Identification of a small molecule for enhancing lentiviral transduction of T cells.

Genetic modification of cells using viral vectors has shown huge therapeutic benefit in multiple diseases. However, inefficient transduction contributes to the high cost of these therapies. Several transduction-enhancing small molecules have previously been identified; however, some may be toxic to the cells or patient, otherwise alter cellular characteristics, or further increase manufacturing complexity. In this study, we aimed to identify molecules capable of enhancing lentiviral transduction of T cells from available small-molecule libraries. We conducted a high-throughput flow-cytometry-based screen of 27,892 compounds, which subsequently was narrowed down to six transduction-enhancing small molecules for further testing with two therapeutic lentiviral vectors used to manufacture GSK's clinical T cell therapy products. We demonstrate enhanced transduction without a negative impact on other product attributes. Furthermore, we present results of transcriptomic analysis, suggesting alteration of ribosome biogenesis, resulting in reduced interferon response, as a potential mechanism of action for the transduction-enhancing activity of the lead compound. Finally, we demonstrate the ability of the lead transduction enhancer to produce a comparable T cell product using a 3-fold reduction in vector volume in our clinical manufacturing process, resulting in a predicted 15% reduction in the overall cost of goods.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

T-cell gene therapy for perforin deficiency corrects cytotoxicity defects and prevents hemophagocytic lymphohistiocytosis manifestations.

BACKGROUND: Mutations in the perforin 1 (PRF1) gene account for up to 58% of familial hemophagocytic lymphohistiocytosis syndromes. The resulting defects in effector cell cytotoxicity lead to hypercytokinemia and hyperactivation with inflammation in various organs. OBJECTIVE: We sought to determine whether autologous gene-corrected T cells can restore cytotoxic function, reduce disease activity, and prevent hemophagocytic lymphohistiocytosis (HLH) symptoms in in vivo models. METHODS: We developed a gammaretroviral vector to transduce murine CD8 T cells in the Prf-/- mouse model. To verify functional correction of Prf-/- CD8 T cells in vivo, we used a lymphocytic choriomeningitis virus (LCMV) epitope-transfected murine lung carcinoma cell tumor model. Furthermore, we challenged gene-corrected and uncorrected mice with LCMV. One patient sample was transduced with a PRF1-encoding lentiviral vector to study restoration of cytotoxicity in human cells. RESULTS: We demonstrated efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene-corrected Prf-/- CD8 T cells into Prf-/- mice. In the tumor model infusion of Prf-/- gene-corrected CD8 T cells eliminated the tumor as efficiently as transplantation of wild-type CD8 T cells. Similarly, mice reconstituted with gene-corrected Prf-/- CD8 T cells displayed complete protection from the HLH phenotype after infection with LCMV. Patients' cells showed correction of cytotoxicity in human CD8 T cells after transduction. CONCLUSION: These data demonstrate the potential application of T-cell gene therapy in reconstituting cytotoxic function and protection against HLH in the setting of perforin deficiency.

Mutations in linker for activation of T cells (LAT) lead to a novel form of severe combined immunodeficiency.

BACKGROUND: Signaling through the T-cell receptor (TCR) is critical for T-cell development and function. Linker for activation of T cells (LAT) is a transmembrane adaptor signaling molecule that is part of the TCR complex and essential for T-cell development, as demonstrated by LAT-deficient mice, which show a complete lack of peripheral T cells. OBJECTIVE: We describe a pedigree affected by a severe combined immunodeficiency phenotype with absent T cells and normal B-cell and natural killer cell numbers. A novel homozygous frameshift mutation in the gene encoding for LAT was identified in this kindred. METHODS: Genetic, molecular, and functional analyses were used to identify and characterize the LAT defect. Clinical and immunologic analysis of patients was also performed and reported. RESULTS: Homozygosity mapping was used to identify potential defective genes. Sanger sequencing of the LAT gene showed a mutation that resulted in a premature stop codon and protein truncation leading to complete loss of function and loss of expression of LAT in the affected family members. We also demonstrate loss of LAT expression and lack of TCR signaling restoration in LAT-deficient cell lines reconstituted with a synthetic LAT gene bearing this severe combined immunodeficiency mutation. CONCLUSION: For the first time, the results of this study show that inherited LAT deficiency should be considered in patients with combined immunodeficiency with T-cell abnormalities.

High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis.

Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/- transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.

Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency.

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8(+) T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8(+) lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

Gene therapy for haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) describes a severe and often fatal immunodysregulatory disorder caused primarily by the uncontrolled activation and proliferation of T cells and macrophages. A number of genetic defects mainly involving defective granule exocytosis and effector cell cytotoxicity have been identified and well characterised at the molecular and cellular level. These conditions have limited therapeutic options and given the predominant restriction of the causative gene to the haematopoietic system, they have become attractive targets for haematopoietic cell gene therapy. Pre clinical studies in murine models of HLH due to perforin deficiency have shown correction of the disease phenotype as a result of autologous haematopoietic stem cell (HSC) gene transfer using lentiviral vectors. In a murine model of X-linked lymphoproliferative disease (XLP1), HSC gene transfer is able to correct the immunological manifestations of the disease. These encouraging murine studies have led to further work to develop clinically applicable strategies. An alternative approach is to correct defective T cells as this approach is safer than HSC gene therapy and may allow early control of the HLH through the engraftment of functional gene modified effector T cells. Both strategies are now in development and a gene therapy option for certain genetic forms of HLH may soon enter clinical trials.

Preclinical demonstration of lentiviral vector-mediated correction of immunological and metabolic abnormalities in models of adenosine deaminase deficiency.

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

The ß-globin locus control region in combination with the EF1α short promoter allows enhanced lentiviral vector-mediated erythroid gene expression with conserved multilineage activity.

Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the ß-globin gene (ß-LCR). We show that the ß-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the ß-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the ß-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Manufacturing of retroviruses.

Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.

Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer.

BACKGROUND: The low stability of gammaretroviral and lentiviral vectors affects their production, making high quality clinical preparations a difficult goal to achieve. Recently, our laboratory has shown that the main inactivation mechanism for both these vectors is the loss of their capacity to perform reverse transcription. The present study aimed to increase the stability of gammaretroviral and lentiviral at 37 degrees C and at 4 degrees C. METHODS: Inactivation studies were performed with gammaretroviral and lentiviral vectors at 37 and 4 degrees C, with and without several stabilizing compounds. The residual viral infectivity and reverse transcription capacity of these samples were tested. RESULTS: The results obtained demonstrate that it is possible to increase the stability of reverse transcription and the infectivity stability of purified gammaretroviral vectors by adding recombinant human albumin (rHSA) to the storage buffer, both at 37 degrees C and at 4 degrees C. For lentiviral vectors, it was observed that further protection was needed. This was achieved by adding lipids to the storage buffer, using a mixture of lipoproteins and rHSA. The difference of stabilization between gammaretroviral and lentiviral vectors was validated by performing stabilization tests with vectors possessing different envelope proteins and produced by different cell lines. CONCLUSIONS: The presented study reveals that it is possible to increase the half-life of purified gammaretroviral and lentiviral vectors at 37 degrees C and at 4 degrees C, but the two vectors have different stabilization requirements: for retroviral vectors, the addition of rHSA is enough and, for lentiviral vectors, it is necessary to add both lipoproteins and rHSA. The increase of the stability of the reverse transcription process was shown to have a high impact with respect to the increase of the stability of infectivity.

Scaleable purification process for gene therapy retroviral vectors.

BACKGROUND: Retroviral vectors (RVs) constitute one of the preferred gene therapy tools against inherited and acquired diseases. Development of scaleable downstream processes allowing purification under mild conditions and yielding viral preparations with high titer, potency and purity is critical for the success of clinical trials and subsequent clinical use of this technology. METHODS: A purification process for murine leukaemia virus (MLV)-derived vector supernatants was developed based on membrane separation and anion-exchange chromatography (AEXc). Initial clarification of the vector stocks was performed using 0.45 microm membranes followed by concentration with 500 kDa molecular weight cut-off (MWCO) membranes; further purification was performed by AEXc using a tentacle matrix bearing DEAE functional ligands. Finally, concentration/diafiltration was performed by 500 kDa MWCO membranes. To validate final product quality the process was scaled up 16-fold. RESULTS: Optimization of microfiltration membrane pore size and ultrafiltration transmembrane pressure allowed the recovery of nearly 100% infectious particles. Further purification of the RVs by AEXc resulted in high removal of protein contaminants while maintaining high recoveries of infectious vectors (77+/-11%). Up-scaling of the process resulted in high titer vector preparations, 3.2x10(8) infectious particles (IP)/ml (85-fold concentration), with an overall recovery reaching 26%. The process yielded vectors with transduction efficiencies higher than the starting material and more than 99% pure, relative to protein contamination. CONCLUSIONS: The combination of membrane separation and AEXc processes results in a feasible and scaleable purification strategy for MLV-derived vectors, allowing the removal of inhibitory contaminants thus yielding pure vectors with increased transduction efficiencies.

Screening of novel excipients for improving the stability of retroviral and adenoviral vectors.

In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.