R. Patrick Weitzel

Nonmyeloablative HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation for Severe Sickle Cell Phenotype

IMPORTANCE Myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is curative for children with severe sickle cell disease, but toxicity may be prohibitive for adults. Nonmyeloablative transplantation has been attempted with degrees of preparative regimen intensity, but graft rejection and graft-vs-host disease remain significant. OBJECTIVE To determine the efficacy, safety, and outcome on end-organ function with this low-intensity regimen for sickle cell phenotype with or without thalassemia. DESIGN, SETTING, AND PARTICIPANTS From July 16, 2004, to October 25, 2013, 30 patients aged 16-65 years with severe disease enrolled in this nonmyeloablative transplant study, consisting of alemtuzumab (1 mg/kg in divided doses), total-body irradiation (300 cGy), sirolimus, and infusion of unmanipulated filgrastim mobilized peripheral blood stem cells (5.5-31.7 × 10(6) cells/kg) from human leukocyte antigen-matched siblings. MAIN OUTCOMES AND MEASURES The primary end point was treatment success at 1 year after the transplant, defined as a full donor-type hemoglobin for patients with sickle cell disease and transfusion independence for patients with thalassemia. The secondary end points were the level of donor leukocyte chimerism; incidence of acute and chronic graft-vs-host disease; and sickle cell-thalassemia disease-free survival, immunologic recovery, and changes in organ function, assessed by annual brain imaging, pulmonary function, echocardiographic image, and laboratory testing. RESULTS Twenty-nine patients survived a median 3.4 years (range, 1-8.6), with no nonrelapse mortality. One patient died from intracranial bleeding after relapse. As of October 25, 2013, 26 patients (87%) had long-term stable donor engraftment without acute or chronic graft-vs-host disease. The mean donor T-cell level was 48% (95% CI, 34%-62%); the myeloid chimerism levels, 86% (95% CI, 70%-100%). Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no graft-vs-host disease. The normalized hemoglobin and resolution of hemolysis among engrafted patients were accompanied by stabilization in brain imaging, a reduction of echocardiographic estimates of pulmonary pressure, and allowed for phlebotomy to reduce hepatic iron. The mean annual hospitalization rate was 3.23 (95% CI, 1.83-4.63) the year before, 0.63 (95% CI, 0.26-1.01) the first year after, 0.19 (95% CI, 0-0.45) the second year after, and 0.11 (95% CI, 0.04-0.19) the third year after transplant. For patients taking long-term narcotics, the mean use per week was 639 mg (95% CI, 220-1058) of intravenous morphine-equivalent dose the week of their transplants and 140 mg (95% CI, 56-225) 6 months after transplant. There were 38 serious adverse events: pain and related management, infections, abdominal events, and sirolimus related toxic effects. CONCLUSIONS AND RELEVANCE Among 30 patients with sickle cell phenotype with or without thalassemia who underwent nonmyeloablative allogeneic HSCT, the rate of stable mixed-donor chimerism was high and allowed for complete replacement with circulating donor red blood cells among engrafted participants. Further accrual and follow-up are required to assess longer-term clinical outcomes, adverse events, and transplant tolerance. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00061568.

Characterization of extracellular DDX4- or Ddx4-positive ovarian cells

To the Editor: A few groups1–5 have now reported that ovarian-derived stem cells (OSCs; also known as oogonial stem cells or oocyte precursor cells) have been isolated from adult mouse5 and rat4 ovaries; these cells are able to undergo meiosis after transplantation back into recipient ovaries and give rise to offspring. These cells have also been isolated from human adult ovaries, where they can give rise to oocytes after xenotransplantation2. The marker used to isolate viable cells with germ cell characteristics, DDX4 in humans or Ddx4 in mice and rats (hereafter referred to collectively as DDX4/Ddx4), is controversial because it was historically considered to be exclusively an intracellular protein distributed in the cytoplasm of germ cells. However, DDX4/Ddx4 was reported to have a C-terminal domain that is expressed extracellularly, whereas the N terminus is expressed intracellularly2,3. DDX4/Ddx4 expression was reported in freshly isolated OSCs and after propagation for 18 months (mouse) and 4 months (human) in defined cultures by immunostaining, reverse-transcription PCR (RT-PCR), and fluorescence-activated cell sorting (FACS)2. Here we further characterize the expression of DDX4/Ddx4 in mouse, rhesus macaque and human ovarian cells using a polyclonal antibody specific to DDX4 (ab13840; Abcam, generously provided by Jonathan Tilly and purchased from Abcam). By immunohistochemical analysis of paraffin-embedded tissue, we observed staining for DDX4/Ddx4 in the expected locations in

Infusion of hemolyzed red blood cells within peripheral blood stem cell grafts in patients with and without sickle cell disease

Peripheral blood stem cell (PBSC) infusions are associated with complications such as elevated blood pressure and decreased creatinine clearance. Patients with sickle cell disease experience similar manifestations, and some have postulated release of plasma-free hemoglobin with subsequent nitric oxide consumption as causative. We sought to evaluate whether the infusion of PBSC grafts containing lysed red blood cells (RBCs) leads to the toxicity observed in transplant subjects. We report a prospective cohort study of 60 subjects divided into 4 groups based on whether their infusions contained dimethyl sulfoxide (DMSO) and lysed RBCs, no DMSO and fresh RBCs, DMSO and no RBCs, or saline. Our primary end point, change in maximum blood pressure compared with baseline, was not significantly different among groups. Tricuspid regurgitant velocity and creatinine levels also did not differ significantly among groups. Our data do not support free hemoglobin as a significant contributor to toxicity associated with PBSC infusions. This study was registered at clinicaltrials.gov (NCT00631787).

Maternal T Regulatory Cell Depletion Impairs Embryo Implantation Which Can Be Corrected with Adoptive T Regulatory Cell Transfer

Maternal immune tolerance of fetal engraftment is critical for the establishment and maintenance of pregnancy, but the exact mechanisms permitting this semi-allograft in the maternal host are not completely understood. Further, failure of the embryo to implant in the uterus accounts for at least 30% of the best prognosis in vitro fertilization cycles when a perfect embryo is transferred to a normal uterus. We hypothesized that T regulatory cells (Tregs), defined by CD4+CD25hi surface expression and the FoxP3+ transcription factor, play an important role in the initiation of the earliest stages of pregnancy, specifically implantation of the embryo. In this study, we evaluated the role of Tregs in the establishment of pregnancy using a conditional depletion of Treg transgenic mouse model. We found that embryo implantation in the syngeneic mating was defective as evidenced by smaller litter sizes after Treg depletion and that embryo implantation could be restored by adoptively transferring Tregs into the mating mice. In allogeneic mating, litter sizes were not different but breeding efficiency was significantly decreased. These data reveal that Tregs are important for the establishment of the earliest stages of pregnancy and may be a potential cause of infertility due to recurrent implantation failure, which may be amenable to cellular or pharmacologic therapy to improve maternal immune tolerance of embryo implantation.

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

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

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

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