8 research outputs found
Study and development of new culture additives capable to promote the entry of lentiviral vectors for gene therapy on target cells
Les vecteurs lentiviraux (LVs) dérivés du virus de l’immunodéficience humaine de type I (VIH-1) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPHs). Cette thèse porte sur la caractérisation de deux nouveaux additifs de culture permettant de promouvoir l’entrée des LVs dans les cellules cibles : Vectofusin-1 et Tat-Beclin1. Dans un premier temps, mon travail a consisté à caractériser la structure-fonction de la nouvelle famille de Vectofusins, dérivés du peptide LAH4. Dans cette étude, Nous avons comparé l’efficacité de transduction des CSPHs en présence de différents mutants peptidiques de la Vectofusin-1. Nous avons montré que la Vectofusin-1 est le peptide le plus performant parmi tous les isomères de Vectofusin pour la transduction des CSPHs avec les pseudotypes VSV-G-LV et GALVTR-LV. Nos résultats montrent l’importance de (i) l’angle polaire à 140° formé par les résidus histidine dans la représentation de Schiffer-Edmundson, (ii) la présence de résidus lysine à l’extrémité N-terminale, et (iii) l’utilisation de leucines comme résidus hydrophobes. Ces paramètres cruciaux pour la fonction biologique des peptides de la famille Vectofusin nous permettent de mieux comprendre leur mécanisme d’action. Cette thèse porte également sur l’étude du nouvel additif de culture Tat-Beclin1, décrit dans la littérature comme étant un inducteur d’autophagie et un inhibiteur puissant de la réplication virale, en particulier du VIH-1. De façon surprenante, nous avons observé que l’utilisation de faibles doses de Tat-Beclin1 augmente fortement les niveaux de transduction de lignées cellulaires et de CSPHs en présence de divers pseudotypes LVs. Au sein de cette étude nous avons montré que ce peptide Tat-Beclin1 est compatible avec une application en thérapie génique grâce à l’absence de cytotoxicité que ce soit ex vivo ou in vivo lors de l’utilisation de modèle murin humanisé NSG pour la greffe de CSPHs. Le peptide Tat-Beclin1 agit au niveau de l’étape d’entrée, en potentialisant l’adhésion et la fusion virale, sans induire l’agrégation des particules virales. Des études du flux autophagique montrent que Tat-Beclin1 n’induit pas d’autophagie aux faibles doses utilisées. Ceci suggère que ce nouvel additif de culture agit à travers des voies moléculaires encore inconnues impliquant probablement des voies de signalisation intracellulaire indépendantes de l’autophagie. En conclusion, ce travail de thèse permet de décrire Tat-Beclin1 et Vectofusin-1 comme des nouveaux additifs de culture performants pour promouvoir la transduction de cellules cibles avec des LVs de thérapie génique.Human immunodeficiency virus type 1 (HIV-1)-derived Lentiviral vectors (LVs) are used for various gene transfer applications, notably hematopoietic gene therapy. Culture additives are necessary to promote an efficient entry of LVs into hematopoietic stem/progenitor cells (HSPCs). This work highlights two culture additives: Vectofusin-1 and Tat-Beclin1. First, we studied the structure-function of the new family of culture additives derived from LAH4: Vectofusins. We designed Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPCs transduction enhancement with LVs pseudotyped with VSV-G and GALVTR envelope glycoproteins. By comparing the efficiency of numerous Vectofusin-1 mutants, it appeared the importance of (i) lysine residues on the N-terminal extremity of Vectofusin-1, (ii) a hydrophilic angle of 140° formed by histidine residues in the Schiffer-Edmundson helical wheel representation, and (iii) hydrophobic residues consisting of leucine are all essential and help to define a minimal active sequence leading to an optimal activity of the Vectofusin-1 peptide. This work also allowed the discovery of a new culture additive called Tat-Beclin1, previously described as an autophagy-inducer peptide and a powerful viral replication inhibitor, especially on HIV-1. In this study, we showed that the use of low doses of Tat-Beclin1 peptide strongly promotes the transduction of cell lines or HSPCs with LVs pseudotyped with various envelope glycoproteins. The use of low doses of Tat-Beclin1 peptide is compatible with a gene therapy application since these experiments showed no cytotoxic effect either ex vivo or in vivo using a humanized mice model of HSPCs engraftment. The Tat-Beclin1 peptide is acting on the viral entry, specifically on the adhesion and fusion steps, but without inducing any aggregation of the viral particles. Studies of the autophagic flux showed that Tat-Beclin1 is not inducing this pathway at the low doses tested. Therefore, Tat-Beclin1 is acting on LVs through signaling pathways that are still to be defined. In conclusion, Tat-Beclin1 and Vectofusin-1 peptides are performant and safe transduction enhancers for retrovirus-based vectors dedicated to gene therapy
Concurrent measures of fusion and transduction efficiency of primary CD34+ cells with human immunodeficiency virus 1-based lentiviral vectors reveal different effects of transduction enhancers.: Concurrent study of lentiviral fusion/transduction
International audienceLentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols
Influence of Mildly Acidic pH Conditions on the Production of Lentiviral and Retroviral Vectors.
International audienceAbstract Human immunodeficiency virus type 1-derived lentiviral vectors (LVs) are becoming major tools for gene transfer approaches. Several gene therapy clinical studies involving LVs are currently ongoing. Industrial production of clinical-grade LVs is therefore an important challenge. Some improvements in LV production protocols have already been possible by acting on multiple steps of the production process like transfection, cell culture, or media optimizations. Yet, the effects of physicochemical parameters such as pH remain poorly studied. Mammalian cell cultures are generally performed at neutral pH, which may not be the optimal condition to produce high quantities of LVs with optimal infectious properties. In this study, we showed that lentiviral transient production in HEK293T cells is inversely dependent on the pH value of the harvesting medium. Infectious and physical titers of LVs pseudotyped with GALVTR or VSV-G glycoproteins are enhanced by two- to threefold at pH 6 compared with neutral conditions. pH 6-produced LVs are highly infectious on cell lines but also on relevant primary target cells like hCD34+ hematopoietic stem/progenitor cells. GALVTR-LV particles produced at pH 6 are highly stable at 37°C and resistant to multiple freeze-thaw cycles. Higher levels of expression of intracellular pr55gag polyproteins are observed within HEK293T producer cells cultured at pH 6. The positive effect of pH 6 conditions is also observed for moloney-derived retroviral vectors produced from NIH-3T3 fibroblasts, arguing that the mildly acidic pH effect is not limited to the lentivirus genus and is reproducible in various producer cell lines. This observation may help us in the design of more effective LV production protocols for clinical applications
IFITM3 Reduces Retroviral Envelope Abundance and Function and Is Counteracted by glycoGag
The viral envelope glycoprotein, known as “Env” in Retroviridae, is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3
Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state
Constraints on Human CD34+ Cell Fate due to Lentiviral Vectors Can Be Relieved by Valproic Acid
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Peptides derived from evolutionarily conserved domains in Beclin-1 and Beclin-2 enhance the entry of lentiviral vectors into human cells
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Vectofusin-1, a potent peptidic enhancer of viral gene transfer forms pH-dependent α-helical nanofibrils, concentrating viral particles
International audienceGene transfer using lentiviral vectors has therapeutic applications spanning from monogenic and infectious diseases to cancer. Such gene therapy has to be improved by enhancing the levels of viral infection of target cells and/or reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1, a recently developed cationic amphipathic peptide with a pronounced capacity to enhance such viral transduction, strongly promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium. To clarify the molecular basis of its action the peptide was investigated on a molecular and a supramolecular level by a variety of biophysical approaches. We show that in culture medium vectofusin-1 rapidly forms complexes in the 10 nm range that further assemble into annular and extended nanofibrils. These associate with viral particles allowing them to be easily pelleted for optimal virus-cell interaction. Thioflavin T fluorescence, circular dichroism and infrared spectroscopies indicate that these fibrils have a unique α-helical structure whereas most other viral transduction enhancers form β-amyloid fibrils. A vectofusin-1 derivative (LAH2-A4) is inefficient in biological assays and does not form nanofibrils, suggesting that supramolecular assembly is essential for transduction enhancement. Our observations define vectofusin-1 as a member of a new class of α-helical enhancers of lentiviral infection. Its fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to Good Manufacturing Practices and scalable gene therapy protocols