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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.

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.

Deletion of the nei gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived crones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49-50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants. (C) 1999 Academic Press.

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.

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.

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 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.