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.

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.

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.

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.

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.

The β-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 β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells.

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.

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.

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).

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.