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

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 152-155 ◽  
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

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.

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

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 152-155 ◽  
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.

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

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 100-108 ◽  
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.

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

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 100-108 ◽  
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 &lt; .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.

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

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2890-2897 ◽  
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)

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

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2890-2897 ◽  
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)

scholarly journals Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using retroviral vectors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 717-722 ◽  
Author(s):  
MA Eglitis ◽  
PW Kantoff ◽  
JD Jolly ◽  
JB Jones ◽  
WF Anderson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Progenitor Cells ◽  
Neomycin Phosphotransferase ◽  
Drug Resistant ◽  
Hematopoietic Progenitor ◽  
Exogenous Genes ◽  
Murine Leukemia

Abstract The Moloney murine leukemia retrovirus-derived vector N2 was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells. Approximately 5% of day seven CFU-GM were resistant to 2,000 micrograms/ml G418, using a supernatant infection protocol in the absence of vector-producing cells. A greater proportion of CFU-GM colonies were recovered relative to uninfected controls as the stringency of selection was diminished. Enzyme activity was detected in drug-resistant colonies, confirming that the resistant colonies obtained after infection with N2 represented cells producing neomycin phosphotransferase. Activity in the CFU-GM colonies approached 50% of that of drug-resistant vector- producing cells on a per cell basis. To test the hypothesis that more rapidly cycling bone marrow cells would be more susceptible to vector infection, we treated progenitor cells obtained from cyclic hematopoietic (CH) dogs with the N2 vector. Despite the increased numbers of hematopoietic progenitor cells obtained from CH dogs, the proportion of G418-resistant CFU-GM did not increase over that obtained with N2-infected normal marrow. These results demonstrate that retroviral vectors can be used to transfer and express exogenous genes in canine hematopoietic progenitor cells.

Sign in / Sign up

Export Citation Format

Share Document