Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells

2015 ◽  
Vol 24 ◽  
pp. 79-85 ◽  
Author(s):  
Camille Lévy ◽  
Els Verhoeyen ◽  
François-Loïc Cosset
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Surface Engineering ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Therapy Target

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 A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
V. Kalidasan ◽  
Wai Hoe Ng ◽  
Oluwaseun Ayodeji Ishola ◽  
Nithya Ravichantar ◽  
Jun Jie Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Delivery ◽  
Delivery System ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Model System ◽  
Selection Marker ◽  
Target Cells ◽  
Primary Cells

AbstractGene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)–deficient mice and corrects their immune and metabolic defects

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 2979-2988 ◽  
Author(s):  
Alessandra Mortellaro ◽  
Raisa Jofra Hernandez ◽  
Matteo M. Guerrini ◽  
Filippo Carlucci ◽  
Antonella Tabucchi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Adenosine Deaminase ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Lentiviral Vectors ◽  
Mixed Chimerism ◽  
Pulmonary Insufficiency ◽  
Ex Vivo Gene Therapy

AbstractAdenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA–/– mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell–activating polypeptide

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2342-2350 ◽  
Author(s):  
Marielle Maurice ◽  
Els Verhoeyen ◽  
Patrick Salmon ◽  
Didier Trono ◽  
Stephen J. Russell ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Genetic Material ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Blood Lymphocytes ◽  
Hiv 1

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1–derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G0 to G1 phases of the cell cycle. Compared to unmodified HIV-1–based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

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.

Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1461-1468 ◽  
Author(s):  
Thierry VandenDriessche ◽  
Zoltán Ivics ◽  
Zsuzsanna Izsvák ◽  
Marinee K. L. Chuah
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
B Cell Lymphoma ◽  
Sleeping Beauty ◽  
Target Cells ◽  
Stable Gene ◽  
Induced Pluripotent

AbstractEffective gene therapy requires robust delivery of the desired genes into the relevant target cells, long-term gene expression, and minimal risks of secondary effects. The development of efficient and safe nonviral vectors would greatly facilitate clinical gene therapy studies. However, nonviral gene transfer approaches typically result in only limited stable gene transfer efficiencies in most primary cells. The use of nonviral gene delivery approaches in conjunction with the latest generation transposon technology based on Sleeping Beauty (SB) or piggyBac transposons may potentially overcome some of these limitations. In particular, a large-scale genetic screen in mammalian cells yielded a novel hyperactive SB transposase, resulting in robust and stable gene marking in vivo after hematopoietic reconstitution with CD34+ hematopoietic stem/progenitor cells in mouse models. Moreover, the first-in-man clinical trial has recently been approved to use redirected T cells engineered with SB for gene therapy of B-cell lymphoma. Finally, induced pluripotent stem cells could be generated after genetic reprogramming with piggyBac transposons encoding reprogramming factors. These recent developments underscore the emerging potential of transposons in gene therapy applications and induced pluripotent stem generation for regenerative medicine.

scholarly journals High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments

1998 ◽  
Vol 72 (6) ◽  
pp. 4882-4892 ◽  
Author(s):  
Karen E. Pollok ◽  
Helmut Hanenberg ◽  
Timothy W. Noblitt ◽  
Wendy L. Schroeder ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
T Lymphocytes ◽  
Adenosine Deaminase ◽  
High Efficiency ◽  
Human Gene ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Human Gene Therapy ◽  
Human T Lymphocytes

ABSTRACT Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.

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