical transplantation model was utilized to study the engraftment and safety of hematopoietic stem and progenitor cells expanded in co-culture with E4ORF-1 gene transduced human umbilical vein endothelial cell as compared to non-expanded cells, documenting polyclonal long-term multilineage hematopoiesis.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow. Here, we compare engraftment of CD34+ cells transplanted in a myeloablative rhesus macaque (RM) model using the OIB method compared with IV delivery. RM CD34+ cells obtained by apheresis were split equally for transduction with lentiviral vectors encoding either green fluorescent protein or yellow fluorescent protein reporters. Following conditioning, one marked autologous population of CD34+ cells was injected directly IB using the OIB method and the other was injected via slow IV push into the same animal (n = 3). Daily flow cytometry of blood quantified the proportion of engrafting cells deriving from each source. Marrow retention was examined using positron emission tomography/computed tomography imaging of 89Zirconium (89Zr)-oxine–labeled CD34+ cells. CD34+ cells injected via the OIB method were retained in the marrow and engrafted in all 3 animals. However, OIB-transplanted progenitor cells did not engraft any faster than those delivered IV and contributed significantly less to hematopoiesis than IV-delivered cells at all time points. Rigorous testing of our OIB delivery system in a competitive RM myeloablative transplant model showed no engraftment advantage over conventional IV infusion. Given the increased complexity and potential risks of IB vs IV approaches, our data do not support IB transplantation as a strategy to improve hematopoietic engraftment.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer–edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer– or control locus–targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)–targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A–based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A–expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment. In contrast to murine aging models reporting persistence of myeloid-biased HSPCs, aged macaques demonstrated persistent output from both B-cell and myeloid-biased clones. Clonal expansions of MP, myeloid-biased, and B-biased clones occurred in aged macaques, providing a potential model for human clonal hematopoiesis of indeterminate prognosis. These results suggest that long-term MP HSPC output is impaired in aged macaques, resulting in differences in the kinetics and lineage reconstitution patterns between young and aged primates in an autologous transplantation setting.

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. Figure 1 Figure 1. Disclosures Latimer: 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.

Steady state bone marrow (BM) is the preferred hematopoietic stem cell (HSC) source for gene therapy in sickle cell disease (SCD) due to the recognized risk of vaso-occlusive crisis during granulocyte colony–stimulating factor mobilization. We previously established clinically relevant HSC gene transfer in the rhesus model following transplantation of mobilized peripheral blood (PB) CD34+ cells transduced with lentiviral vectors. In this study, we examined steady state bone marrow (BM) in the rhesus competitive repopulation model and demonstrate similar gene marking in vitro and in vivo, as compared with mobilized PB CD34+ cells. We then evaluated PB and steady state BM in subjects with SCD and observed a higher frequency of CD34+ cells when compared with controls, likely due to enhanced hematopoiesis. However, CD34+ cell counts were reduced in both the PB and BM in patients treated with hydroxyurea, and hydroxyurea treatment strongly inhibited iPS cell generation from SCD subjects. Our data support that steady state BM is a useful HSC source for SCD gene therapy with similar transduction. The lower CD34+ percentages observed with hydroxyurea treatment warrants withholding hydroxyurea temporarily prior to harvesting HSCs. Our results are important for the design of gene targeting strategies for SCD.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34(+) selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34(+) cell count was lower in SCD BM (P < 0·05), however CD34(+) count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P < 0 05). Greater than 50% of CD34(+) HSPCs from SCD BM are CD34(dim) due to higher erythroid lineage expression (P < 0·01) as single cell CD34(+)CD45(+)GPA(+) (P < 0·01) and CD34(+)CD45(‒)GPA(+) (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34(+) cell transplantation. HbF protein expression peaked in the first 4–9 weeks posttransplant (0.99%–2.53%), and F-cells increased in the first 6–17 weeks posttransplant (8.7%–45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4–7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34(+) cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development. Published by Elsevier Inc. on behalf of ISEH – Society for Hematology and Stem Cells.

Summary Sickle cell disease (SCD) is caused by a 20A > T mutation in the β-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34+ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34+ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus β-to-βs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34+ cells 10–12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34+ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials., Graphical abstract, Highlights Sickle cell disease (SCD) is a blood disease caused by a mutation in the β-globin gene A viral vector-free non-footprint gene correction is developed for SCD CD34+ cells Achieve therapeutic-level SCD gene correction of DNA (∼30%) and protein (∼80%) Demonstrate engraftment of gene-edited CD34+ cells in xenografts and non-human primates, Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene and can be cured by the replacement of hematopoietic stem cells (HSCs). Uchida et al. demonstrate a high-efficiency gene correction method for the SCD mutation and engraftment of gene-edited CD34+ HSCs in xenograft mice and non-human primates.

Lentiviral vectors (LV) have been used for the delivery of genes into hematopoietic stem and progenitor cells (HSPC) in clinical trials worldwide. LV, in contrast to retroviral vectors, have not been associated with insertion site-associated malignant clonal expansions, and thus have been considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis impacting the erythroid, myeloid and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. 9 insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between a lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPC, and are thus concerning, and suggest that strong constitutive promoters should not be included within LV vectors.

Autologous hematopoietic stem cell transplantation (Auto-HSCT) with gene-modification represents a potential cure for multiple genetic diseases, but its broad curative potential, is limited because of morbidity/mortality from cytotoxic chemotherapy-based conditioning. To overcome these limitations, we developed antibody drug conjugates (ADC) targeting CD117 (C-KIT) to specifically deplete the hematopoietic stem and progenitor cells (HSPC). To validate CD117 ADC-mediated depletion prior to HSCT, we developed an optimized non-human primate (NHP) tool anti-CD117 ADC and evaluated it in an auto-gene modified HSCT in a rhesus model. The CD117-ADC is potent on primary human and NHP CD34+ cells in vitro (Figure 1A). Humanized NSG mice treated with a single dose had full depletion of human HSPCs in the bone marrow, while maintaining peripheral immune cells. In rhesus a single administration was fully myeloablative (>99% HSPC depletion) and comparable to HSPC depletion observed following busulfan conditioning (6 mg/kg/day x4). There was no effect of the ADC on the peripheral and bone marrow lymphocytes and the ADC was well tolerated compared to busulfan where the animals had diarrhea, loss of appetite and weight loss. To facilitate use in HSCT, the CD117-ADC was engineered to have a fast clearance and the half-life was We next explored whether the tool CD117-ADC could enable auto gene modified HSCT in the rhesus model. Two rhesus NHP were mobilized with GCSF and plerixafor. The selected CD34+ cells were transduced with β-globin encoded lentivirus and cryopreserved. The tool CD117-ADC was dosed on day -6 and the CD34+ cells were thawed and infused on day 0. Bone marrow aspirates analyzed on the day of infusion (day 0) demonstrated >99% depletion of the HSPCs and maintenance of the bone marrow lymphocytes (Figure 1B). The primates engrafted neutrophils (day 8 and 10) and platelets (day 10 and 11), and the peripheral lymphocytes were maintained throughout the transplant (Figure 1C-D). The gene marking in the granulocytes is comparable to busulfan conditioned animals previously reported (Tisdale, Molecular Therapy 2019). Longer follow up and data from additional animals will be presented. In summary, we have developed a tool CD117 ADC that shows potent activity on NHP CD34+ cells, is fully myeloablative, has a favorable safety profile, spares the immune system and is cleared rapidly as designed. In a rhesus model of auto-gene modified HSCT, a single dose of the ADC enables engraftment of gene modified HSC. These proof of concept studies validate the use of CD117-ADC for targeted HSPC depletion prior to transplant and support its use as a new conditioning agent for auto-gene modified HSCT. This targeted approach for safer conditioning could improve the risk benefit profile for patients undergoing HSCT and enable more patients to benefit from these potentially curative therapies.

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 (5.5 mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months 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.

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented β-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and β-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders., Vectors used in gene therapy for hemoglobin disorders carry globin in a reverse-orientation to prevent the loss of key regulatory elements by RNA splicing, but this limits their efficiency. Here, the authors develop a vector carrying β-globin in a forward orientation and show that it has improved titers and transduction efficiency in humanized mice and nonhuman primates.

Ionizing irradiation is used routinely to induce myeloablation and immunosuppression. However, it has not been possible to evaluate the extent of ablation without invasive biopsy. For lymphoid recovery, peripheral blood (PB) lymphocytes (PBLs) have been used for analysis, but they represent

In mice, clonal tracking of hematopoietic stem cells (HSCs) has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3-10 years), arising in sequential groups and likely representing self-renewing HSCs. The remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid, or both myeloid and lymphoid lineages. Over time, the 4%-10% of clones with robust dual lineage contribution predominated in repopulation. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically relevant model over a long time frame and provides a substantial system-level data set that is a reference point for future work. © 2014 Elsevier Inc.

Autologous hematopoietic stem cell transplantation (Auto-HSCT) with gene-modification techniques represents a potential cure for multiple genetic blood diseases. Despite its broad curative potential, auto-gene modified HSCT is currently limited due to morbidity/mortality from cytotoxic chemotherapy-based conditioning, including risks of secondary malignancies, organ toxicity, and infertility. To overcome these limitations, we have developed antibody drug conjugates (ADC) targeting CD117 (C-KIT) to specifically deplete the hematopoietic stem and progenitor cells (HSPC) prior to auto-gene modified HSCT. We have previously shown that the anti-CD117 ADC is highly effective at killing human CD117+ cells in vitro and in vivo (Pearse et al., Blood 2018 132:3314). To validate CD117 as an appropriate antigen for targeted ADC-mediated depletion prior to HSCT, we developed an optimized non-human primate (NHP) tool anti-CD117 ADC and evaluated it in an auto-gene modified HSCT in the rhesus macaque model. The tool CD117-ADC is potent on primary human and NHP CD34+ cells in vitro with EC50 of 0.2 and 0.09 pM respectively (Figure 1A). Humanized NSG mice treated with the tool CD117-ADC had full depletion of human HSPCs in the bone marrow 21 days after a single administration of the ADC, while maintaining the peripheral immune cells. We next tested the efficacy and safety of the tool CD117-ADC in NHPs. A single administration of the tool CD117-ADC was fully myeloablative (>99% HSPC depletion) and comparable to HSPC depletion observed following busulfan conditioning (6 mg/kg, once daily for 4 consecutive days). There was no effect on the peripheral and bone marrow lymphocytes and the ADC was well tolerated. To facilitate the use in HSCT, the tool CD117-ADC was engineered to have a fast clearance and in this study the half-life was Based on these encouraging results, we explored whether the tool CD117-ADC could enable engraftment of autologous gene modified hematopoietic stem cells in the rhesus macaque model. A single rhesus macaque was mobilized with granulocyte-colony stimulating factor (G-CSF, 20 mcg/kg/day x 5) and plerixafor (1 mg/kg on day 5 of G-CSF) prior to apheresis. The isolated CD34+ cells were transduced with a lentivirus encoding the β-globin gene and cryopreserved. The tool CD117-ADC was dosed on day -6 and the cryopreserved gene modified cells were thawed and infused (3.3 x 106 CD34+ cells/kg) on day 0. A bone marrow aspirate analyzed on the day of infusion (day 0) demonstrated >99% depletion of the HSPCs and preserved of the bone marrow lymphocytes (Figure 1B). The primate engrafted neutrophils and platelets on day 8 and 10 respectively, and the peripheral lymphocytes were maintained throughout the transplant (Figure 1C). The gene marking in the granulocytes was detectable at day 9, and additional follow up and data from additional animals will be presented. In summary, we have developed a tool CD117 ADC that shows potent activity on NHP CD34+ cells. This optimized CD117-ADC is fully myeloablative with a single dose in NHPs, has a favorable safety profile, spares the immune system and is cleared rapidly as designed. In a rhesus model of autologous gene modified HSCT, a single dose of the ADC enables engraftment of auto-gene modified HSC. These proof of concept studies validate the use of CD117-ADC for targeted HSPC depletion prior to transplant and support its use as a new conditioning agent for autologous gene modified HSCT. This targeted approach for safer conditioning could improve the risk benefit profile for patients undergoing stem cell transplant and enable more patients to benefit from these potentially curative therapies. Disclosures Pearse: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonough:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Latimer:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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 (>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 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.

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 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 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.

Sickle cell disease (SCD) is caused by a 20A>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>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.

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34+ cell transplantation. HbF protein expression peaked in the first 4–9 weeks posttransplant (0.99%–2.53%), and F-cells increased in the first 6–17 weeks posttransplant (8.7%–45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4–7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34+ cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development.

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34(+) cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34(+) cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.

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.

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.

In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4+/CD25high/CD127low/FoxP3+ Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8+ T cells were mobilized to a greater extent than CD4+ T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8+ effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.

SummaryHematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

Introduction: Umbilical cord blood (UCB) grafts are the only option for a significant minority of patients who require hematopoietic stem cell transplantation (HSCT) but lack a suitable related or unrelated donor. While UCB can serve as a suitable 'off the shelf' graft for many patients, units with the best HLA match often contain low and sometimes insufficient numbers of CD34+ cells for use in transplantation, particularly for adult patients. Furthermore, UCB grafts contain lower CD34+ cells number compared to BM or PBSC grafts, which leads to longer engraftment times and a higher risk of graft failure. BM and PBSC grafts are usually injected intravenously, homing to the bone marrow after several hours in the circulation. During this time, CD34+ cells are lost in the lungs, liver and spleen with typically < 20% making it to the bone marrow.1 Investigators have sought to overcome the limitation of low cell dose in UCB grafts and loss of CD34+ cells in the circulation by injecting CD34+ cells directly into the bone marrow space. However, we have recently shown that conventional intrabone delivery methods used in investigational trials to transplant UCB, result in low-level retention of hematopoietic progenitor cells in the intrabone space. Recently, we developed an optimized intrabone (OIB) transplant method using computer controlled low pressure and low volume injection (controlled infusion rate Methods: Rhesus macaques received GCSF and plerixafor mobilization prior to apheresis. Products were CD34+ selected using Miltenyi beads. CD34+ cells were split equally for transduction with lentiviral vectors encoding the reporters GFP and YFP. After myeloablative conditioning with 10Gy total body irradiation, half the graft was injected directly intra-bone using the OIB method, with the other half of the autograft simultaneously being injected intravenously via slow iv push. Peripheral blood samples were measured daily by flow cytometry to assess the proportion of engrafting cells deriving from each source. To address whether OIB transplantation could allow engraftment of low CD34+ cell numbers, as found in UCB units, the cell doses transplanted in one rhesus macaque recipient were reduced to only 0.5 x 106CD34+ cells/kg. Results: CD34+ cells injected intrabone utilizing the OIB method engrafted in all 3 animals. However, flow cytometric analysis gating on GFP vs YFP positive neutrophils showed CD34+ cells injected utilizing OIB delivery did not engraft quicker than IV transplanted cells. Sequential monitoring of neutrophils over 30 days showed the contribution to hematopoiesis of OIB delivered cells was significantly lower than the cells injected IV (Table 1.) Conclusions: We developed a novel intrabone delivery system that optimizes the retention of CD34+ cells into the bone marrow space. Although autologous CD34+ cells injected using this OIB transplant method were capable of engrafting in rhesus macaques that had undergone myeloablative conditioning, they did not engraft faster and contributed less to hematopoiesis than CD34+ cells simultaneously transplanted using conventional IV infusion. These data raise questions over whether intrabone delivery, even when using techniques to optimize intrabone retention, has utility in improving CD34+ cell engraftment. References: 1. Van der Loo JC et al. Marrow and spleen seeding efficiencies of all murine hematopoietic stem cell subsets are de- creased by preincubation with hematopoietic growth factors. Blood. 1995; 85:2598-2606 2. Pantin et. al. Optimization of intrabone delivery of hematopoietic progenitor cells in a swine model using cell radiolabeling with 89zirconium. American J Transplant. 2015 Mar;15(3):606-17. Disclosures Davidson: Macrogenics: Employment. Pantin:NIH: Patents & Royalties: a patent application for an intrabone delivery device. Dunbar:National Institute of Health: Research Funding.

Introduction : Although autologous transplantation of peripheral blood stem cells (PBSC) - mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) - has been well characterized, the efficacy of G-CSF/SCF-primed bone marrow stem cell (BMSC) transplantation, however, remains unclear and controversial. In our previous lentiviral vector-mediated PBSC and BMSC transplantation study, we reported efficient and long-term hematopoietic reconstitution by PBSC but not by BMSC - the later being associated with the gradual decline of vector markings in two BMSC-transplanted animals, with a loss of marking occurring in most lineages by 26 or 32 weeks after transplant. Follow-up analysis indicated that low-level yet consistent repopulation by BMSC continued in these animals for a longer period. Here we have compared peripheral blood (PB) markings and vector integration sites (VIS) in PBSC- and BMSC-transplanted animals for up to 12 years and 6 years, respectively. Methods : Young adult rhesus macaques were treated with G-CSF (10 mg/kg of body weight/day) and SCF (200 mg/kg/day) four days before the cell harvest for transplant. Mobilized PB leukapheresis cell products from five rhesus macaques (95E132, 2RC003, RQ5427, RQ3570, and 96E035) were harvested using a CS3000 Plus blood cell separator. Bone marrow (BM) cells from two animals (95E131 and 96E041) were surgically harvested from their femurs and iliac crests under anesthesia. After harvest, PBSC and BMSC were isolated by Ficoll-Hypaque density centrifugation followed by immunoselection of CD34+ cells, and transduced with HIV-based self-inactivating lentiviral vectors expressing EGFP. Vector-marked cells were then autologously transplanted into the host after total body irradiation (10 Gy). No further priming treatment was provided after transplant. PB from the 5 PBSC- and 2 BMSC-transplanted animals were serially collected over time and cryo-preserved for PCR, flow cytometry, and VIS analyses. Results: Both PBSC- and BMSC-transplanted animals showed long-term repopulation for lymphocytes, monocytes, granulocytes, platelets, and red blood cells. PBSC animals showed an average EGFP marking that ranged from 0.32 % to 10.24 %. From these animals, a total of 141 to 4,858 VIS were recovered. We found that the total number of VIS in each animal was proportional to the average EGFP marking in the same animals, and that both of these in turn were linearly correlated with the number of EGFP+ CD34+ cells initially transplanted (1.4 x106 - 28.8 x106 per animal). BMSC-transplanted animals did not show any such patterns. In two BMSC animals, the average EGFP marking levels remained at 0.05 % and 0.10 % until the end point (5 and 6 years) despite the fact that a comparatively large number of EGFP+ CD34+ cells had been transplanted (5.2 x106 and 17.7x106)and a large number of VIS recovered (793 and 680 VIS) in these animals. Temporal VIS analysis of PBSC animals showed that different groups of a large number of PBSC clones repopulated sequentially and reached a point of maximum repopulation at different time points, with some gradually declining after this. BMSC animals also showed a wave-like sequential repopulation similar to the patterns seen in PBSC animals. Unlike PBSC, however, nearly all BMSC clones were detected at a low frequency and at a single time point, except a few larger ones that were detected at multiple time points in a rising and falling pattern. There was no notable difference between the genomic features of VIS in PBSC- and BMSC-repopulating cells. Conclusions : Our data suggest that both the BMSC and PBSC consist of highly heterogeneous stem/progenitor cells that can provide long-term polyclonal repopulation through wave-like, sequential repopulation. Unlike PBSC, however, BMSC transplant was inefficient in PB repopulation resulting in only barely detectable markings in PB. The BMSC clonal profiles reflected the clonal patterns seen in PBSC animals, aside from BMSC animals having primarily low-frequency clones. We have previously shown significant differences in immunophenotype and cell cycle status between PBSC and BMSC, where BMSC were significantly lower in Thy-1 expression and had a higher percentage of cells in the S+G2/M phase of the cell cycle than PBSC. These differences may account for the inefficient differentiation and proliferation capabilities of BMSC compared to PBSC shown in this study. Disclosures Dunbar: National Institute of Health: Research Funding.

Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5′ and the 3′ vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.

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

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

Objective Granulocyte colony-stimulating factor (G-CSF) is frequently used therapeutically to treat chronic or transient neutropenia and to mobilize hematopoietic stem cells. Shortly following G-CSF administration, we observed a dramatic transient drop in circulating neutrophil number. This article characterizes this effect in a rhesus macaque animal model. Methods Hematologic changes were monitored following subcutaneous (SQ) administration of G-CSF. G-CSF was administered as a single SQ dose at 10 μg/kg or 50 μg/kg. It was also administered (10 μg/kg) in combination with stem cell factor (SCF; 200 μg/kg) over 5 days. Flow cytometry was performed on serial blood samples to detect changes in cell surface adhesion protein expression. Results Neutrophil count dramatically declined 30 minutes after G-CSF administration. This decline was observed whether 10 μg/kg G-CSF was administered in combination with SCF over 5 days, or given as a single 10 μg/kg dose. At a single 50 μg/kg dose, the decline accelerated to 15 minutes. Neutrophil count returned to baseline after 120 minutes and rapidly increased thereafter. An increase in CD11a and CD49d expression coincided with the drop in neutrophil count. Conclusion A transient paradoxical decline in neutrophil count was observed following administration of G-CSF either alone or in combination with SCF. This decline accelerated with the administration of a higher dose of G-CSF and was associated with an increase in CD11a and CD49d expression. It remains to be determined whether this decline in circulating neutrophils is associated with an increase in endothelial margination and/or entrance into extravascular compartments.

Modeling human hematopoietic progenitor cell gene therapy in nonhuman primates allows long-term evaluation of safety, maintenance of gene expression, and potential immune response against transgene products. We transplanted autologous G-CSF/SCF-mobilized CD34+ cells transduced with lentiviral vectors expressing EGFP into myeloablated rhesus macaques. To date, more than 4 years posttransplantation, 0.5-8% EGFP expression is maintained in multiple cell lineages. The animals remain healthy with no evidence of hematopoietic abnormalities or malignancies. To assess immune functions, we actively immunized two of our transplanted animals with purified rEGFP proteins and CpG adjuvant and demonstrated stable levels of EGFP+ cell populations maintained for over 29 months despite four active immunizations. We did not detect a persistent anti-EGFP antibody response or anti-EGFP T cell response in these immunized animals. Immune response to an irrelevant antigen was normal. Taken together, our data provide formal support that transplantation of lentivirally transduced CD34+ progenitor cells in myeloablated rhesus macaques induces specific immunological tolerance toward a foreign transgene.

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

The ability to efficiently transduce hematopoietic stem and progenitor cells under serum-free conditions would be desirable for safety and standardization of clinical gene therapy protocols. Using rhesus macaques, we studied the transduction efficiency and engraftment ability of CD34-enriched SCF/G-CSF mobilized progenitor cells (PBSC) transduced with standard amphotropic marking vectors under serum-free and serum-containing conditions. Supernatants were collected from producer cells 16 hours after serum-free medium or medium containing 10% fetal calf serum was added. Vector titers were approximately two- to threefold higher when producer cells were cultured in serum-containing medium. However, retroviral transduction of rhesus CFU-GM was improved using serum-free vector-containing medium. For analysis of engraftment with transduced cells, three macaques had CD34+ peripheral blood stem cells split into two fractions for transduction. One fraction was transduced using serum-free vector-containing medium, and the other fraction was transduced using standard serum-containing medium. The two fractions were re-infused simultaneously following total body irradiation. In all three animals, there was equivalent marking from both vectors for 7-9 months post-transplantation. These data are encouraging regarding the removal of serum-containing medium from clinical hematopoietic cell transduction protocols, given the lack of a detrimental effect on transduction and engraftment with transduced cells.

Introduction Gene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery. Methods Twenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests. Results BM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p Conclusions SCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. Disclosures Luo: bluebird bio Inc.: Employment. Pierciey: bluebird bio: Employment.

The classical model of hematopoietic hierarchies is being reconsidered, based on data from in vitro assays and single cell expression profiling. Recent experiments suggest that erythroid and megakaryocytic lineages might differentiate directly from multipotent hematopoietic stem/progenitor cells (HSPC) or from a highly biased subpopulations of HSPC, rather than transiting through a common MEP or CMP. We examined the clonal ontogeny of the erythroid lineage using genetic barcoding of rhesus macaque HSPC (Wu Cell Stem Cell, 2014; Koelle Blood, 2017), allowing quantitative and sensitive tracking of the in vivo clonal output of thousands of individual HSPC over time following autologous transplantation. CD34+ HSPC were lentivirally-transduced with a high diversity barcode library, with the barcode in an expressed region of the provirus, allowing barcode retrieval from DNA or RNA, with each barcode representing an individual HSPC clone. CD34+ HSPC were purified from bone marrow(BM) of 3 macaques at 3-45 months post-transplant, and plated in CFU assays. 240 colonies each of CFU-E, CFU-G and CFU-GM were plucked individually, and each colony type was pooled before DNA extraction for barcode retrieval, along with purification and barcode retrieval from concurrent BM CD34+ cells and both blood and BM T cells (T), B cells (B), granulocytes (Gr), and monocytes (Mono). The majority of barcodes retrieved from pooled CFU-E were also detected in pooled CFU-G and CFU-GM, along with purified T cells, B cells, Mono and Gr, suggesting a shared unbiased precursor pool. A small fraction of clones unique to CFU-E were identified, however, unique clones were also detected in CFU-G and in CFU-GM pools, likely reflecting low frequency clones that were to be represented randomly in the pooled CFU of each lineage. To overcome the sampling bias inherent in colony assays on any reasonable colony number, we FACS purified CD71+/CD45- nucleated maturing erythroid lineage cells (nRBC) from the BM, and compared nRBC to other lineages purified concurrently from the same BM sample. There was very high correlation of barcode contributions between BM nRBC and other BM-produced lineages, with the highest correlation between nRBC and both Gr and Mono (r> 0.9), whether at earlier or later time points. We investigated whether RNA barcode retrieval could be utilized for clonal tracking, allowing analysis of anucleate circulating RBC and thus a more global analysis of hematopoiesis compared to local BM production at a limited number of sites. We have reported that clonal output from individual HSPC remains highly geographically restricted within the BM for months-years post-transplant. We compared fractional contributions of DNA and RNA barcodes retrieved from the same sample of each lineage. There was very high correlation between DNA and RNA barcode contributions to T, B, NK, Gr and Mono lineages (r= 0.85±0.04), suggesting the differentiation pathway for these lineages does not impact significantly on expression level of barcodes from the proviral promoter, and RNA fractional contributions in these lineages reflect the clonal representation of cells in a sample. However, nRBC DNA and RNA barcode contributions were less closely related (r= 0.62), suggesting that erythroid differentiation was more likely to alter expression from loci in a manner disconnecting RNA barcode expression from clonal representation of cells in a sample, and suggesting that RNA barcode retrieval may not be ideal for comparing erythroid cells to other lineages. However, tracking RNA barcodes can be used to assess clonal stability in circulating RBC over time, and revealed very stable clonal contributions to erythropoiesis for as long as 4 years post-transplant. Finally, we used RNA barcode retrieval to compare clonal contributions between circulating platelets and other lineages. Whether DNA or RNA was used for T, B, Gr, and Mono clonal mapping, at steady state platelet RNA barcodes were clonally closely related to other lineages. But preliminary data suggests that a unique set of clones is newly recruited to contribute only to platelets following inflammatory stimuli. The presence of a separate pool of platelet-biased HSPC contributing following inflammation has been suggested by prior in vitro assays, but our model may provide the first clonal in vivo confirmation of a unique inflammation-related platelet-biased HSPC pool. Disclosures Dunbar: Novartis/GSK to institute: Research Funding.

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

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 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. Disclosures No relevant conflicts of interest to declare.

Background: Hematopoietic stem cell (HSC) gene therapy is a new treatment paradigm that can potentially provide lifelong protection against HIV-1 infection. The basic principle is to genetically modify a patient's own HSC such that the progeny, including CD4+T cells and macrophages, are resistant to HIV-1. Given the expected low levels of the anti-HIV gene-marked cells in clinical studies, it is critical to understand how the vast number of HSC, each bearing a unique phenotype, and their mature progeny contribute to maintaining homeostatic regulation in HIV gene therapy settings. We have recently published novel and detailed insights about the long-term behavior patterns of individual HSC followed for 4 to 12 years post-transplant in our nonhuman primate (NHP) model of lentiviral gene therapy, revealing for the first time in primates the precise time point of HSC repopulation and the functional heterogeneity of HSCs (Kim, Cell Stem Cell, 2014; Goyal, BMC Biology, 2015). Consistent clonal behavior patterns have been observed in human gene therapy (Biasco, Cell Stem Cell, 2016), demonstrating the clinical relevance of our data. Here, in order to develop a systems-level understanding of HSC clonal dynamics in anti-HIV therapy settings requiring efficient immune recovery from T lymphopenia, we further analyzed HSC clonal repopulation after T-cell depletion in one of our NHP animals. Our study generated systems-level datasets useful for understanding the parameters of T-cell repopulation and homeostatic regulation in anti-HIV therapy, as well as other therapies requiring efficient immune recovery from disease- or treatment-induced T lymphopenia. Subjects and Methods: The HSC clonal behaviors in animal 95E132 have been well characterized for 16 years post-transplant. At the 16-year time point, this animal was treated with 8 doses (25 ug/kg/dose) of an anti-CD3e immunotoxin over 4 days. Hematopoietic recovery after T-cell depletion was monitored by a complete blood count, multi-color flow cytometry analysis, TCRv_ spectratyping, and a high-throughput lentiviral-tagging assay (Kim, Journal of Virology, 2010). HSC subtypes and their clonal behaviors were determined based on the clonal profiling of blood lineages, including CD4, CD8, CD20, CD14, and CD18 cells, over time. Results: The peripheral CD3+ T cells recovered in 2-3 months (Fig. 1B). Cytotoxic (CD8+) T-cell recovery was faster than that of T-helper cells. Effector and memory T cells expanded promptly after T-cell depletion, whereas the na•ve T cells had not recovered more than one year after CD3e-immunotoxin treatment. Clonal profiling analysis revealed a few dominant clones in the recovered T-cell compartment, while showing no notable clonal fluctuation in the CD18+ granulocytes over time (Fig. 1C-D). TCRv_ spectratyping showed a skewed T-cell receptor repertoire even a year after immunotoxin treatment. Conclusion: Our data can bolster our understanding of hematopoietic regulation in patients recovering from T lymphopenia. The data showed skewed a T-cell receptor repertoire and clonal dominance in the recovered T-cells after immunotoxin treatment in an aged animal, suggesting that T-cell recovery had occurred primarily as a result of peripheral T-cell expansion. A systems-level, clonal dynamics study of this extreme form of homeostatic regulation provides unique opportunities to identify and characterize the regenerative pathways, as well as the obstacles, that emerge in the process of restoring homeostasis after disease- or treatment-induced T-cell depletion. Figure 1 A. Hematopoietic recovery after CD3e-immunotoxin treatment (red arrow) was assessed at the clonal level. B. CD3+ T cells were effectively ablated by immunotoxin treatment. The T-cell percentage rebounded to the normal range by 2-3 months. C. A ternary diagram showing Myeloid-biased (black circles), Balanced (red circles), and Lymphoid-biased (green circles) HSC subtype clones. The relative position of a circle in the diagram indicates the lineage output potential toward G/M (granulocyte/monocyte), B-cell, and T-cell. The size of a circle indicates the relative frequency of a clone. D. The relative frequencies of the HSC clones in T-cell, B-cell, Granulocytes, and monocytes are shown at 1 month before (-1M), 1 month (1M) and 3 month (3M) immunotoxin treatment. The individual clones (circles) are located at the identical positions in C. Figure 1. A. Hematopoietic recovery after CD3e-immunotoxin treatment (red arrow) was assessed at the clonal level. B. CD3+ T cells were effectively ablated by immunotoxin treatment. The T-cell percentage rebounded to the normal range by 2-3 months. C. A ternary diagram showing Myeloid-biased (black circles), Balanced (red circles), and Lymphoid-biased (green circles) HSC subtype clones. The relative position of a circle in the diagram indicates the lineage output potential toward G/M (granulocyte/monocyte), B-cell, and T-cell. The size of a circle indicates the relative frequency of a clone. D. The relative frequencies of the HSC clones in T-cell, B-cell, Granulocytes, and monocytes are shown at 1 month before (-1M), 1 month (1M) and 3 month (3M) immunotoxin treatment. The individual clones (circles) are located at the identical positions in C. Disclosures Dunbar: GSK/Novartis: Research Funding. Chen:Calimmune: Membership on an entity's Board of Directors or advisory committees.

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.

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

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

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.