Biomedical subjects

D B Kohn

Publications and source records attributed to D B Kohn.

At least 19 recordsLinked to original sources

PubMed2000

Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage- colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses.

Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (10(8)-10(9) infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80(+)). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80(+)). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/10(6) cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/10(6) cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4-immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses. (Blood. 2000;96:1317-1326)

Acute Disease↗

PubMed2000

Gene therapy using hematopoietic stem cells: Sisyphus approaches the crest.

Gene transfer targeting cells of the blood and immune system was one of the first areas of investigation in the field of gene therapy. Despite the encouraging results achieved in early studies using murine bone marrow, the task of gene transfer into human hematopoietic stem cells proved to be far more difficult. As a result, progress has been disappointingly slow and initial clinical trials generally failed to achieve significant levels of gene marking. The continued application of new advances in vectorology and hematopoietic stem cell biology has now led to improvements in preclinical models that are being translated into clinical trials. The progress and remaining problems are discussed in this review article.

Adenoviridae↗

PubMed2000

Intrakines--evidence for a trans-cellular mechanism of action.

Human CXCR4 is the receptor for the CXC chemokine SDF-1alpha and also acts as a coreceptor for T lymphotropic HIV-1 strains. Blocking the surface expression of this receptor via an intrakine approach has recently been shown to efficiently prevent HIV-1 infection of T cells. The CXC-chemokine gene is fused to an endoplasmic reticulum retention signal (KDEL) that retains the newly synthesized chemokine and its receptor within the cell, where both are subsequently degraded. We constructed MoMuLV-based vectors containing the SDF-KDEL construct driven by the "MND" long terminal repeat, using eGFP as a marker gene (MND-SDF-KDEL-IRES-eGFP) and a control vector (MND-X-IRES-eGFP). CEM human T lymphoblastic leukemia cells were transduced with the intrakine vector or the control vector. We detected a marked downregulation of CXCR4 expression in the cells transduced with the intrakine vectors as opposed to the cells transduced with the control vector. However, the eGFP-negative fraction of the cells transduced with the intrakine vector displayed the same CXCR4 downregulation as the eGFP-positive fraction, suggesting an effect in trans. The possibility of this being due to eGFP being silenced while SDF-KDEL was still expressed was excluded by Southern and Northern blot analyses. Upon cultivating the control cells with supernatant of the cells transduced with the intrakine vector, we observed a downregulation of CXCR4 expression on the control cells. Experiments using rhSDF-1alpha showed downregulation by the supernatant to be comparable to that achieved by the exogenous addition of 30 ng/ml SDF-1alpha. To assess the bioactivity of the secreted substance in the supernatant, a chemotaxis assay was performed. The transmigration observed was, once again, within the range of that achieved by the addition of 30 ng/ml SDF-1alpha. We conclude that the intrakine SDF-KDEL, apart from acting within the cell, is also in part secreted and causes the downregulation of the receptor by acting like a secreted chemokine.

Blotting, Northern↗

PubMed2000

Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors.

Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34(+) cells on fibronectin-coated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells.

Antigens, CD34↗

PubMed2000

Expression of human Wiskott-Aldrich syndrome protein in patients' cells leads to partial correction of a phenotypic abnormality of cell surface glycoproteins.

The Wiskott-Aldrich syndrome (WAS) is an uncommon X-linked recessive disease characterized by thrombocytopenia, eczema and immunodeficiency. The biochemical defect of this disorder primarily affects cells derived from bone marrow. To understand better the molecular mechanisms underlying this disease and to evaluate the possibility of correcting the genetic defects in hematopoietic cells, a Moloney murine leukemia virus (MoMLV)- based retroviral vector carrying a functional Wiskott-Aldrich syndrome protein (WASp) cDNA driven by an SV40 promoter (LNS-WASp) was constructed. A packaging cell line containing this vector produced a stable level of WAS protein and maintained a high titer of viral output. Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCL) from WAS patients, which lack expression of the WAS protein, were transduced by the LNS-WASp retroviral vector and showed expression of WASp by Western blot. Analysis of the O-glycan pattern on cell surface glycoproteins from WAS patients' B-LCL showed an altered glycosylation pattern, due to increased activity of beta-1, 6-N-acetylglucosaminyltransferase (C2GnT). Transduction by the retroviral vector carrying the functional WASp cDNA partially restored the abnormal glycosylation pattern, and was accompanied by a decreasing C2GnT activity. These findings imply a functional linkage between the WAS protein and the expression of the glycosyltransferase involved in the O-glycosylation, and also suggest a potential gene therapy via transferring a functional WASp cDNA into hematopoietic cells for Wiskott-Aldrich syndrome. Gene Therapy (2000) 7, 314-320.

Cells, Cultured↗

PubMed2000

T cell depleted haploidentical bone marrow transplantation for the treatment of children with severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is fatal in early childhood if unrecognized and if not treated. The aim was to determine the efficacy of T cell depleted bone marrow transplantation (TCD BMT) in the treatment of children with SCID. Eleven children diagnosed with SCID received histocompatible related donor bone marrow transplantation--HRD BMT (group I). Thirty seven children diagnosed with SCID who did not have histocompatible donors were treated with TCD haploidentical parental bone marrow transplantation (BMT) (group II). TCD was performed by in vitro soybean lectin agglutination followed by E-rosette depletion. Patients were longitudinally assessed for the presence and function of T and B lymphocytes. In group I all children survived. The mean age of children in this group at the time of HRD BMT was 15.4 months. All surviving patients normalized their specific T cell function. Two out of 11 require treatment with intravenous immunoglobulin i.v. Ig. In group II 17 out of 37 (46%) children survived. At the time of TCD BMT the mean age of survivors was 7.5 months, vs. 11.4 months in patients who died. Death was caused most commonly by opportunistic infections, Epstein-Barr virus induced lymphoproliferative disease (EBV-LPD), and graft versus host disease (GvHD). Seventeen out of 17 surviving patients recovered normal numbers of CD3+ cells and antigen specific T cell function. Five out of 17 never recovered their B cell function and require i.v. Ig injections. Early diagnosis, prevention or treatment of opportunistic infections, and enhancement of immune recovery will be necessary to improve survival in patients with SCID treated with TCD BMT.

Adolescent↗

PubMed1999

Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.

3T3 Cells↗

PubMed1999

Combination of CD80 and granulocyte-macrophage colony-stimulating factor coexpression by a leukemia cell vaccine: preclinical studies in a murine model recapitulating Philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a highly aggressive malignancy caused by the bcr-abl translocation oncogene. To explore alternative treatments for Ph+ ALL we tested gene-modified cell vaccines in the BALB/c-derived BM185 leukemia model. We compared the efficacy of BM185 cell vaccine expressing CD80 alone or in combination with IL-2 or GM-CSF. Mice injected with viable BM185 leukemia cells modified to express CD80 and GM-CSF (BM185/CD80+GM-CSF) showed the highest leukemia rejection rates. Cell vaccines consisting of irradiated BM185/CD80+GM-CSF cells administered subcutaneously stimulated a potent cytotoxic T lymphocyte (CTL) response against parental BM185. Histological examination of the vaccination site showed a large concentration of immune cells. Administration of the BM185/CD80+GM-CSF cell vaccine before intravenous challenge with parental cells caused strong inhibition of leukemia development. Vaccination after subcutaneous challenge with BM185 cells caused efficient elimination of leukemia promoting 40-60% long-term survival rates. The immunization efficacy of the BM185/CD80+ GM-CSF cell vaccine was directly correlated with the percentage of cells expressing the transgenes. In all, this preclinical study shows that leukemia cell vaccines coexpressing CD80 and GM-CSF can potentially be explored for immunotherapy in Ph+ ALL patients.

Animals↗

PubMed1999

Constitutive HOXA5 expression inhibits erythropoiesis and increases myelopoiesis from human hematopoietic progenitors.

The role of the homeobox gene HOXA5 in normal human hematopoiesis was studied by constitutively expressing the HOXA5 cDNA in CD34(+) and CD34(+)CD38(-) cells from bone marrow and cord blood. By using retroviral vectors that contained both HOXA5 and a cell surface marker gene, pure populations of progenitors that expressed the transgene were obtained for analysis of differentiation patterns. Based on both immunophenotypic and morphological analysis of cultures from transduced CD34(+) cells, HOXA5 expression caused a significant shift toward myeloid differentiation and away from erythroid differentiation in comparison to CD34(+) cells transduced with Control vectors (P =.001, n = 15 for immunophenotypic analysis; and P <.0001, n = 19 for morphological analysis). Transduction of more primitive progenitors (CD34(+)CD38(-) cells) resulted in a significantly greater effect on differentiation than did transduction of the largely committed CD34(+) population (P =.006 for difference between HOXA5 effect on CD34(+) v CD34(+)CD38(-) cells). Erythroid progenitors (burst-forming unit-erythroid [BFU-E]) were significantly decreased in frequency among progenitors transduced with the HOXA5 vector (P =.016, n = 7), with no reduction in total CFU numbers. Clonal analysis of single cells transduced with HOXA5 or control vectors (cultured in erythroid culture conditions) showed that HOXA5 expression prevented erythroid differentiation and produced clones with a preponderance of undifferentiated blasts. These studies show that constitutive expression of HOXA5 inhibits human erythropoiesis and promotes myelopoiesis. The reciprocal inhibition of erythropoiesis and promotion of myelopoiesis in the absence of any demonstrable effect on proliferation suggests that HOXA5 diverts differentiation at a mulitpotent progenitor stage away from the erythroid toward the myeloid pathway.

Cells, Cultured↗

PubMed1999

A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34(+) cells from the bone marrow of human immunodeficiency virus-1-infected children.

Genetic modification of hematopoietic stem cells with genes that inhibit replication of human immunodeficiency virus-1 (HIV-1) could lead to development of T lymphocytes and monocytic cells resistant to HIV-1 infection after transplantation. We performed a clinical trial to evaluate the safety and feasibility of this procedure, using bone marrow from four HIV-1-infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34(+) cells, performed in vitro transduction with a retroviral vector carrying a rev-responsive element (RRE) decoy gene, and reinfused the cells into these subjects with no evidence of adverse effects. The levels of gene-containing leukocytes in peripheral blood samples in the 1 year after gene transfer/cell infusion have been extremely low. These observations support the potential of performing gene therapy for HIV-1 using hematopoietic cells, but emphasize the need for improved gene transfer techniques.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Stem cell directed gene therapy.

A potential therapeutic approach to HIV-1 infection is the genetic modification of cells of a patient to make them resistant to HIV-1. Hematopoietic stem cells are an attractive target for gene therapy of AIDS because of their ability to generate a broad repertoire of mature T lymphocytes, as well as the monocytic cells (macrophages, dendritic cells and microglia) which are also involved in HIV-1 pathogenesis. A number of synthetic "anti-HIV-1 genes" have been developed which inhibit HIV-1 replication. However, current methods for gene transfer into human hematopoietic stem cells, using retroviral vectors derived from the Moloney murine leukemia virus, have been minimally effective. Clinical trials performed to date in which hematopoietic cells from HIV-1-positive patients have been transduced with retroviral vectors and then reinfused have produced low to undetectable levels of gene-containing peripheral blood leukocytes. New vector delivery systems, such as lentiviral vectors, need to be developed to ensure efficient gene transfer and persistent transgene expression to provide life-long resistance to the cells targeted by HIV-1.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors.

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.

ADP-ribosyl Cyclase↗

PubMed1998

Retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with Gaucher disease: in vivo detection of transduced cells without myeloablation.

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.

Adult↗

PubMed1998

Gene delivery to human B-precursor acute lymphoblastic leukemia cells.

Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1-based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.

Cation Exchange Resins↗

PubMed1998

High-resolution analysis of cytosine methylation in the 5ĺong terminal repeat of retroviral vectors.

Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.

Animals↗

PubMed1998

Immune response to Philadelphia chromosome-positive acute lymphoblastic leukemia induced by expression of CD80, interleukin 2, and granulocyte-macrophage colony-stimulating factor.

We examined the potential of generating an immune response against Philadelphia chromosome-positive acute lymphoblastic leukemia. The immunostimulatory molecules chosen for this study were the cytokines IL-2 and GM-CSF and the costimulatory ligand CD80 (B7.1). We used a murine model based on a BALB/c pre-B cell line, BM185wt, in which leukemia is induced by the p185 BCR-ABL oncogenic product, which reproduces Philadelphia chromosome-positive ALL. BM185wt cells were transduced with retroviral vectors and the transduced clones expressing mIL-2, mGM-CSF, or mCD80 were used for challenge. Expression of the immunomodulators by BM185 cells was correlated with delay in leukemia development in immunocompetent mice, but not in immunodeficient mice, indicating an immune response against the modified leukemia cells. Expression of CD80 caused leukemia rejection in 50% of the cohort, which was associated with the CD4+ and CD8+ T cell-dependent development of anti-leukemia cytotoxic T lymphocytes. Furthermore, mice surviving the BM185/CD80 challenge or preimmunized with irradiated BM185/CD80 cells developed an immune response against subsequent challenge with the parental leukemia. These studies provide evidence that immunotherapeutic approaches can be developed for the treatment of ALL.

Adjuvants, Immunologic↗

PubMed1998

Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2 degreesCFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2 degreesCFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2 degreesCFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2 degreesCFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2 degreesCFU-S from 8 recipients and expression in >50% of the vector-containing 2 degreesCFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.

Animals↗

PubMed1998

An in vitro model of human red blood cell production from hematopoietic progenitor cells.

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

Adult↗