scholarly journals Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 356-364 ◽  
Author(s):  
RF Carter ◽  
AC Abrams-Ogg ◽  
JE Dick ◽  
SA Kruth ◽  
VE Valli ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Autologous Transplantation ◽  
Large Animal Model ◽  
Multiple Infections ◽  
Large Animal ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

scholarly journals Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 356-364 ◽  
Author(s):  
RF Carter ◽  
AC Abrams-Ogg ◽  
JE Dick ◽  
SA Kruth ◽  
VE Valli ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Autologous Transplantation ◽  
Large Animal Model ◽  
Multiple Infections ◽  
Large Animal ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Marrow Cells

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

Sustained Stable Engraftment of MGMT Gene-Modified Long-Term Repopulating Cells in Primary and Secondary Canine Recipients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 202-202
Author(s):  
Brian C. Beard ◽  
Reeteka Sud ◽  
Sabine Gerull ◽  
Laura J. Peterson ◽  
Tobias Neff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Lentiviral Vector ◽  
Large Animal Model ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Mgmt Gene ◽  
In Vivo Selection ◽  
Therapeutic Benefits

Abstract Chemo-protection of gene-modified cells with mutant forms of methylguanine methyltransferase (MGMT) has the potential to increase the efficacy of clinical hematopoietic stem cell gene therapy. The therapeutic benefits of MGMT gene-modified cells are due to two factors: in vivo selection following transplantation to increase the level of cells that carry MGMT and a therapeutic transgene for genetic diseases or inhibitory transgenes for acquired diseases such as AIDS andhematopoietic protection from chemotherapy agents such as BCNU and temozolomide (TMZ) during treatment of malignant diseases. In the allogeneic and autologous canine models we have demonstrated efficient engraftment, in vivo selection, and chemo-protection of MGMT gene-modified cells and increased gene-marking levels to >90%. Unequivocal proof that true ‘stem cells’ are the transduced population leading to long-term engraftment and gene-marking is elusive and the only reliable test is sustained engraftment in the large animal model (i.e. canine or non-human primates). To further test the durability and engraftment potential of MGMT gene-modified canine cells, we investigated three aspects:engraftment and repopulation in a primary recipient,durability during in vivo selection, andengraftment and repopulation in a secondary recipient. Briefly, two DLA-identical allogeneic transplants were carried out with CD34-selected cells using an 18-hour transduction protocol. Cells were transduced with a VSVG-pseudotyped lentiviral vector expressing only the mutant version of MGMT(P140K). Cells were infused into primary recipients (G340 and G403) after pre-transplant conditioning with 200 cGy total body irradiation (TBI). G340 and G403 received multiple rounds of in vivo selection comprising 4 and 3 treatments with O6-benzylguanine (O6BG)/TMZ and 1 and 2 treatments with O6BG/BCNU respectively. Gene-marking stabilized in G340 and G403 after the final round of in vivo selection at about 60% and 4% respectively for at least 3 months before we proceeded to secondary transplantation. The original donors (G346 and G404) were conditioned with 920cGy TBI before receiving whole bone marrow cells from the primary recipients G340 and G403 respectively. Both G346 and G404 recovered neutrophil and platelet counts within expected time frames compared to historical controls. Shortly after transplantation the gene-marking in G346 stabilized around 80% and this level has been maintained for more than 1 year without in vivo selection. The gene-marking in G404 stabilized around 1% shortly after transplantation and this level was maintained until recently when in vivo selection was carried out in both G404 and G403 to increase gene-marking levels. Multiple clones contributed to hematopoietic repopulation in the primary and secondary recipients and we are in the process of tracking shared clones in the animals. These data establish the durability of MGMT(P140K) gene-modified cells and also present convincing evidence that true hematopoietic stem cells were transduced during the 18-hour transduction. This study demonstrates that MGMT(P140K) gene-modified cells repopulate a primary recipient and subsequently survive multiple rounds of in vivo selection while maintaining engraftment and repopulation potential in a secondary recipient.

scholarly journals Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates

Blood ◽  
2016 ◽  
Vol 127 (20) ◽  
pp. 2416-2426 ◽  
Author(s):  
Christopher W. Peterson ◽  
Jianbin Wang ◽  
Krystin K. Norman ◽  
Zachary K. Norgaard ◽  
Olivier Humbert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Nonhuman Primates ◽  
Large Animal Model ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Specific Manner

Key Points This study is the first to show that genome-editing approaches can modify multilineage, long-term repopulating cells in a large animal model. We demonstrate that the persistence of genome-edited hematopoietic stem cells can be tracked in vivo in a mutation-specific manner.

scholarly journals Correction of canine X-linked severe combined immunodeficiency by in vivo retroviral gene therapy

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3091-3097 ◽  
Author(s):  
Suk See Ting–De Ravin ◽  
Douglas R. Kennedy ◽  
Nora Naumann ◽  
Jeffrey S. Kennedy ◽  
Uimook Choi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Large Animal Model ◽  
Large Animal ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem

AbstractX-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor γ chain (γc). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term–surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals Short- and Long-Term Multilineage Repopulating Hematopoietic Stem Cells in Late Fetal and Newborn Mice: Models for Human Umbilical Cord Blood

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 174-181 ◽  
Author(s):  
David E. Harrison ◽  
Clinton M. Astle
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Functional Abilities ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Short And Long Term ◽  
Marrow Cells

Abstract Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult “competitor” marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.

scholarly journals Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Blood ◽  
2009 ◽  
Vol 113 (16) ◽  
pp. 3682-3689 ◽  
Author(s):  
Paris Margaritis ◽  
Elise Roy ◽  
Majed N. Aljamali ◽  
Harre D. Downey ◽  
Urs Giger ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Hemophilia A ◽  
Viral Vector ◽  
Canine Model ◽  
Factor Vii ◽  
Large Animal Model ◽  
Large Animal ◽  
Spontaneous Bleeding ◽  
Human Factor Viii

Abstract Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor–dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.

Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1137-1137
Author(s):  
Tong Wu ◽  
Hyeoung Joon Kim ◽  
Stephanie E. Sellers ◽  
Kristin E. Meade ◽  
Brian A. Agricola ◽  
...  
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Nonhuman Primates ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Colony Pcr ◽  
High Level

Abstract Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.

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