scholarly journals Systematic Comparison of the EF-1 Alpha Short (EFS) and Viral Promoters for Gene Modification of Human Primary Cells for Clinical Applications

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3497-3497 ◽  
Author(s):  
Roy L. Kao ◽  
Eric H. Gschweng ◽  
Avigael Rebecca Lerman ◽  
Sarah M. Larson ◽  
Andy Tu ◽  
...  
Keyword(s):  
T Cells ◽  
Transgene Expression ◽  
Lentiviral Vectors ◽  
Clinical Applications ◽  
Jurkat Cells ◽  
Egfp Expression ◽  
Primary Cells ◽  
Hematopoietic Stem ◽  
Copy Numbers ◽  
Human T Cells

Abstract Optimization of transgene expression is paramount for successful gene modification of primary cells for clinical applications, and careful selection of the viral vector construct is a critical part of this process. Viral promoters based on the U3 region of the Moloney murine leukemia virus (such as MNDU3 and MSCV) are currently the most commonly used for gene transfer in human primary cells. These viral promoter-containing vectors, however, can activate nearby genes, potentially causing toxicity and/or neoplastic transformation. EF1alpha (or its short, intron-less form, EFS) is a promoter that has been recently used in many clinical trials. It is a cellular-derived enhancer/promoter with decreased cross-activation of nearby promoters, therefore hypothetically decreasing the risk of genotoxicity. We have produced vector constructs carrying the internal enhancer/promoters MNDU3, MSCV, or EFS driving clinically relevant transgenes for modification of primary human T lymphocytes and hematopoietic stem cells. Lentiviral vectors containing either the MNDU3 or EFS promoters driving the EGFP reporter gene were used to transduce Jurkat cells and primary human T cells. In Jurkat cells, MNDU3-driven vectors provided 2-3 times higher vector copy integrations with a corresponding higher percentage of EGFP expression, across a wide range of multiplicity of infection (MOI). In primary T cells, however, there was no significant increase in vector copy numbers per cell, but a significant increase in transduction efficiency and geometric mean fluorescence intensity of EGFP expression in cells transduced with MNDU3-driven vectors at all MOI studied, even when corrected for vector copy number. Lentiviral vectors containing either a MNDU3 or EFS promoter driving a first-generation anti-CD19 chimeric antigen receptor (CAR) were used to transduce primary human T cells. We found that integrated vector copy numbers per cell were 0.8 with MNDU3 and 0.5 with EFS, and resultant transgene expression in the transduced populations was 45% with MNDU3 and 22% with EFS. Primary human T cells were also transduced with a lentivirus carrying MSCV or EFS driving a codon-optimized MART-1-specific T cell receptor (TCR) and then analyzed by tetramer staining. MSCV promoter-driven vectors resulted in 33.76%, 33.1%, and 29% higher transgene expression at 5 ng, 10 ng, and 25 ng p24 equivalents compared with T cells transduced with vectors driven by the EFS promoter using the same amount of p24. After correction for integrated vector copy numbers, T cells had more than 2-fold increase in transgene expression when using the MSCV promoter. CD34+ hematopoietic stem cells isolated from human cord blood were transduced using the same high-titer MSCV- or EFS-driven MART-1-specific TCR expression vectors; MSCV-driven lentiviral vectors provided an average vector copy number of 0.5 copies per cell compared to 0.7 copies per cell with the similar EFS-containing vectors. These gene-modified cells were then injected into NOD-scid-IL2rγnull mice, with peripheral blood analyzed by flow cytometry after 8 weeks. HuCD45+/huCD3+/huCD4+ and huCD45+/huCD3+/huCD8+cells had mean transgene expression of 18% and 16% in the MSCV group, compared to 0% and 0% in the EFS group. Together, these results demonstrate more efficient transgene expression is conveyed by the virally-derived MSCV and MNDU3 promoters versus the cellular EFS promoter in gene-modified primary human hematopoietic cells. Higher transgene expression relative to integrated vector copies is consistent with higher promoter function, and transgene expression may be significantly decreased when using the EFS promoter in lentiviral vectors for clinical applications. Further studies are needed to carefully evaluate genotoxic effects of the MNDU3 and MSCV promoters in comparison to the EFS promoter for safe and efficient clinical translation. Disclosures Larson: Millenium Pharmaceuticals, Inc.: Speakers Bureau.

Specific transgene expression in human and mouse CD4+cells using lentiviral vectors with regulatory sequences from theCD4 gene

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3416-3423 ◽  
Author(s):  
Gilles Marodon ◽  
Enguerran Mouly ◽  
Emma J. Blair ◽  
Charlotte Frisen ◽  
François M. Lemoine ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Cd4 T Cells ◽  
Transgene Expression ◽  
Lentiviral Vectors ◽  
Egfp Expression ◽  
Regulatory Sequences ◽  
Cd4 Cells ◽  
Specific Expression ◽  
Hematopoietic Stem

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4+ cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from theCD4 gene—a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 107 infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2–deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4+ T cells in reconstituted mice and in ex vivo–transduced human T cells. Non–T CD4+ dendritic and macrophage cells derived from human CD34+ cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4+ T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4+ cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.

Disruption of HIV-1 Co-Receptors CCR5 and CXCR4 in Primary Human T Cells and Hematopoietic Stem and Progenitor Cells Using Base Editing

2021 ◽  
Author(s):  
Friederike Knipping ◽  
Gregory A. Newby ◽  
Cindy R. Eide ◽  
Amber N. McElroy ◽  
Sarah C. Nielsen ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Base Editing ◽  
Human T Cells ◽  
Stem And Progenitor Cells ◽  
Hiv 1

scholarly journals DNA damage-independent apoptosis induced by curcumin in normal resting human T cells and leukaemic Jurkat cells

Mutagenesis ◽  
2013 ◽  
Vol 28 (4) ◽  
pp. 411-416 ◽  
Author(s):  
Z. Korwek ◽  
A. Bielak-Zmijewska ◽  
G. Mosieniak ◽  
O. Alster ◽  
M. Moreno-Villanueva ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
Jurkat Cells ◽  
Human T Cells

scholarly journals Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1427
Author(s):  
Audrey Page ◽  
Floriane Fusil ◽  
François-Loïc Cosset
Keyword(s):  
Gene Expression ◽  
Transgene Expression ◽  
Lentiviral Vector ◽  
Basic Research ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Clinical Applications ◽  
Long Terminal Repeats ◽  
Vector Systems ◽  
Gene Excision

Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.

scholarly journals MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene.

1995 ◽  
Vol 15 (6) ◽  
pp. 3110-3118 ◽  
Author(s):  
L M Ritter ◽  
M Bryans ◽  
O Abdo ◽  
V Sharma ◽  
N M Wilkie
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Binding Site ◽  
Nuclear Protein ◽  
Inflammatory Responses ◽  
Hematopoietic Cells ◽  
Jurkat Cells ◽  
Negative Regulator ◽  
U937 Cells ◽  
Hematopoietic Stem

Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.

scholarly journals Cytokine Signals Are Sufficient for HIV-1 Infection of Resting Human T Lymphocytes

1999 ◽  
Vol 189 (11) ◽  
pp. 1735-1746 ◽  
Author(s):  
Derya Unutmaz ◽  
Vineet N. KewalRamani ◽  
Shana Marmon ◽  
Dan R. Littman
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Structure Function ◽  
Human Lymphocytes ◽  
Stable Gene ◽  
Mutant Form ◽  
Primary Cells ◽  
Human T Cells ◽  
Transformed Cell Lines ◽  
Hiv 1

Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells. However, certain cell types, such as resting T lymphocytes, are resistant to infection with HIV-1. Establishing parameters for stable gene delivery into primary human lymphocytes and approaches to overcome the resistance of resting T cells to HIV infection may permit potential gene therapy applications, genetic studies of primary cells in vitro, and a better understanding of the stages of the lentiviral life cycle. Here we demonstrate that an HIV-1–derived vector can be used for stable delivery of genes into activated human T cells as well as natural killer and dendritic cells. Remarkably, a sizeable fraction of resting T cells was stably transduced with the HIV-1 vector when cultured with the cytokine interleukin (IL)-2, IL-4, IL-7, or IL-15, or, at a lower level, with IL-6, in the absence of any other stimuli. Resting T cells stimulated with these cytokines could also be infected with replication-competent HIV-1. To test the utility of this system for performing structure–function analysis in primary T cells, we introduced wild-type as well as a mutant form of murine CD28 into human T cells and showed a requirement for the CD28 cytoplasmic domain in costimulatory signaling. The ability to stably express genes of interest in primary T cells will be a valuable tool for genetic and structure–function studies that previously have been limited to transformed cell lines. In addition, the finding that cytokine signals are sufficient to permit transduction of resting T cells with HIV may be relevant for understanding mechanism of HIV-1 transmission and pathogenesis.

Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3106-3106
Author(s):  
Bruno Nervi ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
Gerhard Bauer ◽  
Jon Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Activation Markers ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Human T Cells ◽  
The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

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