Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic hemolytic anemia, painful vaso-occlusive episodes, and end-organ damage. Cellular therapies, including allogeneic hematopoietic cell transplant (allo-HCT) and gene therapy, are potentially curative treatments for SCD. Cases of myelodysplastic syndrome and acute myeloid leukemia (MDS/AML) occurring after cellular therapy for SCD and reports of an increased relative risk of AML in populations with SCD have raised questions about the association between SCD and clonal hematopoiesis (CH), a recognized precursor state for MDS/AML. We used error-corrected DNA sequencing to detect CH variants at a variant allele fraction > 0.0005 in blood samples from 98 individuals with SCD and 72 non-SCD donor controls who underwent HCT at the NIH between July 2004 and June 2023. Baseline CH prevalence was similar in recipients and donors; however, SCD recipients had 3.8-fold higher odds of DNA Damage Response (DDR)-mutant CH, involving TP53, PPM1D, ATM, and CHEK2, compared with non-SCD donors. Following HCT, the proportion of SCD recipients with CH increased, driven primarily by mutations in DNMT3A/TET2 and TP53. Post-HCT CH arose from newly emergent variants undetectable at baseline in both recipient and paired donor samples, engrafted donor-derived CH, and persistent recipient CH. All cases of donor-derived CH involved DNMT3A/TET2 mutations, while persistent-recipient CH involved DDR mutations. Hematologic malignancies were rare but included 3 cases of fatal TP53-mutant MDS/AML. Larger cohort studies are needed to identify risk factors for developing DDR-CH and MDS/AML in SCD and to optimize cellular therapy safety in this population.

Nonmyeloablative (NMA) conditioning is being used increasingly with success in matched related donor (MRD) and alternative donor allogeneic hematopoietic cell transplantation (allo-HCT) in individuals with sickle cell disease (SCD). Advantages include decrease toxicity and applicability in patients otherwise unable to tolerate conditioning regimens due to end-organ damage or age. We aimed to add to published data outcomes of two similar NMA conditioning protocols, termed Protocol 1 (ClinicalTrials.gov ID NCT00061568) and Protocol 2 (ClinicalTrials.gov ID: NCT02105766)) in mainly adult patients with SCD to evaluate the safety, toxicity, and success of these regimens in individuals at high-risk for poor transplantation-related outcomes. We also evaluated the tolerability and outcomes of Protocol 2, which included preconditioning immunodepletion, in patients at even higher risk of T cell-mediated rejection or plasma/B cell-mediated anti-donor erythrocyte antibody production—the latter due to ABO incompatibility or recipient RBC alloimmunization to a donor antigen. Finally, we evaluated the incidence and trajectory of mixed donor myeloid chimerism over time following allo-HCT. In this retrospective analysis of the 2 prospective phase 2 NMA transplant protocols, 91 individuals with SCD or transfusion-dependent β-thalassemia underwent MRD allo-HCT at the National Heart, Lung, and Blood Institute; regimens contained alemtuzumab, low-dose radiation, and sirolimus for graft-versus-host disease (GVHD) prophylaxis with or without preconditioning immunodepletion with pentostatin and oral cyclophosphamide (Protocol 2). In the total cohort of 91 transplantation recipients, outcomes were favorable with timely neutrophil and platelet engraftment (median, 21 days [range, 7 to 67 days] and 21 days [range, 10 to 112 days], respectively), minimal high-grade acute GVHD and no chronic GVHD, overall survival of 90%, sickle-free survival of 85%, and mixed donor myeloid chimerism in 43% at a median follow up of 7.3 years (range, 0.8 to 20 years). Most patients with mixed myeloid chimerism at 2-years post-HCT remained stable in their values. In analyzing each protocol separately, outcomes were comparable except for higher cytomegalovirus reactivation necessitating treatment in Protocol 2 without an associated increase in graft failure. In the combined cohort, graft failure occurred in 11 patients, and hematologic malignancy or abnormal cytogenetics on bone marrow evaluation developed in 7 patients. In a subanalysis of factors that may implicate transplantation outcomes, the number of RBC units transfused post-HCT was significantly higher in recipients with pre-HCT history of alloimmunization to donor RBC antigens. There was no difference in the number of RBC units transfused, duration of transfusion, or red cell engraftment in those with major ABO incompatibility; preconditioning immunodepletion and pretreatment with rituximab likely were helpful. Both NMA allo-HCT protocols were successful in achieving adequate engraftment and sickle-free survival with minimal toxicity, including in individuals with mixed donor myeloid chimerism. The addition of preconditioning immunodepletion was well-tolerated and reduced the rate of graft failure in high-risk recipients.

Non-myeloablative hematopoietic cell transplantation (HCT) is a curative option for individuals with sickle cell disease (SCD). Our traditional goal with this approach has been to achieve a state of mixed donor/recipient chimerism. Recently, we reported an increased risk of hematologic malignancies (HMs) in adults with SCD following graft failure or mixed chimerism. To evaluate the origin of HMs, we performed chimerism analyses of 5 patients with SCD who developed HMs after non-myeloablative HCT. DNA was extracted from sorted peripheral blood or bone marrow cells representing mature cell lineages or leukemic blasts and subjected to chimerism analysis by PCR amplification of polymorphic short tandem repeats. Unlike mature cell lineages in patients with mixed chimerism, which still showed a donor-derived fraction of cells, leukemic blast cells were found to be 99-100% recipient-derived in all patients. Non-myeloablative conditioning allows for the survival of patients' cells that might harbor pre-leukemic clones that possess the capacity to evolve under genotoxic or environmental stress into malignancies; therefore, we have modified our HCT protocols with the goal of full donor chimerism to mitigate the risk of HM development.

Sickle cell disease (SCD) is a genetic disorder leading to subclinical and overt organ damage and early mortality. The only cure for SCD is a hematopoietic cell transplant (HCT). Unfortunately, adults with overt organ damage cannot tolerate myeloablative conditioning. Thus, we and others developed a non-myeloablative (NM) allogeneic regimen to achieve a state of mixed donor/recipient chimerism, given that 20% donor myeloid chimerism (DMC) is sufficient to reverse the SCD phenotype. Traditionally, one consequence of this approach, especially in the haploidentical setting, has been a high risk of graft failure. Further, we recently reported that the risk of hematologic malignancies (HMs) is higher than expected in patients with mixed chimerism or graft failure following NM HCT. Since most of these HMs were diagnosed after graft failure, their origin was assumed to be recipient-derived; however, this has yet to be confirmed. Here, we report, for the first time, the origin of leukemic blasts and myelodysplastic syndrome (MDS) mononuclear cells (MNCs) in patients with SCD after HCT. We utilized frozen biospecimens from patients diagnosed with the most aggressive HMs after HCT. All samples were collected under an NHLBI IRB-approved protocol. Chimerism analysis was performed by PCR amplification of polymorphic short tandem repeats on DNA extracted from peripheral blood (PB) or bone marrow (BM) cellular fractions. Five patients were included in this study: two patients aged 37 years at HCT had MDS 2 and 2.5 years post-HCT (SCD-01 and SCD-02); two patients aged 20 and 34 years at HCT had acute myeloid leukemia (AML) 4 months and 5.5 years post-HCT, respectively (SCD-03 and SCD-04); and one patient aged 39 years at HCT had T cell acute lymphoblastic leukemia (T-cell ALL) 3 years post-HCT ( SCD-05). Three patients (SCD-01, 02, and 03) had graft failure with 0% PB DMC and 0% donor lymphoid chimerism (DLC) accompanied by the return of SCD. SCD-04 had impending graft failure with 16% PB DMC and 18% DLC. SCD-05 had mixed chimerism with 30% PB DMC and 25% DLC. Although there were no available samples for SCD-01, whole BM chimerism was performed at the time of the diagnostic evaluation, revealing 100% recipient chimerism. Further, we confirmed the origin of MDS MNCs from SCD-02 by analyzing the chimerism of DNA extracted from PB MNCs at Y3 post-HCT; cells were 100% recipient-derived. Moreover, from a PB sample of SCD-03 at Y5 post-transplant, we sorted 3 PB cell populations: CD3+ T cells, CD19+ B cells, and CD3-CD19-CD13+CD33+CD34+ leukemic blasts, all showing a 100% recipient-derived origin. In addition, we sorted 2 populations from SCD-04 PB at D100 post-HCT: CD3+ T cells and CD3-CD19-CD13+CD33+CD34+ leukemic blasts. While we detected 16% donor-derived cells in the T cell compartment, consistent with the PB DLC, the blast population showed 99% recipient-derived cells. Lastly, we sorted 3 populations from SCD-05 BM at Y3 post-HCT: CD19+ B cells, CD11b+ Myeloid cells, and CD19-CD11b-CD2+CD5+CD7+CD38+ CD1a-, mainly leukemic blasts. While we detected 11% BM DMC and 22% BM B-cell chimerism, the blast population was 100% recipient-derived. In conclusion, we showed that in patients who experienced MDS, AML, or T-cell ALL following graft failure or mixed chimerism, leukemic blasts or MDS MNCs originated from patient cells, with 99-100% recipient chimerism. While the reasons are unknown why the risk of HMs is higher when the therapeutic goal is mixed chimerism, the goal of our future protocols is full donor chimerism to eliminate the possibilities of evolving pre-existing leukemic clones, or transforming recipient, clonally expanded hematopoietic cells, as a result of exposure to conditioning regimens, erythropoietic stress, or alloreactivity after graft failure. Further evaluation is ongoing.

Hematopoietic stem cell transplantation (HSCT) is the only curative option for patients with sickle cell disease (SCD). We sought to extend our haploidentical HSCT regimen with nonmyeloablative conditioning. The objective of our study was to assess the ability of the immunosuppressive agent CTLA4-Ig to induce mixed chimerism in the haploidentical HSCT setting when combined with sirolimus, post-transplant cyclophosphamide (PT-Cy), or both. We used a mismatched mouse model with BALB/c donors and C57BL/6 recipients. Recipient mice received 200 cGy TBI on day of the transplant and were conditioned with or without sirolimus, PT-Cy, and/or CTLA4-Ig. Our data show that when CTLA4-Ig alone or CTLA4-Ig/PT-Cy are given, donor cells are not detected. However, mixed chimerism is maintained in mice who received either sirolimus/PT-Cy or sirolimus/PT-Cy given together with CTLA4-Ig over 60 days. Additionally our data indicate a significant increase in recovery of donor cells within CD8 T-cell, CD19 B-cell, and CD11b myeloid cell populations in groups conditioned with sirolimus/PT-Cy, or sirolimus/PT-Cy/CTLA4-Ig as compared to groups conditioned with CTLA4-Ig (p=0.003; p=0.002; p=0.049) alone or CTLA4-Ig/PT-Cy (p=0.001; p<0.0001; p=0.001) at 30 days, respectively. These data demonstrate that sirolimus in combination with PT-Cy and CTLA4-Ig is synergistic in an MHC-mismatched mouse model. The novel tri-drug group establishes mixed chimerism while conditioning with CTLA4-Ig alone or CTLA4-Ig/PT-Cy does not. The data also identify induction of stable mixed chimerism in specific cellular subpopulations within the sirolimus/PT-Cy and sirolimus/PT-Cy/CTLA4-Ig groups, indicating that these cells may play an important role in HCST.

Allogeneic stem cell transplant (SCT) is a potentially curative option for many hematologic diseases, yet donor availability remains a challenge, and haploidentical SCT is associated with a higher risk of rejection. We successfully translated a model based on total-body irradiation (TBI) with to humans; however, TBI involves toxicities and risk of malignant side-effects, and we hypothesize that busulfan may be a superior alternative in some settings. G-CSF-mobilized F1/J (C57BL/6-BALB/c hybrid) splenocytes were injected into C57BL/6 hosts who had received 10-30mg/kg IP busulfan the previous day. All recipients received 31 days of 3mg/kg IP sirolimus beginning the day before transplant. No GvHD was observed and counts recovered by week 8 in all groups. Mice conditioned with 30mg/kg busulfan displayed remarkably high donor chimerism (>90%), which was significantly higher than 300cGy TBI-conditioned control mice (64-86%) across all timepoints. Mice receiving 30mg/kg or 20mg/kg busulfan maintained stable chimerism for over 18 months. Mice conditioned with 10mg/kg busulfan showed only transient chimerism through 4 weeks. Proliferation of CD4+ cells in response to donor increases with decreasing busulfan, and mice receiving 30mg/kg responded more strongly than mice receiving TBI. A memory effect was observed in mice that received 10mg/kg busulfan and lost their grafts. Notably, a slightly higher incidence of host-derived Tregs in mice was also observed after graft rejection.

Aims/Hypothesis Non-Fc-binding Anti CD3 antibody has proven successful in reverting diabetes in the non-obese diabetes mouse model of type 1 diabetes and limited efficacy has been observed in human clinical trials. We hypothesized that addition of rapamycin, an mTOR inhibitor capable of inducing operational tolerance in allogeneic bone marrow transplantation, would result in improved diabetes reversal rates and overall glycemia. Methods Seventy hyperglycemic non-obese diabetic mice were randomized to either a single injection of anti CD3 alone or a single injection of anti CD3 followed by 14 days of intra-peritoneal rapamycin. Mice were monitored for hyperglycemia and metabolic control. Results Mice treated with the combination of anti CD3 and rapamycin had similar rates of diabetes reversal compared to anti CD3 alone (25/35 vs. 22/35). Mice treated with anti CD3 plus rapamycin had a significant improvement in glycemia control as exhibited by lower blood glucose levels in response to an intra-peritoneal glucose challenge; average peak blood glucose levels 30 min post intra-peritoneal injection of 2 gr/kg glucose were 6.9 mmol/L in the anti CD3 plus rapamycin group vs. 10 mmo/L in the anti CD3 alone (P<0.05). Conclusions/Interpretation The addition of rapamycin to anti CD3 results in significant improvement in glycaemia control in diabetic NOD mice.

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoform alpha (TRIM5 alpha) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (chi HIV) 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 chi HIV 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 chi HIV vectors. As compared with SIV vectors, chi HIV 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 chi HIV 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, chi HIV vector frequently integrated into gene regions, especially into introns. In summary, our chi HIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This chi HIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.

Abstract Abstract 3118 Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application. We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model. We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis. We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis. We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors. In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. Disclosures: No relevant conflicts of interest to declare.

The third-generation NOD/LtSz-scid/IL2Rγ(null) (NOD/SCID IL2Rγ(null)) mouse represents a significantly improved xenograft model allowing high levels of human leukocyte engraftment over extended follow up. One remaining limitation of this mouse model, however, is the low level of circulating human erythrocytes. We established a practical ex vivo erythroid culture system of xenograft marrow progenitors to enrich for human erythroid progeny. At various time points after transplant, erythroid cells were easily assayed after 17 days of ex vivo culture of xenograft marrow, with nearly all nucleated cells of human origin and approximately 60% human GPA or CD71 positive. We then transplanted cord blood CD34(+) cells marked with a lentiviral vector encoding green fluorescent protein (GFP). Three months later, ex vivo culture of xenograft marrow progenitors showed 41.3% of the cultured erythroid cells were positive for GFP and human CD71, and 56.2% were positive for GFP and human GPA, similar to that of circulating leukocytes at the same time point. Next, G-CSF mobilized peripheral blood CD34(+) cells from a sickle cell trait subject were infused in this mouse model to determine if the hemoglobin pattern could be modeled. CD34(+) cells from the sickle cell trait subject engrafted equally compared to CD34(+) cells from normal subjects, establishing the sickle cell trait phenotype. Lastly, a comparison of adult-derived peripheral blood CD34(+) cells and cord blood-derived CD34(+) cells xenografted mice was made, and long term follow-up demonstrated a recapitulation of the fetal to adult hemoglobin switch. This approach should prove a useful tool for testing strategies for genetic manipulation of erythroid progeny and the study of hemoglobin switching.

BACKGROUND: Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system. STUDY DESIGN AND METHODS: We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony‐forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ null mice. RESULTS: CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3 ± 9.23%) than those frozen in 10% DMSO (75.3 ± 11.0%, p < 0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ null mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO. CONCLUSION: Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.

Xenografting immunodeficient mice after low-dose irradiation has been used as a surrogate human hematopoietic stem cell (HSC) assay; however, irradiation requires strict and meticulous animal support and can produce significant mortality rates, limiting the usefulness of this model. In this work, we examined the use of parenteral busulfan as an alternative conditioning agent. Busulfan led to dose-dependent human HSC engraftment in NOD/LtSz-scid/IL2R gamma(null) mice, with marked improvement in survival rates. Terminally differentiated B and T lymphocytes made up most of the human CD45+ cells observed during the initial 5 weeks post-transplant when unselected cord blood (CB) products were infused, suggesting derivation from existing mature elements rather than HSCs. Beyond 5 weeks, CD34+ enriched products produced and sustained superior engraftment rates compared with unselected grafts (CB CD34+, 65.8% +/- 5.35%, vs. whole CB, 4.27% +/- 0.67%, at 24 weeks). CB CD34+ group achieved significantly higher levels of engraftment than mobilized CD34+ enriched peripheral blood (PB CD34+). At 8 weeks, all leukocyte subsets were detected, yet human red blood cells (RBCs) were not observed. Transfused human red cells persisted in the chimeric mice for up to 3 days; an accompanying rise in total bilirubin suggested hemolysis as a contributing factor to their clearance. Recipient mouse-derived human HSCs had the capacity to form erythroid colonies in vitro at various time points post-transplant in the presence of human transferrin (Tf). When human Tf was administered singly or in combination with anti-CD122 antibody and human cytokines, up to 0.1% human RBCs were detectable in the peripheral blood. This long evasive model should prove valuable for the study of human erythroid cells. STEM CELLS 2009; 27: 175-182

We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV. Here we report an assessment of retroviral integration sites (RISs) recovered out to 3 years post-transplantation. We identified 213 unique RISs from the patient's peripheral blood samples by linear amplification-mediated PCR (LAM-PCR). While vector integration patterns were similar to that previously reported, only 3.76% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). Additionally, common integration sites were enriched among late samples (14.9% vs. 36.8%, respectively). Three RISs were found near or within known oncogenes, but 2 were limited to early timepoints. Interestingly, an integration site near the MDS1 gene was detected in long-term follow-up samples; however, the overall contribution of MDS1 integrated clone remained stably low during follow-up.

Abstract Abstract 3540 Poster Board III-477 A strategy that can induce stable mixed chimerism across human leukocyte antigen (HLA) barriers would be beneficial in extending the application of hematopoietic stem cell transplantation (HSCT) to patients with severe sickle cell disease (SCD) who are in need of this potentially curative procedure. Indeed, we have recently demonstrated the feasibility of an HLA-matched sibling protocol employing low dose total body irradiation (TBI, 300cGy), the lymphocyte depleting agent alemtuzumab, and sirolimus to reverse the phenotype with minimal side effects. Due to the lack of HLA-matched siblings in the majority of patients, our goal is to develop a safe haploidentical regimen. In this work, we focused on determining optimal postgrafting immunosuppression and examined sirolimus and post-transplant cyclophosphamide (PT-cy), agents known to induce transplantation tolerance. To determine the optimal sequence for combining these drugs and whether this combination is synergistic in promoting stable donor chimerism despite the antiproliferative effects of sirolimus, we used a mismatched murine model with BalbC donors and C57Bl6 recipients. Twenty-five to 40 recipient mice received 200cGy TBI and PT-cy (200mg/kg intraperitoneally (IP) 2 days post transplant) with or without sirolimus (3mg/kg IP) for 14 to 30 days starting 1 day before or 4, 6, or 10 days post transplant. We found that in contrast to sirolimus or PT-cy alone, the combination of PT-cy and a limited course of sirolimus resulted in stable mixed chimerism: all mice that received PT-cy and sirolimus starting between 1 day before and 6 days after transplant attained donor chimerism levels ranging from 15-35%. Further, a 14 day course of sirolimus was sufficient to maintain stable mixed chimerism in our model (See Figures 1 and 2). To examine whether this synergistic effect is mediated by regulatory T cells, we administered anti-CD25 monoclonal antibody (CD25 mAb), an agent known to transiently deplete these cells in vivo. Fifteen mice received 200cGy TBI, sirolimus, PT-Cy, and either no CD25 mab, CD25 mab (1mg IP) on 7 and 3 days before and 1 day after transplant, or CD25 mab starting 14 days after transplant. CD25 mab was given biweekly for 5 weeks to mice in both groups. Donor engraftment levels did not differ in the three groups, with donor chimerism levels ranging from 30-40%. Our data show that the anti-proliferative effects of sirolimus do not inhibit the efficacy of the cytotoxic agent cyclophosphamide. Rather, our data demonstrate that the combination of PT-cy and a limited course of sirolimus synergistically promote mixed bone marrow chimerism in a complete mismatched setting. Further, the synergistic effect of this drug combination appears to be mediated independently from CD25+ regulatory T cell expression. In light of our previous success using sirolimus in an HLA-matched HSCT protocol, these findings lay the groundwork for developing PT-cy and sirolimus as a novel, safe, and effective means of promoting stable mixed chimerism in the haploidentical setting and thus greatly enhancing our ability to successfully apply this approach to patients with severe SCD. Figure 1 PT-cy and sirolimus are synergistic. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 30 days starting from 1 day before to 4 days after transplant. Figure 1. PT-cy and sirolimus are synergistic. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 30 days starting from 1 day before to 4 days after transplant. Figure 2 Fourteen days of sirolimus is sufficient to maintain stable mixed chimerism. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 14 days starting from 1 day before to 10 days after transplant. Figure 2. Fourteen days of sirolimus is sufficient to maintain stable mixed chimerism. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 14 days starting from 1 day before to 10 days after transplant. Disclosures: No relevant conflicts of interest to declare.

Inherited disorders of globin synthesis remain desirable targets for hematopoietic stem cell (HSC)-based therapies. Gene transfer using retroviral vectors offers an alternative to allogeneic HSC transplantation by the permanent integration of potentially therapeutic genes into primary autologous HSCs. Although proof of principle has been demonstrated in humans, this approach has been met by formidable obstacles, and large-animal models have become increasingly important for the preclinical development of gene addition strategies. Here we report lentiviral gene transfer of the human beta-globin gene under the control of the globin promoter and large fragments of the globin locus control region (LCR) in the nonhuman primate. Using an HIV-1, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped vector, modified to overcome a species-specific restriction to HIV-1, gene transfer to colony-forming units (CFU) derived from mobilized peripheral blood (PB) rhesus CD34+ cells was 84.4 +/- 2.33%. Erythroid cells derived from transduced rhesus CD34+ cells expressed human beta-globin at high levels as assessed by flow cytometry with a human beta-globin-specific antibody. Two rhesus macaques (RQ3586 and RQ3583) were transplanted with mobilized PB CD34+ cells transduced with our modified HIV vector at a multiplicity of infection of 80. High gene transfer rates to CFUs were achieved in vitro (RQ3586, 87.5%; RQ3583, 83.3%), with efficient human beta-globin expression among erythroid progeny generated in vitro. Early posttransplantation, gene transfer rates of 5% or higher were detectable and confirmed by genomic Southern blotting, with equivalent-level human beta-globin expression detected by flow cytometry. Long-term gene marking levels among mononuclear cells and granulocytes assessed by quantitative polymerase chain reaction gradually decreased to about 0.001% at 2 years, likely due to additional HIV-1 restrictive elements in the rhesus macaque. No evidence of clonal hematopoiesis has occurred in our animals in up to 2 years. Current efforts are aimed at developing a lentiviral vector capable of efficiently transducing both human and rhesus HSCs to allow preclinical modeling of globin gene transfer.

Xenografting immunodeficient mice has been employed as a surrogate human hematopoietic stem cell (HSC) assay, however, erythroid output has not been reliably reported, limiting the usefulness of this model for erythroid disorders. We have previously demonstrated that busulfan preconditioning is sufficient to produce stable, high level engraftment of human cells in NOD/SCID/IL2Rγ null mice. Importantly, this high level engraftment can be achieved with low mortality, substantially reducing the number of animals required for experiments requiring long-term follow-up. Supplementation with human holo-transferrin (Tf) allows the detection human erythrocytes in this chimeric mouse assay at low levels, providing a potential model for the study of disorders affecting human red blood cells (2007 ASH meeting #3594). In the current work, we extend our observations and establish practical in vivo erythroid assay system of human HSCs in the humanized mouse model by the addition of an in vitro culture. Bone marrow (BM) from humanized mice containing 24.8±8.7% human cells (n=6) was first exposed to recombinant human (rHu) SCF+IL3 for 3 days in order to specifically enrich for human cells in the mixed chimera. After 3 days, human cells comprised 68.1±8.5% of the culture. The enriched cells were then cultured with rHu EPO+SCF+TGF-β for 7 additional days and then in rHu EPO+SCF for 7 days. After culture, and 98.9±1.47% of cells were of human origin. After centrifugation, the pellets were visibly red. Cells were assayed with both human CD71+ and GPA+ by flow cytometry: 64.0±7.44% were CD71+ and 69.2±7.02% were GPA+, and human α, β, and γ globin were confirmed by hemoglobin electrophoresis and mass spectrometry. In order to determine the utility of this approach, we tested 3 possible applications of this methodology: gene marking erythroid progeny, modeling of human hemoglobinopathies, and modeling of hemoglobin switching. We first transplanted human cord blood (CB) CD34+ cells after lentiviral transduction with a vector encoding GFP following busulfan conditioning. Three months post-transplant, bone marrow was harvested and placed in the in vitro culture. After in vivo culture, 98.9% of cells were of human origin and 60.7% were CD71+ and 72.3% were GPA+. The majority of CD71+ or GPA+ cells were GFP+ (82.3% and 87.7%, respectively). We subsequently transplanted human PB CD34+ cells derived from individuals with sickle cell trait (SCT) as we have previously demonstrated that these cells, unlike those from individuals with sickle cell disease, can be safely mobilized and processed. Further, the percentage of HbS expressed can be reliably measured. Three months post transplant, HbS was easily detectable by hemoglobin electrophoresis. Finally, we sought to address whether this model accurately reflects human erythropoiesis by examining hemoglobin switching after transplanting either CB expressing HbF or PB HSCs expressing HbA and monitoring the output of HbF and HbA over time. Early post-transplant, bone marrow derived from CB recipients expressed predominantly HbF after culture whereas that derived from PB HSC recipients expressed predominantly HbA. HbF declined during follow up and was replaced by HbA over 6 months of follow up from CB recipients, whereas HbA expression remained stable from PB HSC recipients. The time course of hemoglobin switching is similar to human ontogeny. In summary, our practical approach to model human erythropoiesis in the xenograft mouse should prove useful in the both the study of human erythroid disorders as well as therapeutic interventions.

Objective Myeloablative total body irradiation (TBI) in the setting of autologous transplantation of genetically modified hematopoietic stem cells (HSC) is associated with substantial toxicity. Nonmyeloablative doses of TBI are less toxic, but result in low-level engraftment of genetically modified HSCs. As an alternative to TBI, escalating doses of parenteral busulfan were tested for their hematologic toxicity, their ability to promote donor leukocyte engraftment, and the time window for such engraftment. Materials and Methods Hematologic toxicity of busulfan was assessed in C57BL6 mice after single nonmyeloablative doses of intraperitoneal busulfan ranging from 1 to 40 mg/kg by serial complete blood counts monitored up to 40 days. The level of donor engraftment attainable after nonmyeloablative busulfan was determined by infusion of 20 million congenic murine bone marrow nucleated cells (BMNC) following 5 to 40 mg/kg of busulfan. To determine the effects of delayed HSC infusions, BMNCs were infused 1, 10, 15, and 20 days after a single dose of 10 mg/kg of busulfan. Results Busulfan doses from 1 to 40 mg/kg produced hematologic toxicity that was most pronounced in the 2nd to 3rd week. In transplantation experiments, dose-dependent donor leukocyte engraftment was attained with levels >70% after only 20 mg/kg of busulfan. Similar levels of engraftment were achieved even when infusion of BMNCs was delayed up to 20 days after busulfan injection. Conclusion Nonmyeloablative parenteral busulfan produced transient myelosuppressive effects, clinically relevant levels of engraftment, and an extended time window for HSC infusion in murine hosts.

Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.