Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

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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Efficient MGMTP140K-Mediated In Vivo Selection and Chemoprotection of Long-Term Repopulating Cells in Nonhuman Primates Following Reduced Intensity Conditioning.

Blood ◽

10.1182/blood.v114.22.3572.3572 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3572-3572

Author(s):

Brian C Beard ◽

Grant D Trobridge ◽

Jeannine S McCune ◽

Hans-Peter Kiem

Keyword(s):

Nonhuman Primates ◽

Conditioning Regimen ◽

Reduced Intensity Conditioning ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Reduced Intensity Conditioning Regimen ◽

Hematopoietic Toxicity ◽

Reduced Intensity

Abstract Abstract 3572 Poster Board III-509 Strategies using gene-modified hematopoietic stem cells to treat various severe hematopoietic diseases, including but not limited to hemoglobinopathies, will likely require high levels of gene marking. Here we have established efficient and stable in vivo selection in nonhuman primates using methylguanine methyltransferase (MGMTP140K). In the macaque (Macaca nemestrina) we were able to increase pre-chemotherapy lentiviral gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. Furthermore, stable increases in gene marking were also observed in red blood cells (RBCs) and platelets (PLTs) with a pre-chemotherapy gene marking level of 5.6% and 6.7%, respectively, and a post-chemotherapy gene marking level of 15.2% and 64.0%, respectively. Importantly, the chemotherapy regimen was well tolerated, and engraftment was polyclonal as determined by analyzing long-term repopulating clones by LAM-PCR. In order to minimize extra-hematopoietic toxicity we have began to test a more clinically applicable conditioning regimen in the macaque model. This reduced intensity conditioning regimen should allow treatment of patients with severe hematopoietic or infectious diseases, who may not tolerate a high dose conditioning regimen. We tested targeted busulfan for conditioning to provide sufficient myelosuppression and to facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene modified cells (∼1.7 × 107 CD34-selected cells/kg), there was moderate cytopenia with ANC <500/mL for 7 days and thrombocytopenia with a nadir of 18,000/mL. Following stable hematopoietic recovery, we observed gene marking, determined by RT-PCR, in total white blood cells as a provirus copy number of 0.04 (∼4% gene marking) that, following a single cycle of O6BG (x2) and BCNU, rose to 0.16 (∼16% gene marking). Currently, gene marking has been stable for more than 9 months following chemotherapy. The treatment was well tolerated with only transient elevated liver enzymes following O6BG/BCNU treatment and no additional extra-hematopoietic toxicity has been observed. Clonality studies before and after in vivo selection is underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach. In summary, we have attained efficient and stable in vivo selection of long-term repopulating cells in nonhuman primates, and have extended this approach to use a reduced intensity conditioning regimen that should be well tolerated in patients with many hematopoietic diseases. Disclosures: No relevant conflicts of interest to declare.

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Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.88.11.4626 ◽

1991 ◽

Vol 88 (11) ◽

pp. 4626-4630 ◽

Cited By ~ 175

Author(s):

R. Scharfmann ◽

J. H. Axelrod ◽

I. M. Verma

Keyword(s):

Gene Transfer ◽

Mouse Fibroblast ◽

In Vivo Expression

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Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer

Blood ◽

10.1182/blood.v75.3.787.bloodjournal753787 ◽

1990 ◽

Vol 75 (3) ◽

pp. 787-797 ◽

Cited By ~ 1

Author(s):

JA Nolta ◽

LS Sender ◽

JA Barranger ◽

DB Kohn

Keyword(s):

Bone Marrow ◽

Leukemia Virus ◽

Retroviral Vector ◽

Northern Blotting ◽

Gene Transduction ◽

Hematopoietic Growth Factors ◽

3T3 Fibroblasts ◽

Gc Gene

A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.

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Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells.

Journal of Virology ◽

10.1128/jvi.65.11.6365-6370.1991 ◽

1991 ◽

Vol 65 (11) ◽

pp. 6365-6370 ◽

Cited By ~ 29

Author(s):

G H Smith ◽

D Gallahan ◽

J A Zwiebel ◽

S M Freeman ◽

R H Bassin ◽

...

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Expression Of Genes ◽

In Vivo Expression

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Expression of human glucocerebrosidase in murine long-term bone marrow cultures after retroviral vector-mediated transfer

Blood ◽

10.1182/blood.v75.3.787.787 ◽

1990 ◽

Vol 75 (3) ◽

pp. 787-797 ◽

Cited By ~ 34

Author(s):

JA Nolta ◽

LS Sender ◽

JA Barranger ◽

DB Kohn

Keyword(s):

Bone Marrow ◽

Leukemia Virus ◽

Retroviral Vector ◽

Northern Blotting ◽

Gene Transduction ◽

Hematopoietic Growth Factors ◽

3T3 Fibroblasts ◽

Gc Gene

Abstract A retroviral vector (N2-SV-GC) was constructed by inserting a normal human glucocerebrosidase (GC) cDNA under control of the SV40 early region promoter into the Moloney murine leukemia virus-derived N2 vector. N2-SV-GC produced human GC in murine 3T3 fibroblasts at levels in the range of the endogenous murine GC as determined by enzymatic assay and Western blot analysis. The N2-SV-GC retroviral vector was used for studies of gene transduction of murine hematopoietic progenitor cells (HPC). Infection of bone marrow cultured for 2 to 10 days in medium containing hematopoietic growth factors was significantly more efficient than infection of freshly isolated marrow cells (24% to 32% G418-resistant CFU-GM v 15%, respectively). The marrow infected by N2-SV-GC was maintained in long-term bone marrow culture (LTBMC) and had a stable level of G418-resistant HPC over 2 months of serial assays. The human GC gene of the vector was persistently expressed in the nonadherent cell fraction of the murine LTBMC as determined by Northern blotting, Western blotting, and immunohistochemical staining using a monoclonal antibody specific for human GC. N2-SV-GC also expressed the human GC gene in day 12 CFU-S. LTBMC represents a novel system for retroviral vector-mediated gene transduction of HPC and may accurately predict the activities of vectors in vivo.

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Long-term in vivo expression of trastuzumab following intramuscular electrotransfer of the encoding DNA in mice

Journal for ImmunoTherapy of Cancer ◽

10.1186/2051-1426-3-s1-o5 ◽

2015 ◽

Vol 3 (S1) ◽

Cited By ~ 1

Author(s):

Kevin Hollevoet ◽

Nick Geukens ◽

Greetje Vande Velde ◽

Paul Declerck

Keyword(s):

In Vivo Expression

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Retroviral vector-mediated in vivo expression of low-density-lipoprotein receptors in the Watanabe heritable hyperlipidemic rabbit

Somatic Cell and Molecular Genetics ◽

10.1007/bf01232823 ◽

1991 ◽

Vol 17 (3) ◽

pp. 287-301 ◽

Cited By ~ 13

Author(s):

David A. Dichek ◽

Gary L. Bratthauer ◽

Zafarul H. Beg ◽

Kathryn D. Anderson ◽

Kurt D. Newman ◽

...

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Retroviral Vector ◽

Low Density ◽

Lipoprotein Receptors ◽

In Vivo Expression ◽

Hyperlipidemic Rabbit

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Long-Term Mixed Chimerism After Ex Vivo/In Vivo T Cell-Depleted Allogeneic Hematopoietic Cell Transplantation in Patients With Myeloid Neoplasms

Frontiers in Oncology ◽

10.3389/fonc.2021.776946 ◽

2021 ◽

Vol 11 ◽

Author(s):

Leo Ruhnke ◽

Friedrich Stölzel ◽

Uta Oelschlägel ◽

Malte von Bonin ◽

Katja Sockel ◽

...

Keyword(s):

T Cell ◽

Cell Transplantation ◽

Peripheral Blood ◽

Hematopoietic Cell Transplantation ◽

Ex Vivo ◽

Hematopoietic Cell ◽

Myeloid Neoplasms

In patients who have undergone allogeneic hematopoietic cell transplantation (HCT), myeloid mixed donor chimerism (MC) is a risk factor for disease relapse. In contrast, several studies found favorable outcome in patients with lymphoid MC. Thus far, most studies evaluating MC focused on a short-term follow-up period. Here, we report the first case series of long-term survivors with MC. We screened 1,346 patients having undergone HCT for myeloid neoplasms at our center from 1996 to 2016; 443 patients with data on total peripheral blood mononuclear cells (PBMC)/CD4+/CD34+ short tandem repeat (STR) donor chimerism (DC) and follow-up ≥24 months post-HCT were included. We identified 10 patients with long-term MC (PBMC DC <95% at ≥12 months post-HCT). Median follow-up was 11 years. All patients had received combined ex vivo/in vivo T cell-depleted (TCD) peripheral blood stem cells; none experienced ≥grade 2 acute graft-versus-host disease (GVHD). The mean total PBMC, CD4+, and CD34+ DC of all patients were 95.88%, 85.84%, and 90.15%, respectively. Reduced-intensity conditioning (RIC) was associated with a trend to lower mean total DC. Of note, two patients who experienced relapse had lower CD34+ DC but higher CD4+ DC as compared with patients in continuous remission. Bone marrow evaluation revealed increased CD4+/FOXP3+ cells in patients with MC, which might indicate expansion of regulatory T cells (Tregs). Our results support known predictive factors associated with MC such as RIC and TCD, promote the value of CD34+ MC as a potential predictor of relapse, highlight the potential association of CD4+ MC with reduced risk of GVHD, and indicate a possible role of Tregs in the maintenance of immune tolerance post-HCT.

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