Effects of CD2 locus control region sequences on gene expression by retroviral and lentiviral vectors

Area of Science:

• Gene Therapy
• Molecular Biology
• Retroviral Vector Engineering

Background:

• Locus control regions (LCRs) are crucial for establishing open chromosomal domains and regulating gene expression.
• Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors are used for gene delivery, but their expression can be variable.
• T cells are a key target for gene therapy, requiring stable and predictable transgene expression.

Purpose of the Study:

• To investigate the potential of the human CD2 LCR to regulate gene expression mediated by Mo-MLV-based retroviral vectors in T cells.
• To assess the impact of the CD2 LCR on reporter gene expression (EGFP) in lymphoid and nonlymphoid cell lines.
• To evaluate the efficacy of CD2 LCR-containing lentiviral vectors in primary T cells and in vivo models.

Main Methods:

• Inclusion of the full-length (2.1 kb) and truncated (1.0 kb) human CD2 LCR into Mo-MLV-based retroviral vectors carrying the enhanced green fluorescence protein (EGFP) reporter gene.
• Transduction of lymphoid and nonlymphoid cell lines, with deletion of the viral enhancer in the Mo-MLV long terminal repeat to detect LCR activity.
• Generation of lentiviral vectors containing the CD2 LCR for use in primary T cells and subsequent in vivo studies in immunodeficient mice.

Main Results:

• A full-length CD2 LCR, but not a truncated version, modulated EGFP expression, yielding homogeneous, unimodal expression in T-cell lines that remained stable over time.
• Viral titer was significantly reduced in Mo-MLV vectors containing the LCR compared to those without.
• Lentiviral vectors with the CD2 LCR achieved higher titers and demonstrated an advantage in maintaining transgene expression in vivo by preventing EGFP down-modulation in primary T cells.

Conclusions:

• The full-length human CD2 LCR can confer stable, homogeneous transgene expression from retroviral and lentiviral vectors in T lymphocytes.
• While LCR inclusion reduced Mo-MLV vector titers, lentiviral vectors showed promise for higher titers and improved in vivo gene expression stability.
• The CD2 LCR presents a potential strategy for enhancing and stabilizing gene expression in T cells for therapeutic applications.