scholarly journals Efficient Pseudotyping of Different Retroviral Vectors Using a Novel, Codon-Optimized Gene for Chimeric GALV Envelope

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1471
Author(s):  
Manuela Mirow ◽  
Lea Isabell Schwarze ◽  
Boris Fehse ◽  
Kristoffer Riecken
Keyword(s):  
T Cells ◽  
High Titer ◽  
Leukemia Virus ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Good Manufacturing Practice ◽  
Lower Amount ◽  
Efficient Production ◽  
Virus Envelope ◽  
Human T Cells

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 106 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.

Development of an Effective Safety Switch for Selective Elimination of Human T Cells In Vivo after Adoptive Transfer.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5488-5488
Author(s):  
Tom van Meerten ◽  
Henk Rozemuller ◽  
Wendy J.M. Mackus ◽  
Paul W.H.I. Parren ◽  
Jan G.J. van de Winkel ◽  
...  
Keyword(s):  
T Cells ◽  
Adoptive Transfer ◽  
Regulatory Elements ◽  
Retroviral Vectors ◽  
Chimeric Antibody ◽  
Human T Cells ◽  
Cd20 Expression ◽  
Cd20 Antibody ◽  
Cd20 Antibodies

Abstract Adoptive transfer of T cells is frequently associated with unwanted side effects. In order to tackle these effects one could introduce a safety switch into the cells that permits their selective in vivo elimination. The human CD20 gene in combination with CD20 antibodies was recently proposed as a novel safety switch. In such a system, T cells may be genetically modified with a CD20-encoding vector prior to adoptive transfer. If necessary, CD20-transgenic cells can be eliminated in vivo through administration of CD20 antibodies, such as the chimeric antibody rituximab (RTX) that is currently used to treat CD20+ lymphoma. RTX activates the complement system and recruits immune effector cells, resulting in rapid death of CD20+ cells. Recently, a novel human CD20 antibody, Humab 7D8, was shown to have superior activity over RTX. In this study a set of CD20-encoding retroviral vectors was generated, which either lacked or contained one or both of two regulatory elements: the woodchuck posttranscriptional regulatory element (WPRE) to increase CD20 expression, and the chicken hypersensitivity site 4 insulator element (INS) to achieve a position independent expression of CD20 and to increase the safety profile of the vector by preventing activation of cellular (onco)genes by the retroviral enhancer. We found that the level of CD20 expression obtained with vectors containing INS was 2-fold lower than with vectors lacking INS. Additional inclusion of WPRE restored the level to that of the vector without INS. In addition, INS greatly enhanced the homogeneity of CD20 expression in T cells. Moreover, after 3 months in culture, all cells generated with CD20-INS had retained CD20 expression, while 60% of cells transduced with the control CD20 vector had lost CD20 expression. Complement dependent cell kill (CDC) of both RTX and HuMab 7D8 was dependent on the level of CD20 expression (p<0.01). However, while very low CD20-expressing cells were completely resistant against RTX they could be effectively killed by HuMab 7D8. For maximal kill of CD20-high cells, a 100-fold lower dose of HuMab 7D8 was required, compared to RTX. In vivo efficacy was studied through bioluminescent imaging of luciferase+ CD20-transgenic T cells. After transfer of CD20+ cells in immune deficient RAG2−/−gamma c−/− mice, both CD20 antibodies were capable of eliminating >99% of CD20+ cells, prolonging survival of mice from 20 till 42 days. In conclusion, we developed a safe vector that leads to homogeneous and stable expression of CD20 on human T cells. These cells can be killed effectively in vivo with HuMab 7D8, a recently developed CD20 antibody. This system will be applicable to other approaches that require inclusion of a safety switch in ex vivo modified cells.

Targeting Human T Cells by Retroviral Vectors Displaying Antibody Domains Selected from a Phage Display Library

2000 ◽  
Vol 11 (2) ◽  
pp. 293-303 ◽  
Author(s):  
Martin Engelstadter ◽  
Maria Bobkova ◽  
Michael Baier ◽  
Jorn Stitz ◽  
Nicola Holtkamp ◽  
...  
Keyword(s):  
T Cells ◽  
Phage Display ◽  
Retroviral Vectors ◽  
Phage Display Library ◽  
Human T Cells

Use of CMV gB/pp65 eVLPs to harness the immunogenicity of foreign viral antigens to target solid tumors.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 165-165
Author(s):  
David E Anderson ◽  
Tanvir Ahmed ◽  
Catalina Soare ◽  
Anne-Catherine Fluckiger ◽  
Barthelemy Ontsouka ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Healthy Subjects ◽  
De Novo ◽  
Ex Vivo ◽  
High Titer ◽  
Leukemia Virus ◽  
Surface Expression ◽  
Immunogold Labelling ◽  
Hek 293

165 Background: Human cytomegalovirus (CMV) is a ubiquitous, generally asymptomatic virus that is present in over 90% of GBM and breast cancer tumors which have comparatively low mutational burdens and predicted neoantigens relative to melanoma or lung cancer. Memory CD4+ and CD8+T cells are most frequently directed against the gB and pp65 antigens, respectively. Thus, CMV gB and pp65 represent highly immunogenic “foreign” antigen components of a vaccine against GBM and breast cancer. Methods: Enveloped virus-like particles (eVLPs) are produced after transfection of HEK 293 cells with a plasmid encoding murine leukemia virus Gag plasmid fused in-frame with CMV pp65 antigen, which gives rise to particles. Co-transfected CMV gB plasmid enables particles budding from the cell surface to incorporate the gB protein into the lipid bilayer. Surface expression of gB and internal expression of pp65 have been confirmed by CryoEM and immunogold labelling. Production and purification of multiple batches at a GMP-compliant manufacturer has demonstrated consistent purity and yields that meet regulatory requirements. Results: eVLPs restimulate IFN-g secreting CD4+ and CD8+ T cells in ex vivo PBMCs from healthy subjects (n=8) at mean frequencies of 0.27% and 1.28%, respectively. eVLP formulation with GM-CSF augments IFN-g and CCL3 secretion in stimulated PBMCs from healthy subjects (n=5), GBM patients (n=5), and breast cancer (n=1) patients to comparable levels. This vaccine candidate also induces de novo CD4+ and CD8+responses in mice. The vaccine induces high titer antibody responses against CMV gB, and the potential for ADCC activity against gB-expressing GBM tumor cells is currently being investigated. Conclusions: CMV gB/pp65 eVLPs address several shortcomings of past theraeputic vaccines, including: 1) the poor immunogenicity of "self" tumor-associated antigens, 2) failure to induce robust CD4+ responses that support CD8+ tumor-specific responses, 3) inclusion of only one or a few epitopes that rapidly encourage tumor cell immunoselection of variants that no longer express the target(s). An IND submission to FDA for a phase I/IIa trial in GBM patients is planned for H1 2017.

Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 885-893 ◽  
Author(s):  
Valérie Dardalhon ◽  
Sara Jaleco ◽  
Cosette Rebouissou ◽  
Christophe Ferrand ◽  
Nadia Skander ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Murine Leukemia Virus ◽  
Mononuclear Cells ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Murine Leukemia

Abstract Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA+) and memory (CD45RO+) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA+ T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

Effects of CD2 locus control region sequences on gene expression by retroviral and lentiviral vectors

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3607-3617 ◽  
Author(s):  
Stefano Indraccolo ◽  
Sonia Minuzzo ◽  
Francesca Roccaforte ◽  
Rita Zamarchi ◽  
Walter Habeler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Lines ◽  
Reporter Gene ◽  
Control Region ◽  
Lentiviral Vectors ◽  
Locus Control Region ◽  
Retroviral Vectors ◽  
Egfp Expression ◽  
Immunodeficient Mice

Abstract Locus control region (LCR) sequences are involved in the establishment of open chromosomal domains. To evaluate the possibility of exploiting the human CD2 LCR to regulate gene expression by Moloney murine leukemia virus (Mo-MLV)–based retroviral vectors in T cells, it was included in vectors carrying the enhanced green fluorescence protein (EGFP) reporter gene; then transduction in vitro of lymphoid and nonlymphoid cell lines was performed. Deletion of the viral enhancer in the Mo-MLV long terminal repeat was necessary to detect LCR activity in the context of these retroviral vectors. It was found that a full-length (2.1 kb), but not a truncated (1.0 kb), CD2 LCR retained the ability to modulate reporter gene expression by Mo-MLV–derived retroviral vectors, leading to a homogeneous, unimodal pattern of EGFP expression that remained unmodified in culture over time, specifically in T-cell lines; on the other hand, viral titer was strongly reduced compared with vectors not carrying the LCR. Lentiviral vectors containing the CD2 LCR could be generated at higher titers and were used to analyze its effects on gene expression in primary T cells. Subcutaneous implantation of genetically modified cells in immunodeficient mice showed that retroviral vectors carrying the CD2 LCR conferred an advantage in terms of transgene expression in vivo, compared with the parental vector, by preventing the down-modulation of EGFP expression. These findings suggest a potential application of this LCR to increase gene expression by retroviral and lentiviral vectors in T lymphocytes.

scholarly journals Retroviral targeting of proliferating endothelial cells.

2005 ◽  
Vol 52 (3) ◽  
pp. 731-735 ◽  
Author(s):  
Alexander Gornikiewicz ◽  
Anna Zommer ◽  
Raimund Jakesz ◽  
Michael Gnant ◽  
Christine Brostjan
Keyword(s):  
Endothelial Cells ◽  
Tumor Tissue ◽  
Leukemia Virus ◽  
Chimeric Protein ◽  
Retroviral Vectors ◽  
Genetically Engineered ◽  
Moloney Murine Leukemia Virus ◽  
Human Endothelial Cells ◽  
Virus Envelope ◽  
Surface Molecules

Tumor growth requires the formation of new blood vessels by endothelial cells. Thus, surface molecules -- such as angiogenin receptors -- that are selectively expressed on growing endothelium represent an attractive target for directed delivery of compounds to tumor tissue. We attempted to obtain genetically engineered retroviral vectors targeted to the endothelium by inserting the human angiogenin sequence into Moloney murine leukemia virus envelope glycoprotein. Abundant expression of the chimeric protein could be verified. However, while being selective for proliferating human endothelial cells, the recombinant retroviral particles displayed low transduction efficiencies and thus have to be further improved.

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.

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