Transduction of Primitive Human Marrow and Cord Blood-Derived Hematopoietic Progenitor Cells With Adeno-Associated Virus Vectors

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 1882-1894 ◽  
Author(s):  
Saswati Chatterjee ◽  
Wei Li ◽  
Christie Ann Wong ◽  
Grace Fisher-Adams ◽  
Di Lu ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Early Time ◽  
Placental Alkaline Phosphatase ◽  
Adeno Associated Virus ◽  
Long Term Culture ◽  
Vector Integration ◽  
Wide Range

We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34+ and CD34+38− clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34+ and CD34+38− cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34+, CD34+38−LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.

scholarly journals Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease.

1993 ◽  
Vol 178 (2) ◽  
pp. 529-536 ◽  
Author(s):  
T Moritz ◽  
D C Keller ◽  
D A Williams
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Severe Combined Immunodeficiency ◽  
Genetic Material ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Human Cord Blood

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.

A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 696-696
Author(s):  
Zejin Sun ◽  
Yan Li ◽  
Jingling Li ◽  
Shanbao Cai ◽  
Yi Zeng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Transduction Efficiency ◽  
Gene Transfer Efficiency

Abstract Abstract 696 Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited. In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×105 cells into NOD/SCID/gamma-chainnull yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials. Disclosures: No relevant conflicts of interest to declare.

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 The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances theirex vivoexpansion

2007 ◽  
Vol 140 (1) ◽  
pp. 90-98 ◽  
Author(s):  
Karen Li ◽  
Vincent E. C. Ooi ◽  
Carmen Ka Yee Chuen ◽  
Audrey Carmen Lam ◽  
Linda Shiou Mei Ooi ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mannose Binding Lectin ◽  
Long Term Culture ◽  
Mannose Binding ◽  
Binding Lectin

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)

A High Dose of Erythropoietin Induces the Expansion of CD34+CD133+ Cells in Human Umbilical Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4176-4176
Author(s):  
Mitsuho Noguchi ◽  
Haruko Tashiro ◽  
Ryosuke Shirasaki ◽  
Moritaka Gotoh ◽  
Kazuo Kawasugi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cord Blood ◽  
Cell Count ◽  
Progenitor Cells ◽  
Mononuclear Cell ◽  
Culture System ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Long Term Culture

Abstract Objectives: We compared the responsiveness to the high dose stimulation of human EPO between the transplant grafts in an in vitro culture system to clarify the characteristics of cord blood and to make clear the repopulation characteristics on human hematopoietic stem/progenitor cells. Methods: Cells (cord blood, bone marrow and peripheral blood stem/progenitor cells) were cultured for six days with G-CSF (100 ng/ml), EPO (10 U/ml), or without additives, and were sorted with antihuman CD133-1 monoclonal antibody. The obtained fractions were further labeled with antihuman CD34 monoclonal antibody and CD133-2 antibody as well as the lineage markers, and their expressions were analyzed. Cells were also assayed with ordinal colony-formation. For the analysis of long-term culture-initiating cells (LTC-ICs), Dexter’s long-term culture method was carried out. The expression of the specific proteins was also analyzed with RT-PCR method. Results. The ratio of the CD34+ cell count to the total cultured mononuclear cell count was not changed between CB, BM and PBSC. However, the ratio of the CD133+ cell count to the total cultured mononuclear cell count was 1.4-fold that of when CB cells were cultured with a high dose of EPO. No significance was observed for BM and PBSC. The count of CD34+CD133+ cells was 1.9-fold that of when CB was cultured with EPO. Dexter’s long-term culture system demonstrated that CB cultured with EPO contained more LTC-ICs and colony-forming cells with a high proliferation potential than that cultured without additives. Discussion: These results indicate that the CB has unique characteristics to respond to a high dose of EPO.

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