A tissue-specific, activation-inducible, lentiviral vector regulated by human CD40L proximal promoter sequences

Abstract

The application of new protocols for gene therapy against monogenic diseases requires the development of safer therapeutic vectors, particularly in the case of diseases in which expression of the mutated gene is subject to fine regulation, as it is with CD40L (CD154). CD40L, the gene mutated in the X-linked hyper-immunoglobulin M syndrome (HIGM1), is tightly regulated to allow surface expression of its product only on T cells stimulated by antigen encounter. Previous studies in an HIGM1 animal model showed that transduction of progenitor cells corrected the syndrome but caused a thymic lymphoproliferative disease because of the unregulated expression of the transgene by constitutive vectors. To develop a tissue-specific, activation-inducible, lentiviral vector (LV) for gene therapy to counter HIGM1, we have constructed two self-inactivating LVs, pCD40L-eGFP and pCD40L-CD40L, regulated by a 1.3 kb fragment of the human CD40L proximal promoter. The expression of pCD40L-eGFP LV is restricted to cells in which mRNA transcripts of the endogenous CD40L gene can be detected. Moreover, the expression of the reporter gene in primary T lymphocytes depends on the activation state of the cells. Remarkably, primary HIGM1 lymphocytes transduced with pCD40L-CD40L LV expressed CD40L only after T-cell stimulation. Therefore, the CD40L-promoter-driven vectors are able to achieve a near-physiological expression pattern that follows very closely that of the endogenous CD40L gene.

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

“…(Brown et al , 1998; Sacco et al , 2000) In addition, viral vectors that can express the CD40L gene in a physiologically regulated manner have not been achieved in vivo . (Romero et al , 2011) Retroviral vectors also pose a significant risk of insertional oncogenesis, emphasizing the need for other therapeutic options.…”

Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome

“…23 Retroviral delivery of CD40L to bone marrow or thymocytes in a mouse model of X-HIGM was sufficient to restore adaptive immunity 23 ; however, the majority of mice developed thymic lymphoproliferative disorders likely due to unregulated overexpression of CD40L, demonstrating the need for future gene therapy to recapitulate tightly regulated endogenous CD40L expression. Lentiviral transfer of an expression cassette using endogenous CD40LG promoter elements to drive expression of CD40L complementary DNA (cDNA) in human T cells facilitated activation-dependent expression, 22 and transferring a trans-splicing pre-messenger RNA (mRNA) fragment of CD40LG to mouse bone marrow also corrected X-HIGM deficiencies, 24 although the latter approach is unlikely to be therapeutically effective due to the low efficiency of trans-splicing. Both gene-transfer approaches share 2 challenges: the possibility for multimerization of the transgene/spliced product with defective endogenous CD40L 25 thus not fully correcting the defect, and the risk of insertional mutagenesis and/or gene silencing.…”

“…Upon TCR activation, preformed CD40L is rapidly translocated to the cell surface, accompanied by increased transcriptional activity of CD40LG. human 21,22 or mouse hematopoietic cells. 23 Retroviral delivery of CD40L to bone marrow or thymocytes in a mouse model of X-HIGM was sufficient to restore adaptive immunity 23 ; however, the majority of mice developed thymic lymphoproliferative disorders likely due to unregulated overexpression of CD40L, demonstrating the need for future gene therapy to recapitulate tightly regulated endogenous CD40L expression.…”

Gene therapy for primary immunodeficiency

“…Previous attempts to correct this disorder in a murine model led to thymic lymphoproliferative disease as a result of unregulated transgene expression [33]. A lentiviral vector containing sequences from the CD40-ligand promoter has recently been developed which demonstrates not only tissue-specific CD40-ligand gene expression but also enhanced expression in activated T cells, thus following the physiological expression pattern [34].…”