1234. HIV-1 Accessory Proteins Enhance the Levels of Gene Transfer to Murine Hematopoietic Progenitor Cells Long-Term Hemoglobin Synthesis in Mice Rescued from In Utero

Abstract Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR* virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 x 10(6) CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU- GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.

Download Full-text

Human Immunodeficiency Virus-1 Infection Interrupts Thymopoiesis and Multilineage Hematopoiesis In Vivo

Blood ◽

10.1182/blood.v91.8.2672.2672_2672_2678 ◽

1998 ◽

Vol 91 (8) ◽

pp. 2672-2678 ◽

Cited By ~ 71

Author(s):

Morgan Jenkins ◽

Mary Beth Hanley ◽

Mary Beth Moreno ◽

Eric Wieder ◽

Joseph M. McCune

Keyword(s):

Human Immunodeficiency Virus ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitors ◽

Hematopoietic Progenitor ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Human Immunodeficiency Virus 1

It is still uncertain whether multilineage hematopoietic progenitor cells are affected by human immunodeficiency virus-1 (HIV-1) infection in vivo. The SCID-hu Thy/Liv model is permissive of long-term multilineage human hematopoiesis, including T lymphopoiesis. This model was used to investigate the effects of HIV-1 infection on early hematopoietic progenitor function. We found that both lineage-restricted and multilineage hematopoietic progenitors were depleted from grafts infected with either a molecular clone or a primary isolate of HIV-1. Depletion of hematopoietic progenitors (including CD34+ cells, colony-forming units in methylcellulose, and long-term culture-initiating cells) occurred several days before the onset of thymocyte depletion, indicating that the subsequent rapid decline in thymocyte numbers was due at least in part to loss of thymocyte progenitors. HIV-1 proviral genomes were not detected at high frequency in hematopoietic cells earlier than the intrathymic T-progenitor cell stage, despite the depletion of such cells in infected grafts. Proviral genomes were also not detected in colonies derived from progenitor cells from infected grafts. These data demonstrate that HIV-1 infection interrupts both lineage-restricted and multilineage hematopoiesis in vivo and suggest that depletion of early hematopoietic progenitor cells occurs in the absence of direct viral infection.

Download Full-text

Adenoviral vector–mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Blood ◽

10.1182/blood.v96.1.100.013k41_100_108 ◽

2000 ◽

Vol 96 (1) ◽

pp. 100-108 ◽

Cited By ~ 1

Author(s):

Karen L. MacKenzie ◽

Neil R. Hackett ◽

Ronald G. Crystal ◽

Malcolm A. S. Moore

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Progenitor Cells ◽

Adenoviral Vector ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Long Term Culture ◽

Cd34 Cells ◽

Infected Cells

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34+ cells and primary colony forming unit–cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34+ cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P < .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Download Full-text

Adenoviral vector–mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Blood ◽

10.1182/blood.v96.1.100 ◽

2000 ◽

Vol 96 (1) ◽

pp. 100-108 ◽

Cited By ~ 27

Author(s):

Karen L. MacKenzie ◽

Neil R. Hackett ◽

Ronald G. Crystal ◽

Malcolm A. S. Moore

Keyword(s):

Gene Therapy ◽

Gene Transfer ◽

Progenitor Cells ◽

Adenoviral Vector ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Long Term Culture ◽

Cd34 Cells ◽

Infected Cells

Abstract Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34+ cells and primary colony forming unit–cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34+ cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P < .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Download Full-text

Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Blood ◽

10.1182/blood.v84.9.2890.2890 ◽

1994 ◽

Vol 84 (9) ◽

pp. 2890-2897 ◽

Cited By ~ 94

Author(s):

C von Kalle ◽

HP Kiem ◽

S Goehle ◽

B Darovsky ◽

S Heimfeld ◽

...

Keyword(s):

Gene Transfer ◽

Progenitor Cells ◽

Leukemia Virus ◽

Hematopoietic Progenitor Cells ◽

Transduction Efficiency ◽

Hematopoietic Progenitor ◽

Long Term Culture ◽

Marrow Cells

Abstract Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Blood ◽

10.1182/blood.v84.9.2890.bloodjournal8492890 ◽

1994 ◽

Vol 84 (9) ◽

pp. 2890-2897 ◽

Cited By ~ 1

Author(s):

C von Kalle ◽

HP Kiem ◽

S Goehle ◽

B Darovsky ◽

S Heimfeld ◽

...

Keyword(s):

Gene Transfer ◽

Progenitor Cells ◽

Leukemia Virus ◽

Hematopoietic Progenitor Cells ◽

Transduction Efficiency ◽

Hematopoietic Progenitor ◽

Long Term Culture ◽

Marrow Cells

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term marrow culture

Blood ◽

10.1182/blood.v74.1.152.bloodjournal741152 ◽

1989 ◽

Vol 74 (1) ◽

pp. 152-155 ◽

Cited By ~ 1

Author(s):

FG Schuening ◽

R Storb ◽

RB Stead ◽

S Goehle ◽

R Nash ◽

...

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Progenitor Cells ◽

Average Rate ◽

Retroviral Vectors ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Reductase Gene ◽

Successful Transfer

Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR* virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 x 10(6) CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU- GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.

Download Full-text