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Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus . Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20–98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus , significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapy-treated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation.

International audience; Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

International audience; Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.

Cell therapy with T cells genetically modified with lentiviral vectors (LVs) carrying therapeutic genes is growing as a promising tool for tumor treatment. Noticeably, the clinical production of these cells relies on the availability of either LVs, whose production is cumbersome from a safety, cost and reproducibility standpoint, or transduction enhancers to get optimal transduction rates. Development of LV packaging cells represents therefore an obliged goal in LV-based gene therapy, allowing reduction of the manufacturing cost and increasing the overall quality of the vectors. Similarly, the identification of the optimal transduction conditions is crucial. We investigated the efficiency of the recently described transduction enhancer Vectofusin-1® in promoting transduction of human ex-vivo activated T cells with a RD114-TR-pseudotyped SIN-GFP LV derived from MolMed's proprietary RD3-MolPack-SIN-GFP producer cells. Vectofusin-1® is an histidine-rich cationic amphipathic peptide that has been recently shown to increase LV gene transfer in hematopoietic stem cells without inducing toxicity through improvement of adhesion and fusion of LVs with the cellular membrane. Vectofusin-1® is easily synthetized, purified, and can be added to the viral supernatant without a pre-coating step, thus requiring less overall manipulation. RD3-MolPack-SIN-GFP was generated exploiting our RD-MolPack technology which is based on the RD114-TR envelope glycoprotein that, in contrast to the most utilized VSV-G envelope, permits generation of constitutive packaging cells for the production of SIN-LV. We selected a producer clone growing continuously in disposable two-compartment bioreactor for longer than 3 months and releasing LV with a titer, on average, of 1-5 × 106 TU/ml. Here, we show that the addition of Vectofusin-1® significantly improved RD3-MolPack-SIN-GFP LV transduction rate, without spinoculation, of either CD3/CD28 or OKT-3 activated T cells (70% GFP+) compared to the absence of adjuvant (6% GFP+) at MOI 1. The analysis of the vector copy number (VCN) integration confirmed these results (VCN=4 in the presence of Vectofusin-1® vs VCN=0.09 in the absence of Vectofusin-1®, respectively). When Vectofusin-1® was compared with Retronectin® and polybrene a similar effect was registered. No major differences in the immune phenotype and expansion rate were observed between cells transduced with Vectofusin-1® and cells transduced without any adjuvant. In summary, we conclude that combination of SIN-LV produced by RD3-MolPack cells and Vectofusin-1® might be considered for clinical manufacturing of LV-transduced T cells.

Hereditary genetic disorders have been shown to be effectively treated by re-introduction of the wild-type gene product via viral modification of autologous CD34+ progenitor cells. However, current manufacturing processes are performed manually or at best by using semi-automated procedures. A standardized production of cellular therapeutic agents and their genetic modification is a major requirement to move cellular and gene therapies from their current translational setting into routine clinical use. A closed and highly automated manufacturing procedure would lead to large improvements in product performance, product safety and operator safety. We have recently developed a functionally closed and fully automated cell processing device, the CliniMACS Prodigy®, which enables complex cellular products to be manufactured.In this functionally closed system, the following steps have been automated: concentration and washing of blood products, magnetic labeling and enrichment of CD34+ cells, cultivation and pre-activation of the enriched CD34+ cells, transduction with lentiviral vectors and resuspension of the final cell product in buffer for infusion.Automated cell preparation, cell labeling and magnetic CD34+ cell separation was achieved from both mobilized and non-mobilized apheresis samples as well as bone marrow aspirates. CD34+ cells were enriched to purities exceeding 90%. Small scale experiments (10e5-10e7 purified CD34+ cells) performed with a newly developed automated transduction process, incorporating a single transduction cycle, resulted in high transduction efficiencies (up to 90%). A scale-up of the procedure was performed using larger numbers of cells (>10e7) enriched from mobilized apheresis material. CD34+ cells were transduced with a clinical-grade lentiviral vector using a two hit protocol already described in a clinical setting and used as standard for a control experiment run in parallel (Scaramuzza et al. 2013, Mol. Ther). Equivalent gene marking efficiencies were obtained. Moreover, gene modified cells maintained full engraftment potential as demonstrated by biodistribution studies in NSG mice.These preliminary data demonstrate that automated cell processing and genetic manipulation of haematopoetic progenitor cells can be efficiently performed in a closed system. Work in progress aims to further optimize this procedure and improve the efficiency of CD34+ cell transduction to levels superior to those currently achieved by manual protocols.

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols.

The efficacy of several clinical approaches of adoptive T cell therapy (ACT) can be compromised by the insurgence of severe unfavorable events as graft-versus host disease (GvHD) secondary to bone marrow transplantation unless ex-vivo manipulation endow transplantable T cells with safety system. Introduction of drug-induced suicide genes, as for example the herpes simplex virus thymidine kinase (HSV-TK) transgene in T cells during bone marrow transplantation for leukemia and lymphoma treatment, represents a valid approach to control gene-modified cell proliferation and survival overtime. Transplanted modified cells are selectively deleted in vivo by specific activation of an otherwise inactive prodrug in those cells. Despite the well-documented utility of this approach, some limitations arose in terms of poor prodrug activation kinetics, escape from drug selection and immunological rejection.Engineered variants targeting the active site of the human deoxyCytidine Kinase (dCK) suicide enzyme were described as a powerful alternative to current strategies being able to activate a large variety of nucleoside analogue-based prodrugs, having low immunogenicity and permitting in vivo imaging of genetically modified cells. Lentiviral vector (LV)-mediated delivery of the triple mutant dCK.R104M.D133A.S74E (dCK.DM.S74E) fused to the truncated low density nerve growth factor receptor (LNGFR) selection marker renders human cells highly sensitive to the prodrugs bromovinyl-deoxyuridine (brivudine or BVdU), l-deoxythymidine (LdT), and l-deoxyuridine (LdU) via induction of apoptosis. MolMed has developed the proprietary RD-MolPack technology for the stable production of both second and third generation LVs, pseudotyped with the RD114-TR envelope that, in contrast to inducible systems based on VSV-G envelope, permits the generation of a simple, constitutive, no-toxic LV packaging system. We thus exploited the RD-MolPack technology to obtain several colonies of RD-MolPack-trLNGFR/dCK.DM.S74E producer cells by stable transfection of a SIN transfer vector plasmid carrying the trLNGFR/dCK.DM.S74E transgene followed by zeocin selection. The average titer calculated on CEM A3.01 target cells of several colonies is 1.9 × 105 ± 8.15 × 104 TU/106 cells/day, in line with the previous results obtained with the previously established RD2-MolPack-Chim3 and RD3-MolPack-GFP producer cells. These preliminary data suggest that the human suicide gene trLNGFR/dCK.DM.S74E can be constitutively produced in LV stable packaging cells without toxicity and its use can be therefore considered a valid option in the scenario of prodrug convertase enzymes.

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

Our previous results have demonstrated that T-cell lines and primary T lymphocytes transduced with a Tat-dependent HIV-based lentiviral vectors expressing the mutant isoform of the vif gene, F12-vif, are protected from HIV-1 infection. F12-Vif is a 192-aa natural variant polypeptide owing 14 unique amino acid substitutions. The substitutions are randomly scattered along the entire sequence with the exception of a 5-aa cluster located at positions 127, 128, and 130|[ndash]|132. None of the 14 aa substitutions is present in the SOCS box that recruits the E3 ubiquitin ligase responsible of APOBEC3G (AP3G) degradation during HIV infection. In line with this notion, we have shown that the antiviral function of F12-Vif is not due to a dominant negative feature of the mutant in regards to the Vif-mediated degradation of AP3G rather to some other unknown means. Therefore, in the effort to elucidate the F12-Vif mechanism of action, we started to identify the protein domain of F12-Vif responsible of HIV-1 inhibition. To this end, we have constructed three chimeric genes (Chim1, Chim2 and Chim3) composed by wild-type and F12-vif regions. T cell lines and cord blood derived CD4+T lymphocytes were transduced with the lentiviral vectors expressing the chimeric genes and then challenged with both X4 and R5 HIV-1 strains. We show that 45 amino acids in the C-terminal domain of the F12-Vif mutant are sufficient to exert anti-viral effect in transduced cells. In contrast to F12-Vif, Chim3 does not allow the rescue of the replication of a vif-deficient HIV-1 in the context of either X4 or R5 tropism in non permissive cells. This specific feature renders Chim3 a truly dominant negative protein more suitable than F12-Vif for an anti-HIV gene therapy approach.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.

The F12-vif is a mutant isoform of the viral infectivity factor (vif) gene, derived from the natural variant of the F12-HIV proviral DNA, that we are currently testing as transgene in an anti-HIV gene therapy approach. Wild type Vif is normally required for high HIV-1 infectivity because it counteracts the action of the cellular inhibitor APOBEC3G (AP3G). AP3G is a cytidine deaminase that, in the absence of Vif, is incorporated into HIV-1 particles and induces strong G to A hypermuations in the nascent proviral DNA. Vif overcomes the action of AP3G by inducing, without the need of any other viral components, its proteasome-dependent degradation. We have previously established that T lymphocytes transduced with either retroviral or lentiviral vectors carrying F12-vif are protected from HIV-1 infection. Contrary to our hypothesis that F12-Vif functioned as a dominant negative mutant by rescuing the innate antiviral activity of AP3G, we have demonstrated that the dominant negative feature of F12-Vif does not affect the stability of AP3G. To elucidate the mechanism of F12-Vif action, we have looked at the first steps of HIV-1 life cycle in T-cells mock tansduced and transduced with F12-Vif. We have found that F12-Vif expressing cells integrate equal number of HIV-1 proviral DNA, but release HIV-1 particles few in number and with reduced infectivity compared to control cells. In addition, we have observed from 3 to 8 fold more F12-Vif into viral particles compared to wild type Vif. Finally, to reveal the protein domain of F12-Vif responsible of HIV-1 inhibition, we have constructed three chimeric proteins composed by wild type and F12-Vif domains. Results of this analysis will be presented.

Viral infectivity factor (Vif) is required for high HIV-1 infectivity in non-permissive cells, which include the natural targets of HIV-1, whereas it is dispensable in several cell lines called permissive. This requirement depends on the ability of Vif to counteract the action of the cellular inhibitor APOBEC3G, whose expression is restricted to non-permissive cells. APOBEC3G is a cytidine deaminase which is incorporated, in the absence of Vif, into viral particles and induces massive G-to-A hyper mutation in the nascent plus strand cDNA during reverse transcription in new target cells. Vif overcomes the action of APOBEC3G by inducing, without the need of any other viral components, its proteasome-dependent degradation. We have constructed, in the context of a potential new approach of anti-HIV-1 gene therapy, a second generation, VSV-G pseudo-typed lentiviral vector carrying the F12-vif gene, which is transcriptionally regulated by the HIV 5 LTR. F12-Vif derives from the natural variant F12-HIV provirus, which confers homologous interference to HIV-1 super-infection. We have transduced several T cell lines and primary T lymphocytes with F12-vif and WT-vif vectors and then challenged them with either CCR5 or CXCR4 HIV-1 strains. Here, we show that the level of p24 Ag released in the culture medium of infected cells is 5 logs lower in cells expressing F12-Vif, compared to the mock transduced and the control WT-Vif expressing cells. To understand whether F12-Vif inhibits HIV-1 replication in a dominant negative fashion, we have infected with Δvif HIV-1 non-permissive cells, transduced with the Vif vectors. Of note, similarly to WT-Vif, F12-Vif rescues viral replication, suggesting that in the absence of WT-Vif, F12-Vif vicariates its function. Finally, to investigate whether F12-Vif interferes with APOBEC3G function, we have monitored the expression of endogenous APOBEC3G in transduced non-permissive cells infected with either wild type or Δvif HIV-1 strains. Results of this analysis will be presented.