Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4843-4852 ◽  
Author(s):  
Cecilia Frecha ◽  
Caroline Costa ◽  
Didier Nègre ◽  
Emmanuel Gauthier ◽  
Stephen J. Russell ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Measles Virus ◽  
Lentiviral Vectors ◽  
Cell Cycle Entry ◽  
Efficient Gene ◽  
Quiescent T Cells

AbstractA major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells such as primary T cells, which hampers their application for gene therapy. Here we generated high-titer LVs incorporating Edmonston measles virus (MV) glycoproteins H and F on their surface. They allowed efficient transduction through the MV receptors, SLAM and CD46, both present on blood T cells. Indeed, these H/F-displaying vectors outperformed by far VSV-G-LVs for the transduction of IL-7–prestimulated T cells. More importantly, a single exposure to these H/F-LVs allowed efficient gene transfer in quiescent T cells, which are not permissive for VSV-G-LVs that need cell-cycle entry into the G1b phase for efficient transduction. High-level transduction of resting memory (50%) and naive (11%) T cells with H/F-LVs, which seemed to occur mainly through SLAM, was not at cost of cell-cycle entry or of target T-cell activation. Finally, the naive or memory phenotypes of transduced resting T cells were maintained and no changes in cytokine profiles were detected, suggesting that T-cell populations were not skewed. Thus, H/F-LV transduction of resting T cells overcomes the limitation of current lentiviral vectors and may improve the efficacy of T cell–based gene therapy.

IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2167-2174 ◽  
Author(s):  
Els Verhoeyen ◽  
Valerie Dardalhon ◽  
Odile Ducrey-Rundquist ◽  
Didier Trono ◽  
Naomi Taylor ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Interleukin 7 ◽  
Efficient Gene ◽  
Cognate Antigen ◽  
Therapy Target

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell–based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3–displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)–displaying HIV-1–derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7–displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7–displaying vectors could constitute interesting tools for T-cell–targeted gene therapy.

scholarly journals Bax alpha perturbs T cell development and affects cell cycle entry of T cells.

1996 ◽  
Vol 15 (24) ◽  
pp. 6991-7001 ◽  
Author(s):  
H. J. Brady ◽  
G. Gil-Gómez ◽  
J. Kirberg ◽  
A. J. Berns
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Cell Cycle Entry

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

scholarly journals Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Thais B. Bertolini ◽  
Jamie L. Shirley ◽  
Irene Zolotukhin ◽  
Xin Li ◽  
Tsuneyasu Kaisho ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Immune Responses ◽  
Cell Responses ◽  
Cpg Motifs ◽  
Vector Genome

Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector’s DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.

scholarly journals Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3494-3494
Author(s):  
Jiyuan Liao ◽  
Yasushi Soda ◽  
Ai Sugawara ◽  
Yoshie Miura ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Gene Transfer ◽  
Research Funding ◽  
Measles Virus ◽  
Gene Transduction ◽  
Efficient Gene ◽  
Car T ◽  
Ipsc Generation ◽  
Equity Ownership

Abstract By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed. In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction. Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%). Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. Disclosures Liao: neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2107-2107
Author(s):  
E.L.S. Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.

scholarly journals IL-7 administration drives T cell–cycle entry and expansion in HIV-1 infection

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6304-6314 ◽  
Author(s):  
Irini Sereti ◽  
Richard M. Dunham ◽  
John Spritzler ◽  
Evgenia Aga ◽  
Michael A. Proschan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Liver Function Tests ◽  
Maximum Tolerated Dose ◽  
Controlled Study ◽  
Cell Phenotype ◽  
Gamma Chain ◽  
Cell Cycle Entry

Abstract Interleukin 7 (IL-7) is a common gamma chain receptor cytokine implicated in thymopoiesis and in peripheral expansion and survival of T lymphocytes. The safety and activity of recombinant human IL-7 (rhIL-7) administration were therefore examined in HIV-infected persons. In this prospective randomized placebo-controlled study, a single subcutaneous dose of rhIL-7 was well tolerated with biologic activity demonstrable at 3 μg/kg and a maximum tolerated dose of 30 μg/kg. Injection site reactions and transient elevations of liver function tests were the most notable side effects. Transient increases in plasma HIV-RNA levels were observed in 6 of 11 IL-7–treated patients. Recombinant hIL-7 induced CD4 and CD8 T cells to enter cell cycle; cell-cycle entry was also confirmed in antigen-specific CD8 T cells. Administration of rhIL-7 led to transient down-regulation of the IL-7 receptor alpha chain (CD127) in both CD4+ and CD8+ T cells. Single-dose rhIL-7 increased the numbers of circulating CD4+ and CD8+ T cells, predominantly of central memory phenotype. The frequency of CD4+ T cells with a regulatory T-cell phenotype (CD25high CD127low) did not change after rhIL-7 administration. Thus, rhIL-7 has a biologic and toxicity profile suggesting a potential for therapeutic trials in HIV infection and other settings of lymphopenia. This clinical trial has been registered at http://www.clinicaltrials.gov under NCT0099671.

Deoxyadenosine Inhibits T-Cell Activation in ADA-SCID Patients through a cAMP/PKA-Dependent Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1250-1250
Author(s):  
Barbara Cassani ◽  
Massimiliano Mirolo ◽  
Federica Cattaneo ◽  
Ulrike Benninghoff ◽  
Filippo Carlucci ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Adenosine Receptors ◽  
Cytokine Production ◽  
Mapk Pathway ◽  
Intracellular Camp ◽  
Clear Understanding ◽  
Hematopoietic Stem

Abstract Adenosine deaminase (ADA) is a key enzyme of the purine salvage pathway, metabolizing adenosine (Ado) and deoxyadenosine (dAdo). ADA deficiency results in increased levels of Ado and dAdo in plasma and cells, leading to severe combined immunodeficiency (SCID). However, the role of intracellular accumulation of dAdo and/or an aberrant extracellular signaling, mediated by G-coupled adenosine receptors, in the pathogenesis of the disease remains to be elucidated. Retroviral-mediated gene transfer of ADA into hematopoietic stem/progenitor cells has been recently shown to be an effective treatment for ADA-SCID children, thus providing a unique model to investigate the influence of purine metabolism in the survival and functions of T lymphocytes. Using untransformed bulk or CD4+ T-cell lines established from ADA-SCID patients before and after gene therapy, we investigated the biochemical pathways responsible for the pathogenesis of the disease and the ability of gene therapy to restore normal metabolic and immunological functions. We found that the expression of functional ADA in gene corrected T cells resulted in the restoration of SAHH activity and in the normalization of apoptosis induced by exposure to Ado/dAdo in defective cells. Interestingly, while a nucleoside transporter inhibitor could prevent Ado-induced cell toxicity, dAdo exerted its cytotoxic effect via the accumulation of intracellular cAMP, mediated by the engagement of Gs-protein coupled adenosine receptors. Functional studies revealed that both proliferative responses and Th1/Th2 cytokine production were impaired in ADA-deficient cells, but were restored in T cells generated after gene therapy. Such impairment was consequent to a defective TCR signalling, as indicated by the intrinsically reduced activation of p44/p42 MAPK pathway after anti-CD3/anti-CD28 mAb stimulation. Furthermore, a reduced transcription of cytokine genes was observed in ADA−/− CD4+ T cells, which was associated to a defective activation of early transcription factor CREB and possibly to an altered nuclear recruitment of NF-kB, as predicted by the decreased phosphorylation of IkBa. Remarkably, in ADA-deficient T cells, but not in gene corrected or healthy donor cells, exposure to non-toxic concentrations of dAdo resulted in a strong inhibition of p44/p42 MAPK activation and complete abrogation of TCR/CD28-driven proliferation and cytokine production. The effects of dAdo were reverted in the presence of a cAMP/PKA inhibitor, highlighting a key role for PKA hyperactivation and involvement of A2 receptors in causing T-cell dysfunctions. Collectively, these findings provide a more clear understanding of the physiopathology of ADA-SCID and confirm the efficacy of ADA-gene transfer in restoring normal metabolic pathways and immunological functions.

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