Biomedical subjects

H P Kiem

Publications and source records attributed to H P Kiem.

At least 19 recordsLinked to original sources

PubMed2005

Low-dose cyclophosphamide conditioning for haematopoietic cell transplantation from HLA-matched related donors in patients with Fanconi anaemia.

Allogeneic haematopoietic cell transplantation (HCT) is effective therapy for Fanconi anaemia (FA). FA patients do not tolerate conditioning with 200 mg/kg of cyclophosphamide (Cy), typically used in aplastic anaemia. We previously published results of studies in which Cy doses were gradually reduced from 200 to 100 mg/kg. Here we update results of the initial studies and report data on 30 new patients conditioned with Cy either at 80 mg/kg (n = 7) or at 60 mg/kg (n = 23), given over 4 days before HCT from human leucocyte antigen-matched related donors. Methotrexate and cyclosporine were given for graft-versus-host disease (GVHD) prophylaxis. All seven patients given Cy at 80 mg/kg and 21 of 23 given Cy at 60 mg/kg had sustained engraftment, while two patients, both with clonal cytogenetics abnormalities, experienced graft failure. Grades 2-3 acute GVHD rates were 57% and 14% for patients given the higher and lower Cy doses, respectively (P = 0.001). Four patients given Cy at 80 mg/kg and 22 given Cy at 60 mg/kg were alive at a median of 47 (44-58) months and 16 (3-52) months, respectively. Cy at 60 mg/kg has acceptable toxicities, low rates of GVHD, and is sufficient for engraftment of related grafts in most FA patients.

Adolescent↗

PubMed2001

Engraftment of DLA-haploidentical marrow with ex vivo expanded, retrovirally transduced cytotoxic T lymphocytes.

Genetically modified donor T cells with an inducible "suicide" gene have the potential to improve the safety and availability of allogeneic hematopoietic stem cell transplantation by enhancing engraftment and permitting control of graft-versus-host disease (GVHD). However, several clinical studies of gene-modified T cells have shown limited to no in vivo function of the ex vivo expanded T cells. Using the well-established dog model of allogeneic marrow transplantation, the question was asked if retrovirally transduced, donor derived, ex vivo expanded cytotoxic T lymphocytes (CTLs) that are recipient specific could enhance engraftment of dog leukocyte antigen (DLA)-haploidentical marrow following a single dose of 9.2 Gy total body irradiation and no postgrafting immunosuppression. In this setting, only 4 of 11 control recipients of DLA-haploidentical marrow without added CTLs engrafted. CTLs did not enhance engraftment of CD34(+) selected peripheral blood stem cells. However, recipient-specific CTLs enhanced engraftment of DLA-haploidentical marrow in 9 of 11 evaluable recipients (P =.049). All dogs that engrafted developed multiorgan GVHD. To facilitate in vivo tracking, 8 dogs received CTLs transduced with a retroviral vector encoding green fluorescent protein (GFP) and neomycin phosphotransferase (neo). Recipients that engrafted had sharp increases in the numbers of circulating GFP(+) CTLs on days +5 to +6 after transplantation. GFP(+) CTLs isolated from blood were capable of recipient-specific lysis. At necropsy, up to 7.1% of CD3(+) cells in tissues were GFP(+) and polymerase chain reaction in situ hybridization for neo showed infiltration of transduced CTLs in GVHD-affected organs. These results show that ex vivo expanded, transduced T cells maintained in vivo function and enhanced marrow engraftment.

Animals↗

PubMed2001

Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.

Previous studies have shown that the choice of envelope protein (pseudotype) can have a significant effect on the efficiency of retroviral gene transfer into hematopoietic stem cells. This study used a competitive repopulation assay in the dog model to evaluate oncoretroviral vectors carrying the envelope protein of the endogenous feline virus, RD114. CD34-enriched marrow cells were divided into equal aliquots and transduced with vectors produced by the RD114-pseudotype packaging cells FLYRD (LgGLSN and LNX) or by the gibbon ape leukemia virus (GALV)-pseudotype packaging cells PG13 (LNY). A total of 5 dogs were studied. One dog died because of infection before sustained engraftment could be achieved, and monitoring was discontinued after 9 months in another animal that had very low overall gene-marking levels. The 3 remaining animals are alive with follow-ups at 11, 22, and 23 months. Analyses of gene marking frequencies in peripheral blood and marrow by polymerase chain reaction revealed no significant differences between the RD114 and GALV-pseudotype vectors. The LgGLSN vector also contained the enhanced green fluorescent protein (GFP), enabling us to monitor proviral expression by flow cytometry. Up to 10% of peripheral blood cells expressed GFP shortly after transplantation and approximately 6% after the longest follow-up of 23 months. Flow cytometric analysis of hematopoietic subpopulations showed that most of the GFP-expressing cells were granulocytes, although GFP-positive lymphocytes and monocytes were also detected. In summary, these results show that RD114-pseudotype oncoretroviral vectors are able to transduce hematopoietic long-term repopulating cells and, thus, may be useful for human stem cell gene therapy.

Amino Acid Transport System ASC↗

PubMed2001

Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects.

Toxicities have limited the use of allogeneic hematopoietic cell transplantation (HCT) to younger, medically fit patients. In a canine HCT model, a combination of postgrafting mycophenolate mofetil (MMF) and cyclosporine (CSP) allowed stable allogeneic engraftment after minimally toxic conditioning with low-dose (200 cGy) total-body irradiation (TBI). These findings, together with the known antitumor effects of donor leukocyte infusions (DLIs), led to the design of this trial. Forty-five patients (median age 56 years) with hematologic malignancies, HLA-identical sibling donors, and relative contraindications to conventional HCT were treated. Immunosuppression involved TBI of 200 cGy before and CSP/MMF after HCT. DLIs were given after HCT for persistent malignancy, mixed chimerism, or both. Regimen toxicities and myelosuppression were mild, allowing 53% of eligible patients to have entirely outpatient transplantations. Nonfatal graft rejection occurred in 20% of patients. Grades II to III acute graft-versus-host disease (GVHD) occurred in 47% of patients with sustained engraftment. With median follow-up of 417 days, survival was 66.7%, nonrelapse mortality 6.7%, and relapse mortality 26.7%. Fifty-three percent of patients with sustained engraftment were in complete remission, including 8 with molecular remissions. This novel allografting approach, based on the use of postgrafting immunosuppression to control graft rejection and GVHD, has dramatically reduced the acute toxicities of allografting. HCT with the induction of potent graft-versus-tumor effects can be performed in previously ineligible patients, largely in an outpatient setting. Future protocol modifications should reduce rejection and GVHD, thereby facilitating studies of allogeneic immunotherapy for a variety of malignancies. (Blood. 2001;97:3390-3400)

Adult↗

PubMed2001

Gene transfer into baboon repopulating cells: A comparison of Flt-3 Ligand and megakaryocyte growth and development factor versus IL-3 during ex vivo transduction.

Oncoretroviral vectors require division of target cells for successful transduction. In the case of hematopoietic repopulating cells this can be achieved by cytokine stimulation using growth factor combinations which facilitate gene transfer and maintain engraftment. Interleukin-3 (IL-3) has been widely used in growth factor combinations, although more recent data in the mouse showed reduced engraftment in the presence of IL-3. Here, we used a competitive repopulation assay to study the influence of IL-3 and the early acting cytokines megakaryocyte growth and development factor (MGDF) and Flt3-ligand (Flt3-L) on gene transfer efficiency during ex vivo transduction of hematopoietic repopulating cells. In a direct comparison, baboon CD34-enriched cells were transduced on CH-296 fibronectin fragment in the presence of either IL-6, stem cell factor (SCF), Flt3-L, and MGDF or IL-3, IL-6, and SCF. Animals were followed for up to 55 weeks, and analysis of peripheral blood leukocytes by semiquantitative polymerase chain reaction showed that both cytokine combinations achieved marking of repopulating cells. A trend toward increased gene marking, especially early after transplant (P = 0.06), was seen with the combination of IL-6, SCF, Flt3-L, and MGDF. However, the highest gene marking was achieved when IL-3 was combined with early acting cytokines, suggesting that the difference observed in this study was probably due to the addition of MGDF and Flt3-L and not due to a negative effect of IL-3 on engraftment.

Animals↗

PubMed2001

Purified canine CD34+Lin- marrow cells transduced with retroviral vectors give rise to long-term multi-lineage hematopoiesis.

Human CD34+ cells have been shown to retain long-term hematopoietic engrafting potential in preclinical and clinical studies. However, recent studies of human and murine CD34- stem cells suggest that these are functionally important early progenitors. Using autologous transplantation, we investigated whether canine CD34 and CD34- marrow cells could be transduced and give rise to long-term hematopoiesis. CD34+Lin- and CD34-Lin- cell populations purified by fluorescence-activated cell sorting were separately cocultivated with retroviral vectors LN (CD34+Lin-) and LNY (CD34-Lin-), which carry the neomycin (neo) gene. After myeloablative total body irradiation (920 cGy), 3 dogs received transplants of both CD34+Lin- cells and CD34-Lin- cells and 2 dogs received only CD34-Lin- cells. Untransduced autologous marrow cells were given to ensure hematopoietic recovery. Using CFU-C assays, transduction efficiencies of CD34+Lin- cells ranged from 6% to 18% with no CFU-C formation from CD34-Lin- cells. PCR-based detection of the neo gene from WBCs was used to detect transduced cells weekly after transplantation. Additional PCR studies in 3 dogs given both CD34+Lin- and CD34-Lin- cells were performed on monocytes, granulocytes, and T cells (2 dogs, one at 7.5 months and the other at 9 months) and granulocytes (1 dog at 12 months). LN was detected up to 12 months posttransplantation in WBCs and mono-myeloid and lymphoid populations from 3 dogs receiving transplants of transduced CD34+Lin- cells. LNY was not detected at any time after transplantation in 5 dogs that received transduced CD34-Lin- cells. Whereas canine CD34+Lin- marrow cells contributed to long-term multilineage hematopoiesis, progeny of CD34-Lin- progenitor cells were not detected after transplantation in these experiments.

Animals↗

PubMed2001

Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34(+) cells emphasize limited transduction at low receptor levels.

Successful gene therapy for the treatment of heritable or acquired diseases typically requires high efficiency gene transfer and sustained transgene expression. Indirect evidence on the basis of RNA analysis and in vivo competitive repopulation experiments in animal models suggests a correlation between transduction efficiency and the abundance of retrovirus receptors on the hematopoietic target cell. However, transduction by oncoretroviral vectors is also subject to other factors such as target cell cycle status and the composition of the virus-containing medium, making it difficult to determine the level of receptor expression required for efficient transduction. In the present study we investigated the impact of receptor expression level on transduction by a vector with a gibbon ape leukemia virus (GALV) envelope protein in a tetracycline-inducible tissue culture model that allowed for the cell cycle-independent, regulated expression of the GALV receptor (Pit1) in otherwise non-susceptible NIH 3T3 cells. Up-regulation of receptor RNA expression by 4.5-fold resulted in a mean 150-fold increase in transduction efficiency. We then analyzed cell surface expression of the Pit1 receptor using a fusion protein consisting of GALV SU portion of the viral envelope protein linked to the human IgG Fc. These experiments showed that tetracycline-regulated receptor induction resulted in a dose-dependent increase in binding of fusion protein. At maximum induction fusion protein binding increased up to five-fold which paralleled the increase in RNA expression, and correlated with the improved transduction efficiency. Finally, studies of pseudotype-specific fusion protein binding to human CD34-enriched cells revealed increased expression of retrovirus receptors after cytokine stimulation, although overall receptor expression in CD34(+)cells remained lower than in fibroblast cell lines efficiently transduced by amphotropic and GALV vectors.

Antigens, CD34↗

PubMed2001

Severe canine hereditary hemolytic anemia treated by nonmyeloablative marrow transplantation.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase (PK) deficiency can be corrected by marrow allografts from healthy littermates after a conventional high-dose myeloablative conditioning regimen. The nonmyeloablative conditioning regimen used here, which consisted of a sublethal dose of 200 cGy total body irradiation before and immunosuppression with mycophenolate mofetil and cyclosporine after a dog leukocyte antigen (DLA)-identical littermate allograft, has been found to be effective in establishing stable mixed donor/host hematopoietic chimerism in normal dogs. We explored the feasibility of nonmyeloablative marrow allografts for the treatment of canine PK deficiency and studied the effect of stable allogeneic mixed hematopoietic chimerism on the natural course of the disease. Five affected dogs received transplants, of which 3 dogs had advanced liver cirrhosis and myelofibrosis. Both complications were presumed to be due to iron overload. All 5 dogs showed initial engraftment. Two rejected their grafts after 6 weeks but survived with completeautologous marrow recovery and return of the disease. One died from liver failure on day 27 with 60% donor engraftment. Two dogs have shown sustained mixed donor/host chimerism for more than a year with 85% and 12% donor hematopoietic cells, respectively. Overall clinical response correlated with the degree of donor chimerism. The dog with the low degree of chimerism achieved partial resolution of hemolysis, but the disease symptoms persisted as manifested by increasing iron overload resulting in progression of marrow and liver fibrosis. The dog with the high degree of donor chimerism achieved almost complete resolution of hemolysis with a decrease of marrow iron content and resolution of marrow fibrosis. These observations suggest that mixed hematopoietic chimerism can be relatively safely established in dogs with PK deficiency even in the presence of advanced liver cirrhosis. However, although effective in correcting or delaying the development of myelofibrosis, a low degree of mixed chimerism was not sufficient to prevent continued hemolysis of red blood cells of host origin. Complete donor chimerism appears necessary to achieve a long-term cure.

Anemia, Hemolytic, Congenital↗

PubMed2001

All-trans retinoic acid facilitates oncoretrovirus-mediated transduction of hematopoietic repopulating stem cells.

A major limiting factor in achieving high levels of gene transfer into hematopoietic stem cells is the ability to retain significant repopulating activity of the stem cells during the ex vivo exposure to oncoretroviral vectors. Recently, we reported that pharmacological levels (1 microM) of all-trans retinoic acid (ATRA) enhanced the maintenance of in vivo repopulating hematopoietic stem cells during liquid suspension culture. Therefore, we investigated the use of ATRA to improve transduction of hematopoietic repopulating cells. Hematopoietic precursors cultured and transduced with a GFP-containing oncoretroviral vector with or without ATRA were transplanted immediately post-transduction (day 3 post-culture initiation) or following extended culture without further transduction (day 7 post-culture initiation). Mice transplanted with 3-day ATRA-treated cells had four-fold more donor cells than the untreated cells. In contrast, there were more GFP-expressing donor cells in recipients of cells cultured without ATRA (31.31 +/- 8.47% no ATRA vs. 16.52 +/- 9.35% ATRA). After 7 days of culture, however, the repopulating ability of the hematopoietic precursors was the same for both treatment groups, but the ATRA-treated cells had significantly more green fluorescence protein (GFP)-expressing donor cells (5.57 +/- O.53% no ATRA vs. 13.67 +/- 2.14% ATRA). Secondary recipients of marrow from recipients of the 3 day cultured cells had similar donor cell levels, but the percentage of GFP-expressing cells within the donor cell population was higher in the recipients of ATRA-treated cells (3.25 +/- 0.70% no ATRA vs. 7.97 +/- 2.71% ATRA). Our data show that the addition of ATRA to cultures of hematopoietic precursors resulted in increased gene transfer into murine hematopoietic repopulating cells. These data suggest that ATRA may be useful in clinical gene therapy protocols using oncoretroviral vectors.

Animals↗

PubMed2001

Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of gene-modified autologous T cells in a nonhuman primate model.

The in vivo persistence of gene-modified cells can be limited by host immune responses to transgene-encoded proteins. In this study we evaluated in a nonhuman primate model whether the administration of a nonmyeloablative regimen consisting of low-dose total-body irradiation with 200 cGy followed by immunosuppression with mycophenolate mofetil and cyclosporin A for 28 and 35 days, respectively, could be used to facilitate persistence of autologous gene-modified T cells when a transgene-specific immune response had already been established or to induce long-lasting tolerance in unprimed recipients. Two macaques (Macaca nemestrina) received infusions of T cells transduced to express either the enhanced green fluorescent protein and neomycin phosphotransferase genes or the hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes. In the absence of immunosuppression, both macaques developed potent class I major histocompatibility complex-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses that rapidly eliminated the gene-modified T cells and that persisted long term as memory CTL. Treatment with the nonmyeloablative regimen failed to abrogate preexisting memory CTL responses but interfered with the induction of transgene-specific CTL and facilitated in vivo persistence of gene-modified cells in an unprimed host. However, sustained tolerance to gene-modified T cells was not achieved with this regimen, indicating that further modifications will be required to permit sustained persistence of gene-modified T cells.

Animals↗

PubMed2001

Nonmyeloablative hematopoietic cell transplantation. Replacing high-dose cytotoxic therapy by the graft-versus-tumor effect.

Conventional allografting produces considerable regimen-related toxicities that generally limit this treatment to patients younger than 55 years and in otherwise good medical condition. T cell-mediated graft-versus-tumor (GVT) effects are known to play an important role in the elimination of malignant disease after allotransplants. A minimally myelosuppressive regimen that relies on immunosuppression for allogeneic engraftment was developed to reduce toxicities while optimizing GVT effects. Pre-transplant total-body irradiation (200 cGy) followed by post-transplant immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) permitted human leukocyte antigen (HLA)-matched sibling donor hematopoietic cell engraftment in 82% of patients (n = 55) without prior high-dose therapy. The addition of fludarabine (90 mg/m2) facilitated engraftment in all 28 subsequent patients. Overall, fatal progression of underlying disease occurred in 20% of patients after transplant. Non-relapse mortality occurred in 11% of patients. Toxicities were low. Grade 2-4 acute graft-versus-host disease (GVHD) associated with primary engraftment developed in 47% of patients, and was readily controlled in all but two patients. Donor lymphocyte infusions (DLI) were not very effective at converting a low degree of mixed donor/host chimerism to full donor chimerism; however, the addition of fludarabine reduced the need for DLI. With a median follow-up of 244 days, 68% of patients were alive, with 42% of patients in complete remission, including molecular remissions. Remissions occurred gradually over periods of weeks to a year. If long-term efficacy is demonstrated, such a strategy would expand treatment options for patients who would otherwise be excluded from conventional allografting.

Adult↗

PubMed2000

Differential engraftment of genetically modified CD34(+) and CD34(-) hematopoietic cell subsets in lethally irradiated baboons.

OBJECTIVE: To test gibbon ape leukemia virus (GALV) pseudotype vector transduction of marrow subpopulations that contribute to hematopoietic reconstitution in vivo. MATERIALS AND METHODS: Autologous CD34(+) Lin(-), CD34(+) Lin(+), and CD34(-) Lin(-) marrow cells, transduced by coculture with PG13/LN, PG13/LNX, and PG13/LNY vector-producing cells, respectively, were transplanted in three female baboons. Two female baboons also were transplanted with fresh allogeneic CD34(-)Lin(-) marrow cells from MHC-matched male siblings and, to ensure survival, with autologous CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells transduced with PG13/LN and PG13/LNX, respectively. The LN, LNX, and LNY vectors are identical except for different length sequences at the 3' end of the bacterial neomycin phosphotransferase (neo) gene. RESULTS: LN(+) and LNX(+) cells from CD34(+)Lin(-) and CD34(+)Lin(+) cells, respectively, but no LNY(+) from CD34(-)Lin(-) cells were detectable in blood and marrow of all animals after transplant. LN(+), CD34(+)Lin(-) cells contributed to reconstitution of the T, B, and myeloid lineages. LNX(+), CD34(+)Lin(+) cells contributed only to B and myeloid lineages. Male cells, CD34(-)Lin(-), were detected by polymerase chain reaction in blood and marrow of the two allogeneic transplanted animals at estimated frequencies of </=0.001% 1 month after transplant in both animals. Male cells became undetectable in one animal and have remained detectable, with declining frequency, in the other for more than 15 months. In this animal, no male CD34(+) or colony-forming cells have been detected. CONCLUSIONS: CD34(+)Lin(-) and CD34(+)Lin(+) marrow cells can serve as targets for GALV pseudotype retrovirus-mediated gene transfer. CD34(+)Lin(-) cells contribute to reconstitution of all hematopoietic lineages. Autologous CD34(-)Lin(-) cells were either not transduced by GALV pseudotype retrovirus vectors using current approaches or did not contribute significantly to reconstitution, as suggested by allogeneic transplants.

Animals↗

PubMed2000

Highly efficient gene transfer into preterm CD34 hematopoietic progenitor cells.

OBJECTIVE: Retrovirus-mediated gene transfer has been shown to transduce CD34(+) cells from term gestation umbilical cord blood with relatively high efficiency. The purpose of this study was to compare the efficiencies of retrovirus-mediated gene transfer into early (23-28 weeks' gestation) and term (37-41 weeks' gestation) umbilical cord blood CD34(+) hematopoietic progenitor cells. STUDY DESIGN: CD34(+) cells were purified from cyropreserved early (23-28 weeks' gestation) and term (37-40 weeks' gestation) umbilical cord blood specimens with fluorescence-activated cell sorting. The CD34(+) cells were then transduced in virus-containing medium (gibbon ape leukemia virus pseudotype vector LAPSN [PG13]) in wells coated with the recombinant human fibronectin fragment CH-296 and in the presence of multiple hematopoietic growth factors (interleukin 6, stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and megakaryocyte growth and development factor) and protamine sulfate. The LAPSN (PG13) virus-containing medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia virus pseudotype vector LAPSN (PG13) contains the human placental alkaline phosphatase and neomycin phosphotransferase (neo ) genes. The efficiency of gene transfer was assessed by histochemical staining of colony-forming cells in agar for expression of heat-stable alkaline phosphatase. RESULTS: Gene transfers, as assessed by alkaline phosphatase staining of colony-forming cells (granulocyte-macrophage colony-forming units and erythroid burst-forming units), were similar for CD34(+) hematopoietic progenitor cells from early (58.4% +/- 11.8%) and term (63.2% +/- 12.5%) gestation fetal umbilical cord blood. CONCLUSION: CD34(+) hematopoietic progenitor cells from midgestation fetal blood can be transduced with high efficiency using techniques optimized for postnatal samples with a gibbon ape leukemia virus pseudotype vector. The early fetus may be a preferable target for gene therapy because of the higher number of circulating CD34(+) and CD38(-) cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Because in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the efficiency of gene transfer into marrow-repopulating cells in vivo, studies that examine clinically applicable approaches to in utero gene therapy in appropriate animal models are still needed.

Alkaline Phosphatase↗

PubMed2000

Expansion and transduction of nonenriched human cord blood cells using HS-5 conditioned medium and FLT3-L.

Cord blood (CB) stem cell transplantations have been associated with delayed hematopoietic engraftment. This has most likely been due to the limited numbers of hematopoietic short-term repopulating cells in CB. Ex vivo expansion of CB has been attempted, and expansion of CD34-enriched CB has been successful; however, CD34 enrichment procedures are in general associated with substantial cell loss. Thus, we have studied culture conditions for expansion of nonenriched CB. Nonenriched CB cells were cultured for 21 days in the presence of conditioned medium from the HS-5 stromal cell line and FLT3-L or alternatively in the presence of FLT3-L, stem cell factor (SCF), megakaryocite growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF) (FSMG), either on fibronectin fragment CH-296-coated dishes or on uncoated dishes. With all four culture conditions, the number of mononuclear cells initially decreased until day 7 and then increased until the end of the expansion cultures. Overall expansion using HS-5 and FLT3-L resulted in superior expansion of MNC and CFU-C (44-/34-fold) for both cultures with and without CH-296 compared to FSMG (18-/17-fold). Expansion on CH-296 was less efficient than expansion on tissue culture-treated wells without CH-296 for both conditions. We then studied the best time for transduction on nonenriched CB. In contrast to enriched CD34 cells, we found for both conditions, HS-5/FLT3-L and growth factor cocktail, higher transduction efficiencies when cells were transduced on day 7 as compared to day 2. Gene transfer rates up to 45% were achieved with both conditions, which corresponded with the increased number of cells in S phase on day 7 compared to day 2. We conclude that HS-5 and FLT-3L allow efficient expansion and transduction of nonenriched CB.

Cell Culture Techniques↗

PubMed2000

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes.

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.

Animals↗

PubMed2000

Progress towards hematopoietic stem cell gene therapy.

The introduction of recombinant genetic material into human cells for therapeutic purposes offers tremendous potential. However, almost from the beginning, the application of gene therapy has been characterized by the striking discrepancy between its promise and realization. Over the past 15 years, much has been learned about the various gene transfer systems and the requirements for efficient hematopoietic stem cell gene transfer. In the current review, we will summarize recent improvements in hematopoietic stem cell gene transfer, describe some of the promising results from recent clinical applications and the impediments that remain.

Animals↗

PubMed1999

The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs.

A competitive repopulation assay in the dog was used to develop improved gene transfer protocols for hematopoietic stem cell gene therapy. Using this assay, we previously showed improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched marrow cells were cocultivated on gibbon ape leukemia virus (GALV)-based retrovirus vector-producing cells. In the present study, we have investigated the use of fibronectin fragment CH-296 and 2 growth factor combinations to further improve gene transfer efficiency. CD34-enriched marrow cells from each dog were prestimulated for 24 hours and then divided into 3 equal fractions. Two fractions were placed into flasks coated with either CH-296 or bovine serum albumin (BSA) and virus-containing medium supplemented with growth factors, and protamine sulfate was replaced 4 times over a 48-hour period. One fraction was cocultivated on irradiated PG13 (GALV-pseudotype) packaging cells for 48 hours. In 2 animals, cells of the different fractions were transduced in the presence of human FLT-3 ligand (FLT3L), canine stem cell factor (cSCF), and human megakaryocyte growth and development factor (MGDF), and in 2 other dogs, transduction was performed in the presence of FLT3L, cSCF, and canine granulocyte-colony stimulating factor (cG-CSF). The vectors used contained small sequence differences, allowing differentiation of cells genetically marked by the different vectors. After transduction, nonadherent and adherent cells from all 3 fractions were pooled and infused into lethally irradiated dogs. Polymerase chain reaction and Southern blot analysis were used to determine the persistence of the transferred vectors in the peripheral blood and marrow cells after transplantation. The highest levels of gene transfer were obtained when cells were transduced in the presence of FLT3L, cSCF, and cG-CSF (gene transfer levels of more than 10% for more than 8 months so far). Compared with the 2 animals that received cells transduced with FLT3L, cSCF, and MGDF, gene transfer levels were significantly higher when dogs received cells that were transduced in the presence of cG-CSF. Transduction on CH-296 resulted in gene transfer levels that were at least as high as transduction by cocultivation. In summary, the overall levels of gene transfer obtained with these conditions should be sufficiently high to allow stem cell gene therapy studies aimed at correcting genetic diseases in dogs as a model for human gene therapy.

Animals↗

PubMed1999

Stable mixed hematopoietic chimerism in dogs given donor antigen, CTLA4Ig, and 100 cGy total body irradiation before and pharmacologic immunosuppression after marrow transplant.

Stable mixed chimerism can be established in dogs given a sublethal dose of 200 cGy total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP) for 28 and 35 days, respectively, after dog leukocyte antigen-identical marrow transplantation. Most likely, the role of pretransplant TBI was to provide host immunosuppression, since stable mixed chimerism was also achieved in MMF/CSP-treated dogs when 450 cGy irradiation, targeted to cervical, thoracic, and upper abdominal lymph nodes, was substituted for TBI. When TBI was reduced from 200 to 100 cGy, all grafts were rejected within 3 to 12 weeks. Here, we asked whether stable engraftment after 100 cGy TBI could be accomplished by first reducing the intensity of host immune responsiveness with help of the fusion peptide CTLA4Ig, which blocks T-cell costimulation through the B7-CD28 signal pathway. Accordingly, recipient T cells were activated with intravenous (IV) injections of 10(6) donor peripheral blood mononuclear cells (PBMC)/kg per day on days -7 to -1 before 100 cGy TBI, with concurrent administration of CTLA4Ig 4 mg/kg/d IV. All 7 dogs so treated showed initial mixed chimerism. Two rejected their allografts after 8 and 20 weeks, respectively, and survived with autologous marrow recovery; 1 mixed chimera was unevaluable because of death at 3 weeks from intussusception; and 4 showed persisting mixed chimerism, including unirradiated marrow and lymph node spaces, for now more than 46 to 70 weeks after transplant. Data support the hypothesis that stable marrow allografts can be established by combining nonmyeloablative pretransplant host immunosuppression with posttransplant host and donor cell immunosuppression using MMF/CSP.

Abatacept↗