Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies. To date, a comprehensive evaluation of T-cell depletion and CD3 proliferation for all lymphoid tissues has not been conducted. Here, two rhesus macaques were administered A-dmDT390-SCFBdb (CD3-IT) intravenously at 25 μg/kg twice daily for four days. Samples were collected one day prior to and four days post administration. Flow cytometry and immunofluorescence staining were used to evaluate treatment efficiency accurately. Our preliminary results suggest that CD3-IT treatment may induce higher depletion of CD3 and CD4 T-cells in the lymph nodes and spleen, but is ineffective in the colon and thymus. The data showed a better elimination tendency of CD4 T-cells in the B-cell zone relative to the germinal center in the lymph nodes. Further, CD3-IT treatment may lead to a reduction in germinal center T follicular helper CD4 cells in the lymph nodes compared to healthy controls. The number of proliferating CD3 T-cell indicated that repopulation in different lymphoid tissues may occur four days post treatment. Our results provide insights into the differential efficacy of CD3-IT treatment and T-cell proliferation post treatment in different lymphoid tissues. Overall, CD3-IT treatment shows potential efficacy in depleting T-cells in the periphery, lymph nodes, and spleen, making it a viable preconditioning regimen for cell or organ transplantation. Our pilot study provides critical descriptive statistics and can contribute to the design of larger future studies.

Abstract 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 HSPC, 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-HUVEC) 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.

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 cocultured 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.

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.

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 (89)Zirconium (Zr-89)-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.

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.

The classical model of hematopoietic hierarchies is being reconsidered on the basis of data from in vitro assays and single cell expression profiling. Recent experiments suggested that the erythroid lineage might differentiate directly from multipotent hematopoietic stem cells / progenitors or from a highly biased subpopulation of stem cells, rather than transiting through common myeloid progenitors or megakaryocyte-erythrocyte progenitors. We genetically barcoded autologous rhesus macaque stem and progenitor cells, allowing quantitative tracking of the in vivo clonal output of thousands of individual cells over time following transplantation. CD34+ cells were lentiviral-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 stem or progenitor cell clone. Barcode profiles from bone marrow CD45-CD71+ maturing nucleated red blood cells were compared with other lineages purified from the same bone marrow sample. There was very high correlation of barcode contributions between marrow nucleated red blood cells and other lineages, with the highest correlation between nucleated red blood cells and myeloid lineages, whether at earlier or later time points post transplantation, without obvious clonal contributions from highly erythroid-biased or restricted clones. A similar profile occurred even under stressors such as aging or erythropoietin stimulation. RNA barcode analysis on circulating mature red blood cells followed over long time periods demonstrated stable erythroid clonal contributions. Overall, in this nonhuman primate model with great relevance to human hematopoiesis, we documented continuous production of erythroid cells from multipotent, non-biased hematopoietic stem cell clones at steady-state or under stress.

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.

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. Dunbar: National Institute of Health: Research Funding.

Gene therapies using integrating retrovirus vectors to modify hematopoietic stem and progenitor cells have shown great promise for the treatment of immune system and hematologic diseases. However, activation of proto-oncogenes via insertional mutagenesis has resulted in the development of leukemia. We have utilized cellular bar coding to investigate the impact of different vector designs on the clonal behavior of hematopoietic stem and progenitor cells (HSPCs) during in vivo expansion, as a quantitative surrogate assay for genotoxicity in a non-human primate model with high relevance for human biology. We transplanted two rhesus macaques with autologous CD34+ HSPCs transduced with three lentiviral vectors containing different promoters and/or enhancers of a predicted range of genotoxicities, each containing a high-diversity barcode library that uniquely tags each individual transduced HSPC. Analysis of clonal output from thousands of individual HSPCs transduced with these barcoded vectors revealed sustained clonal diversity, with no progressive dominance of clones containing any of the three vectors for up to almost 3 years post-transplantation. Our data support a low genotoxic risk for lentivirus vectors in HSPCs, even those containing strong promoters and/or enhancers. Additionally, this flexible system can be used for the testing of future vector designs.

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 <0.2ml/min, total volume <5ml) under CT guidance. Utilizing porcine and rhesus macaque models, we have shown that OIB delivery of CD34+ cells improves intrabone retention, preventing circulation of CD34+ cells through the lungs, which is observed with conventional non-optimized intrabone methods.2 Here we conducted experiments using an autologous myeloablative transplant model in rhesus macaques comparing engraftment of gene-marked CD34+ cells transplanted intravenously, with cells transplanted using OIB delivery.

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.

HSCs alone possess the ability to both self-renew and differentiate into all mature blood lineages, thereby maintaining immune function, tissue perfusion and hematopoietic homeostasis throughout the lifetime of the organism. HSCs are therapeutically valuable for transplantation in the treatment of hematologic malignances. They are a rare population in the bone marrow (BM), and methods for direct isolation and expansion of a pure population of functional human HSCs remain elusive. The development of therapeutic options to manipulate and maintain human HSCs is of major clinical interest, however, to date, no such therapy has proven effective in large-scale clinical trails.

Lentiviral vectors have been used for the delivery of genetic material into hematopoietic stem and progenitor cells (HSPC) in over 100 patients enrolled in clinical trials worldwide. Lentiviral vectors, in contrast to γ-retroviral vectors, have not been associated with insertion site-associated malignant clonal expansions, and thus have been considered safer for use in HSC gene therapies. Here, however, we present a case of abberant, markedly dysplastic clonal hematopoiesis impacting the erythroid, myeloid and megakaryocytic lineages in a rhesus macaque transplanted with lentivirally barcoded hematopoietic stem and progenitor cells. Autologous CD34+ HPSC from macaque ZL34 were transduced with a highly diverse lentiviral barcoded library and reinfused following total body irradiation. The lentiviral vector contained a copGFP reporter gene and a murine stem-cell virus (MSCV) promoter. Transduction parameters (MOI:25) and cell dose (5.4 * 106 CD34+ cells/kg, 27.1% GFP+ infusate) were similar to those of 14 other macaques transplanted with the identical or similar barcoded lentiviral vectors that manifested highly polyclonal (thousands clones/animal) and stable hematopoiesis following transplantation (Koelle, Blood 2017; Wu Cell Stem Cell, 2014). ZL34 initially engrafted with normal kinetics and blood count recovery. However, beginning 4-5 months (m) post-transplant, increased numbers of eosinophils were noted in the blood, and platelet counts decreased from the normal range to less than 30,000/uL. By 7m, strikingly elevated numbers of nucleated red blood cells (nRBCs) appeared in the blood, persisting at levels of up to 6000/uL up to the present, 16m post-transplant. Fetal hemoglobin levels were markedly increased to >76% as determined by HPLC. The bone marrow was hypercellular and markedly dysplastic, with uninuclear/small megakaryoctyes, erythroid predominance, and a left-shift in all lineages but no increased blasts. Karyotype was normal. Barcode retrieval from purified blood myeloid, T, B, NK and nRB cell populations via low cycle PCR and Illumina sequencing revealed marked clonal dominance of 7 barcodes in the nRBC lineage and in the dysplastic myeloid lineages beginning at 5 months post-transplant. All 7 barcodes were confirmed to be present in the abnormal clone by CFU assays. High-throughput insertion site retrieval followed by insertion-specific PCR assay matched 6 barcodes to insertions, uncovered two additional mutated barcodes in the dominant clone not previously retrieved, and thus so far confirm 8-9 insertions in the dominant nRBC/myeloid clone. These insertions are adjacent to or within several potential genes of interest given the phenotype, including KITLG (SCF) and PLAG1. Bulk and single cell RNA-seq studies are being performed to uncover putative insertion-mediated upregulation or downregulation of genes near the 8 dominant integration sites. In conjunction with our previous barcoding studies and unpublished erythropoiesis studies, these results suggest that lentiviral insertion(s) into an HSPC resulted in clonal expansion, markedly abnormal myeloid and erythroid proliferation and differentiation, and a profoundly abnormal myeloproliferative/myelodysplastic phenotype. As this is possibly the first clear case of a lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal HSPC, these results are concerning, and suggest that other methods such as targeted gene correction are important to pursue for gene therapy of bone marrow diseases. Disclosures Dunbar: Novartis/GSK to institute: Research Funding.

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 similar to 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.

A common complaint attributed to apheresis is chill and hunger. Most likely this is a result of heat loss when blood is externalized from the body. This energy loss has not been quantitated. To determine caloric loss we monitored temperature loss using sterilized Omega hypodermic type K thermocouple probes inserted 20cm from the subject into the draw and return line, the centrifuge chamber, and the body. Inlet, return, centrifuge, and body temperature were recorded at one minute intervals using an Omega RDXL4SD Data Logger. Either a 38oC water bath (sham subject, n=2 per device) or rhesus macaque (weight 5-7 kg) undergoing apheresis served as subject. By definition one calorie (4.2 joules) is the amount of energy required to heat 1mL of water 1oC. By knowing that the heat capacity of blood is 3.6 joules/mL, we can calculate calorie loss per procedure. A total of 15 small volume apheresis procedures were performed with the Fenwal CS3000 on 12 rhesus macaques using a modified single small volume chamber procedure (Donahue et al., BLOOD 87:1644-53, 1996), 7 procedures were performed on 3 rhesus macaques using a Spectra Optia, and 6 procedures were performed on 3 rhesus macaques using a Fenwal Amicus device. Using water, the average temperature difference between inlet and return line dropped logarithmically (R2>0.89) for all three instruments as flow rate was increased (Fig. 1). Similarly the calorie loss/min logarithmically increased (R2>0.89) (Fig. 2) as flow rate increased. For CS3000 rhesus apheresis procedures, the drop in temperature between draw and return lines was 8.7(SD 1.5) oC at a flow rate of approximately 11mL/min, 9.5(0.9) oC for the Optia at a flow rate of 10mL/min, and 10.3(1.2) oC for the Amicus at a flow rate of 11mL/min. This drop was not significantly different between instruments (p=0.10). Procedures did differ in the amount of blood processed. For the CS3000, the blood volume processed per procedure (1013(18) mL) was significantly less (p<0.001) than either the Optia (1425(201) mL) or Amicus (1473(36) mL). In addition, centrifuge chamber temperature differed between the CS3000 (33.1(1.6) oC) and the Optia (28(1.9) oC) or Amicus (26.0(1.0)oC) (p<0.001) as the CS3000 has a chamber heating element. For the CS3000, calorie loss per procedure was 7712(1823) calories, for the Optia 11827(1987) calories, and the Amicus 13199(1354) calories. The significant difference (p<0.001) in caloric loss between the CS3000 and the other instruments was due to the larger blood volume processed using the Optia and Amicus. To prevent calorie loss we developed a method using a 50oC heated water bath in which a segment of the return line (38 cm or more) was placed in the water bath to equilibrate draw and return line temperatures without altering extracorporeal blood volume. On performing this procedure, we successfully matched draw and return line temperatures. This report documents for the first time caloric loss during apheresis procedures. In addition, it introduces a method to limit such a loss. Although adults >50kg may tolerate apheresis-associated caloric loss with only mild to moderate symptoms, pediatric patients, small animals, and debilitated and aging subjects, may not. Here we demonstrate the extent of calorie loss and a means to prevent it.

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.

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 <2% of cells in lymphoid tissues (LTs). Using a combination of single-photon emission computed tomography imaging and a radiotracer ((99m)Tc-labeled rhesus immunoglobulin G1 anti-CD4R1 (Fab')2), we sequentially imaged CD4(+) cell recovery in rhesus macaques following total body irradiation (TBI) and reinfusion of vector-transduced, autologous CD34(+) cells. Our results present for the first time a sequential, real-time, noninvasive method to evaluate CD4(+) cell recovery. Importantly, despite myeloablation of circulating leukocytes following TBI, total depletion of CD4(+) lymphocytes in LTs such as the spleen is not achieved. The impact of TBI on LTs and PBLs is discordant, in which as few as 32.4% of CD4(+) cells were depleted from the spleen. In addition, despite full lymphocyte recovery in the spleen and PB, lymph nodes have suboptimal recovery. This highlights concerns about residual disease, endogenous contributions to recovery, and residual LT damage following ionizing irradiation. Such methodologies also have direct application to immunosuppressive therapy and other immunosuppressive disorders, such as those associated with viral monitoring.

The high risk of insertional oncogenesis reported in clinical trials using integrating retroviral vectors to genetically modify hematopoietic stem and progenitor cells (HSPCs) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of "suicide genes" in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the "inducible Caspase-9" (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75% and 94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches using iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development.