scholarly journals Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1170
Author(s):  
Christina Fichter ◽  
Anupriya Aggarwal ◽  
Andrew Kam Ho Wong ◽  
Samantha McAllery ◽  
Vennila Mathivanan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Cd4 T Cells ◽  
Transgene Expression ◽  
Gene Editing ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Viral Envelope ◽  
Biologically Active ◽  
Cell Therapies

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.

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.

CD86 and CD54 Co-Expression on VSV-G Pseudotyped HIV-1 Based Vectors Improves Transduction and Activation of Human Primary CD4+ T Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1754-1754
Author(s):  
Brian Paszkiet ◽  
Andrew Worden ◽  
Yajin Ni ◽  
Saran Bao ◽  
Franck Lemiale ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Large Scale ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Lentiviral Vectors ◽  
Normal Donor ◽  
Activation Markers ◽  
Gene Therapy Trial

Abstract We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.

scholarly journals Gene Therapy Applications of Non-Human Lentiviral Vectors

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1106
Author(s):  
Altar M. Munis
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Viral Vector ◽  
Lentiviral Vector ◽  
Cell Therapies ◽  
Dividing Cells ◽  
Cell Genome ◽  
Hiv 1

Recent commercialization of lentiviral vector (LV)-based cell therapies and successful reports of clinical studies have demonstrated the untapped potential of LVs to treat diseases and benefit patients. LVs hold notable and inherent advantages over other gene transfer agents based on their ability to transduce non-dividing cells, permanently transform target cell genome, and allow stable, long-term transgene expression. LV systems based on non-human lentiviruses are attractive alternatives to conventional HIV-1-based LVs due to their lack of pathogenicity in humans. This article reviews non-human lentiviruses and highlights their unique characteristics regarding virology and molecular biology. The LV systems developed based on these lentiviruses, as well as their successes and shortcomings, are also discussed. As the field of gene therapy is advancing rapidly, the use of LVs uncovers further challenges and possibilities. Advances in virology and an improved understanding of lentiviral biology will aid in the creation of recombinant viral vector variants suitable for translational applications from a variety of lentiviruses.

scholarly journals Optimizing CD4+ T-Cells Transduction Protocol for Gene Therapy of HIV-1 Infected Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4429-4429
Author(s):  
Amani Ouedrani ◽  
Lounes Djerroudi ◽  
Isabelle Hmitou ◽  
Marina Cavazzana ◽  
Fabien Touzot
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Effector Memory ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Hiv 1

Abstract Gene therapy represents an alternative and promising strategy that could provide a path to a curative therapy for HIV-1 infection. One approach involves the introduction of protective gene into a cell, thereby conferring protection against HIV. We plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma. The patients will received autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells. CD34+ and CD4+ will be ex vivo transduced by the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. We developed a transduction process for CD4+ T-cells using the TransAct™ reagent (Miltenyi Biotec, Bergisch Gladbach , Germany) for CD4+ T-cells activation. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an equivalent efficacy of transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, CD4+ T-cells ex vivo transduced after activation with the TransAct™ reagent display very few changes in their surface marker with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 15-25% of cells suggesting a more "gentle " activation of the transduced CD4+ T-cells. Our transduction process had no significant impact in TCRβ repertoire diversity as evaluated by high-throughput sequencing and analyzis of diversity through the Gini-Simpson index or the Shannon index. Finally, transduced CD4 + T-cells retained the ability to to be primed towards the TH1, TH2 and TH17 pathways suggesting that the transduction protocol used did not alter the functional properties of the target cells. Disclosures No relevant conflicts of interest to declare.

Functional Correction of Lymphocytes and Dendritic Cells of Patients with WAS or XLT, Using Lentiviral Vectors Targeting the Expression of WAS Protein to Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3042-3042
Author(s):  
Sabine Charrier ◽  
Loic Dupre ◽  
Mike Blundell ◽  
Samantha Scaramuzza ◽  
Laurence Jeanson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Dendritic Cells ◽  
Transgene Expression ◽  
Hematopoietic Cells ◽  
Lentiviral Vectors ◽  
Similar Degree ◽  
Mrna Levels ◽  
Promoter Sequences ◽  
Was Gene

Abstract The WAS gene is mutated in Wiskott-Aldrich syndrome (WAS) and in X-linked thrombopenia (XLT). These diseases associate platelet defects with variable immune dysfunction, as a result of abnormal signalling and impaired cytoskeletal regulation in hematopoeitic cells. Conceivably, severe forms of WAS could be treated by gene therapy because retroviral or lentiviral-mediated WAS gene transfer restores protein expression and function in several WAS protein-deficient models. The purpose of the present study was to improve the safety and efficacy of such lentiviral vectors. Endogenous WAS promoter elements were used to restrict transgene expression to the target cell population and to provide the possibility of regulated expression in these cells. Sequences 0.5 or 1.6 kb upstream of transcription start, were operational in the transfer vector and restricted transgene expression to hematopoietic cells. Vectors utilizing either one of the WAS promoter sequences or the ubiquitously-active PGK-1, SFFV or EF1-a promoters, were compared. Equivalent levels of WAS protein were induced in lymphocytes and dendritic cells (DC), although slightly inferior mRNA levels were obtained in B cells using WAS promoters. At the functional level, all vectors restored a similar degree of proliferation and IL-2 production in T cells and equivalent numbers of podosome cytoskeletal structures in DC. The 0.5 kb or 1.6 kb-long WAS promoter sequences functioned similarly in WAS B lymphocytes or in a model of WASP-deficient T cells generated by RNA interference. No toxicity was induced by over-expression of these vectors in CD34+ cells. Altogether, the data show that lentiviral vectors with WAS promoters function efficiently in several lineages of patient cells and support further development of a hematopoietic-restricted approach for the gene therapy of WAS.

Transduction and Expansion of HIV+ CD4 T Cells with an HIV-1 Based Lentiviral Vector and Immobilized CD3/CD28 Antibodies Maintains the Diversity of the TCR Vβ Repertoire.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1759-1759 ◽  
Author(s):  
Franck Lemiale ◽  
Mario Pereira ◽  
Laurent Humeau ◽  
Boro Dropulic
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Negative Selection ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Gene Therapy Approach ◽  
Significant Difference

Abstract Recently, we initiated the first ex vivo HIV-based gene therapy trial in humans with HIV+ CD4+ T cells. In this protocol, a modified lentiviral vector carrying an anti-HIV payload is used to modify CD4+ T cells isolated from HIV-infected patients by apheresis and CD8 negative selection. The T cells are activated in the presence of vector and expanded using immobilized CD3/CD28 antibodies, and then infused back into the patient. T cell receptor (TCR) repertoire analysis has value for safety monitoring of adoptive T cell transfers in the detection of aberrant clonal expansions or deletions. In this study, the TCR Vβ repertoire was assessed using a flow cytometry based assay at various time points in the selection/transduction/expansion process of CD4+ T cells. PBMC isolated from whole blood of HIV+ patients were CD4-selected using a CD8 negative selection, followed by enrichment by CD3 antibody. CD4+ purified cells were transduced with the lentiviral vector, VRX496, in the presence of retronectin, and then co-cultured with CD3/CD28 coated M450 Dynabeads for ten days. The TCR Vβ repertoire was assessed in throughout the process using a FACS-based assay that employs a panel of 20 monoclonal antibodies recognizing most of the 24 Vβ families in PBMC and CD4+ T cells. Repertoires from subjects with normal polyclonal TCR profiles were conserved, as shown by the absence of any significant change in any Vβ family. Moreover, the transduction/expansion of CD4+ T cells from a patient with a previously skewed TCR profile allowed the improvement of the TCR Vβ repertoire. Finally, no significant difference was observed in the repertoire of cells transduced with VRX496 versus mock-transduced cells. These data demonstrate stability of the repertoire diversity and thus provide important support information in favor of the safety of a gene therapy approach involving lentiviral vector mediated modification and expansion of CD4+ T-cells.

scholarly journals Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

2015 ◽  
Vol 96 (8) ◽  
pp. 2381-2393 ◽  
Author(s):  
Chang Li ◽  
Xinmeng Guan ◽  
Tao Du ◽  
Wei Jin ◽  
Biao Wu ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Gene Editing ◽  
Hiv 1
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