Retroviral Vectors for High-Level Transgene Expression in T Lymphocytes

Abstract

Efficient expression of genes transferred by retroviral vectors is a prerequisite for gene therapy, especially when the biological effect depends on the amount of transgene product. High-level gene expression is desirable for several gene therapy approaches involving T lymphocytes. We evaluated standard retroviral vectors with cis-regulatory control elements of the Moloney murine leukemia virus (Mo-MLV) with or without the human T cell-specific CD2 enhancer. For comparison, vectors containing the long terminal repeat (LTR) of myeloproliferative sarcoma virus (MPSV) and an improved 5' untranslated region were used (MP71 vectors), with or without the woodchuck hepatitis virus posttranscriptional regulatory element (PRE). All vectors expressed the enhanced green fluorescent protein (GFP) to measure transgene expression. In mouse T cells MP71 vectors with and without the PRE yielded an up to 10-fold higher expression level compared with the Mo-MLV-based vectors currently used for gene transfer into T lymphocytes. A high multiplicity of infection (MOI) of standard Mo-MLV vectors could not reach expression levels obtained with a low MOI of MP71 vector. Ex vivo-transduced mouse T lymphocytes maintained the vector-dependent differences in level of transgene expression in Rag-1-deficient mice when adoptively transferred. In four human T cell lines and human primary T lymphocytes MP71 vectors yielded an up to 75-fold higher GFP expression level in comparison with the standard Mo-MLV vector. In contrast to mouse T cells, the integration of the PRE into MP71 vectors induced in human T cells a further significant increase in transgene expression level. Southern blot analysis of CEM T cells revealed that the superior performance of MP71 vectors was not due to a higher rate of viral integration. In summary, MP71 vectors are useful tools for stable, high-level gene expression in T lymphocytes, for example, in the expression of T cell receptor genes.

TCR-Redirected Human T Cells Inhibit Hepatitis C Virus Replication: Hepatotoxic Potential Is Linked to Antigen Specificity and Functional Avidity

“…All medium and supplements were purchased from Invitrogen (Carlsbad, CA). [18][19][20][21][22][23][24][25][26][27] were from ProImmune (Oxford, U.K.). For intracellular cytokine staining (ICS) multicolor FACS staining, TCR-transduced PBMCs were incubated overnight with indicated stimuli.…”

CD8α/α homodimers fail to function as co‐receptor for a CD8‐dependent TCR

“…To identify which variable gene segment is used in our CTL, the cDNA were amplified using a set of primers which bind to different Va and Vb genes in combination with a set of constant primers as previously described [24]. The isolated hiTCRa, hiTCRb and loTCRa chain genes were then cloned into the pMP71Gpre retroviral vector [25] using the Not1 and EcoR1 restriction sites and Not1 and BsrG1 restriction sites for the loTCRb chain gene.…”

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

“…MF containing SFFV LTR that had previously been reported to drive a high level of gene expression in hematopoietic cells produced the lowest level in mouse primary neural stem cells (Figure 3). 9,10 In summary, the MSCV LTR appears to work efficiently in mouse primary neural stem cells as well as NIH3T3 cells. Figure 1 Schematic representation of retroviral vectors.…”