Arthur Nienhuis | University of Tennessee Health Science Center (original) (raw)
Papers by Arthur Nienhuis
Blood
In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing eryth... more In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and β-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders tha...
Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodeg... more Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disor- der galactosialidosis (GS). PPCA/ mice display a disease course similar to that of severe human GS, resulting in nephropa- thy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overex- pressing the corrective enzyme in either erythroid cells or monocytes/macro- phages
Journal of Clinical Investigation, 1991
We have established a recombinant HIV gene transfer system based on transient expression of the H... more We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets.
Increased fetal hemoglobin (HbF) levels diminish the clinical severity of -thalas- semia and sick... more Increased fetal hemoglobin (HbF) levels diminish the clinical severity of -thalas- semia and sickle cell anemia. A treatment strategy using autologous stem cell- targeted gene transfer of a -globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentivi- ral-based -globin vectors for expression in transduced erythroid cell lines. Com- pared with -globin, oncoretroviral vec- tors containing either a
Molecular Therapy, 2006
Lentiviral vectors efficiently transduce non-dividing hematopoietic stem cells and are being eval... more Lentiviral vectors efficiently transduce non-dividing hematopoietic stem cells and are being evaluated for gene therapy of Wiskott-Aldrich syndrome. The potentially positive or negative influence of vector elements on cellular gene expression via insertional mutagenesis has become a major consideration in therapeutic vector design to enhance safety. The chicken beta-Globin 5' DNase I Hypersensitive Site (5'cHS4) insulator possesses both chromatin barrier and enhancer-blocking functions. In the context of evaluating various vector designs in preclinical studies, we inserted the 5'cHS4 insulator into a self-inactivating lentiviral vector backbone containing Green Florescent Protein (GFP) coding sequences under the control of a truncated MSCV promoter. Human lympholeukemia (Jurkat) T cells were transduced with lentiviral vector particles, passaged for 12 days, sorted for GFP expression by FACS and plated to obtain single cell clones. Our studies focused on integration site analysis by ligation mediated PCR (LM-PCR) of Jurkat T cell clones most of which contained a single copy, a few had 2 copies and 2 clones had three copies. Two hundred and three integrations sites were defined in clones containing the control vector and 223 in clones containing the vector with insulator. Southern blot analysis confirmed vector genome integrity in>95% of the clones. All chromosomes, including the previously identified integration hot-spot region, 11q13, were targeted approximately equivalently with both the insulator containing and control vectors and the same consensus oligonucleotide palindrome was identified at the LTR-genomic junction for both vectors. However, we discovered that the brief period of culture as a population was sufficient to allow the emergence of dominant clones containing the control vector. Fourteen sets of 2 clones, 14 sets of 3 clones, 4 sets of 4 clones, 1 set of six clones, 1 set of 10 clones and 1 set of 12 clones contained the same integration site in each clone set with the control vector. The insertion site found in twelve clones was into a zinc finger gene. In contrast, the same integration sites for the insulator-containing vector were found in 18 sets of 2 clones and in 3 sets of 3 clones. We infer that certain integrations conferred a proliferative advantage by affecting gene expression through insertional mutagenesis leading to clonal dominance during the 12 day culture period prior to sorting. This effect was diminished by adding the insulator and occurred despite the fact that the insulator had no or minimal effect, as shown in a subset of clones, on the average mean fluorescence intensity (MFI=102±111 versus 106±74[5'cHS4+]) and only modestly reduced the variability of GFP expression amongst individual clones as indicated by a decrease in the co-efficient of variation from 69±19 to 52±13[5'cHS4]. Our data support the addition of an insulator to vectors used in gene therapy trials to enhance safety.
Science (New York, N.Y.), Jan 3, 1992
Experiments were performed to determine if retroviral-mediated transfer of the human multidrug re... more Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo. When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed. This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy.
Blood, Jan 15, 2000
Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient onco... more Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector-mediated gene transfer. Human hematopoietic cell lines and cord blood-derived CD34(+) and CD34(+), CD38(-) cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34(+) cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive f...
Blood, 1997
We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to a... more We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a contro...
Blood, 1994
Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) f... more Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta-galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expre...
Blood, 1994
Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recover... more Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus-mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral-blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenec...
Blood, 1995
Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the... more Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA-1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T-lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks pos...
Blood, 1984
One hundred seventy adult patients with acute lymphoblastic leukemia (ALL) or acute undifferentia... more One hundred seventy adult patients with acute lymphoblastic leukemia (ALL) or acute undifferentiated leukemia (AUL) were entered into a prospective multicenter therapy trial at 25 hospitals. The aim of the trial was to improve remission duration by using a modified form of an intensified induction regimen that was successful in childhood ALL, to define immunologic subtypes of ALL by use of cell-surface markers, and to extract other possible prognostic factors. The overall complete remission rate was 77.8%. The median overall survival time was 26 months, being 4 months for nonresponders and 32 months for responders. The median remission duration for the 126 patients with complete remission was 20 months. Prognostically favorable factors for remission duration were response to chemotherapy within 4 weeks, age less than 35 years, a low initial leukocyte count, and the immunologic subtypes c-ALL with early response to therapy and T-ALL, where 61% and 58%, respectively, are still in comp...
Proceedings of the National Academy of Sciences of the United States of America, 1988
Efficient transfer of the beta-globin gene into primitive hematopoietic progenitors was achieved ... more Efficient transfer of the beta-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human beta-globin gene and the neomycin (G418)-resistance (neoR) gene were constructed. These gave titers of 10(6) or more neoR colony-forming units/ml when packaged in psi 2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human beta-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse beta-globin genes. Infected bone marrow cells were also injected into genetically anemic W/Wv recipients without prior irradiatio...
The Journal of clinical investigation, 1985
The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients ... more The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients with sickle cell anemia or severe thalassemia. Each patient received the drug intravenously for 5 or 7 d. 5-Azacytidine caused a four- to sixfold increase in gamma-messenger RNA concentration in bone marrow cells of eight of the nine patients and decreased the methylation frequency of a specific cytosine residue in the gamma-globin gene promoter in all nine patients. Within 2 d of the start of drug treatment there was a rise in the percentage of reticulocytes containing fetal hemoglobin (HbF; F-reticulocytes) without a significant change in the total number of reticulocytes, which suggested that there was a direct action of 5-azacytidine on erythroid precursors. Late erythroid progenitors (CFU-E), present in bone marrow after 2 d of drug administration, formed colonies containing an increased amount of HbF as compared with control colonies. Moreover, the number of CFU-E derived colonies ...
tion of the transduced cells, up to 55% EGFP-expressing granulocytes were ob- tained in the perip... more tion of the transduced cells, up to 55% EGFP-expressing granulocytes were ob- tained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lympho- cytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,
Blood
In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing eryth... more In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and β-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders tha...
Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodeg... more Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disor- der galactosialidosis (GS). PPCA/ mice display a disease course similar to that of severe human GS, resulting in nephropa- thy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overex- pressing the corrective enzyme in either erythroid cells or monocytes/macro- phages
Journal of Clinical Investigation, 1991
We have established a recombinant HIV gene transfer system based on transient expression of the H... more We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets.
Increased fetal hemoglobin (HbF) levels diminish the clinical severity of -thalas- semia and sick... more Increased fetal hemoglobin (HbF) levels diminish the clinical severity of -thalas- semia and sickle cell anemia. A treatment strategy using autologous stem cell- targeted gene transfer of a -globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentivi- ral-based -globin vectors for expression in transduced erythroid cell lines. Com- pared with -globin, oncoretroviral vec- tors containing either a
Molecular Therapy, 2006
Lentiviral vectors efficiently transduce non-dividing hematopoietic stem cells and are being eval... more Lentiviral vectors efficiently transduce non-dividing hematopoietic stem cells and are being evaluated for gene therapy of Wiskott-Aldrich syndrome. The potentially positive or negative influence of vector elements on cellular gene expression via insertional mutagenesis has become a major consideration in therapeutic vector design to enhance safety. The chicken beta-Globin 5' DNase I Hypersensitive Site (5'cHS4) insulator possesses both chromatin barrier and enhancer-blocking functions. In the context of evaluating various vector designs in preclinical studies, we inserted the 5'cHS4 insulator into a self-inactivating lentiviral vector backbone containing Green Florescent Protein (GFP) coding sequences under the control of a truncated MSCV promoter. Human lympholeukemia (Jurkat) T cells were transduced with lentiviral vector particles, passaged for 12 days, sorted for GFP expression by FACS and plated to obtain single cell clones. Our studies focused on integration site analysis by ligation mediated PCR (LM-PCR) of Jurkat T cell clones most of which contained a single copy, a few had 2 copies and 2 clones had three copies. Two hundred and three integrations sites were defined in clones containing the control vector and 223 in clones containing the vector with insulator. Southern blot analysis confirmed vector genome integrity in>95% of the clones. All chromosomes, including the previously identified integration hot-spot region, 11q13, were targeted approximately equivalently with both the insulator containing and control vectors and the same consensus oligonucleotide palindrome was identified at the LTR-genomic junction for both vectors. However, we discovered that the brief period of culture as a population was sufficient to allow the emergence of dominant clones containing the control vector. Fourteen sets of 2 clones, 14 sets of 3 clones, 4 sets of 4 clones, 1 set of six clones, 1 set of 10 clones and 1 set of 12 clones contained the same integration site in each clone set with the control vector. The insertion site found in twelve clones was into a zinc finger gene. In contrast, the same integration sites for the insulator-containing vector were found in 18 sets of 2 clones and in 3 sets of 3 clones. We infer that certain integrations conferred a proliferative advantage by affecting gene expression through insertional mutagenesis leading to clonal dominance during the 12 day culture period prior to sorting. This effect was diminished by adding the insulator and occurred despite the fact that the insulator had no or minimal effect, as shown in a subset of clones, on the average mean fluorescence intensity (MFI=102±111 versus 106±74[5'cHS4+]) and only modestly reduced the variability of GFP expression amongst individual clones as indicated by a decrease in the co-efficient of variation from 69±19 to 52±13[5'cHS4]. Our data support the addition of an insulator to vectors used in gene therapy trials to enhance safety.
Science (New York, N.Y.), Jan 3, 1992
Experiments were performed to determine if retroviral-mediated transfer of the human multidrug re... more Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo. When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed. This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy.
Blood, Jan 15, 2000
Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient onco... more Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector-mediated gene transfer. Human hematopoietic cell lines and cord blood-derived CD34(+) and CD34(+), CD38(-) cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34(+) cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive f...
Blood, 1997
We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to a... more We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a contro...
Blood, 1994
Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) f... more Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta-galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expre...
Blood, 1994
Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recover... more Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus-mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral-blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenec...
Blood, 1995
Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the... more Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA-1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T-lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks pos...
Blood, 1984
One hundred seventy adult patients with acute lymphoblastic leukemia (ALL) or acute undifferentia... more One hundred seventy adult patients with acute lymphoblastic leukemia (ALL) or acute undifferentiated leukemia (AUL) were entered into a prospective multicenter therapy trial at 25 hospitals. The aim of the trial was to improve remission duration by using a modified form of an intensified induction regimen that was successful in childhood ALL, to define immunologic subtypes of ALL by use of cell-surface markers, and to extract other possible prognostic factors. The overall complete remission rate was 77.8%. The median overall survival time was 26 months, being 4 months for nonresponders and 32 months for responders. The median remission duration for the 126 patients with complete remission was 20 months. Prognostically favorable factors for remission duration were response to chemotherapy within 4 weeks, age less than 35 years, a low initial leukocyte count, and the immunologic subtypes c-ALL with early response to therapy and T-ALL, where 61% and 58%, respectively, are still in comp...
Proceedings of the National Academy of Sciences of the United States of America, 1988
Efficient transfer of the beta-globin gene into primitive hematopoietic progenitors was achieved ... more Efficient transfer of the beta-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human beta-globin gene and the neomycin (G418)-resistance (neoR) gene were constructed. These gave titers of 10(6) or more neoR colony-forming units/ml when packaged in psi 2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human beta-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse beta-globin genes. Infected bone marrow cells were also injected into genetically anemic W/Wv recipients without prior irradiatio...
The Journal of clinical investigation, 1985
The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients ... more The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients with sickle cell anemia or severe thalassemia. Each patient received the drug intravenously for 5 or 7 d. 5-Azacytidine caused a four- to sixfold increase in gamma-messenger RNA concentration in bone marrow cells of eight of the nine patients and decreased the methylation frequency of a specific cytosine residue in the gamma-globin gene promoter in all nine patients. Within 2 d of the start of drug treatment there was a rise in the percentage of reticulocytes containing fetal hemoglobin (HbF; F-reticulocytes) without a significant change in the total number of reticulocytes, which suggested that there was a direct action of 5-azacytidine on erythroid precursors. Late erythroid progenitors (CFU-E), present in bone marrow after 2 d of drug administration, formed colonies containing an increased amount of HbF as compared with control colonies. Moreover, the number of CFU-E derived colonies ...
tion of the transduced cells, up to 55% EGFP-expressing granulocytes were ob- tained in the perip... more tion of the transduced cells, up to 55% EGFP-expressing granulocytes were ob- tained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lympho- cytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,