Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells

Area of Science:

• Molecular Biology
• Immunology
• Gene Therapy

Background:

• Gene and cell therapies offer potential treatments for incurable diseases.
• Efficient delivery of genetic material to resistant cell types, like resting CD4+ T cells, is crucial for gene therapy success, particularly for HIV.
• Lentiviral vectors are a common tool, but their efficacy can be limited by intracellular restriction factors.

Purpose of the Study:

• To develop and optimize a lentiviral gene therapy platform for efficient genetic modification of resting CD4+ T cells.
• To overcome intracellular restriction factors, such as Sterile Alpha Motif and HD domain 1 (SAMHD1), that limit lentiviral transduction and integration.
• To enhance gene delivery and expression in resting CD4+ T cells for potential applications in HIV gene therapy.

Main Methods:

• Tuning lentiviral vector envelopes and expression for enhanced fusogenic delivery into resting CD4+ T cells.
• Investigating the impact of Sterile Alpha Motif and HD domain 1 (SAMHD1) on lentiviral cargo delivery and integration.
• Re-engineering lentiviral vectors to deliver biologically active mRNA for cytoplasmic transgene expression, bypassing reverse transcription and integration steps.
• Utilizing unconcentrated MS2 lentivirus-like particles (MS2-LVLPs) for mRNA delivery.

Main Results:

• Lentiviral delivery into resting CD4+ T cells exceeded 80% after optimizing viral envelope and expression.
• Intracellular restriction factors, including SAMHD1, limited lentiviral gene delivery and integration, with a maximal gene expression of 35% observed.
• Re-engineering vectors for mRNA delivery resulted in gene expression in up to 65% of resting CD4+ T cells.
• MS2-LVLPs demonstrated efficient gene expression without concentration.

Conclusions:

• The optimized lentiviral platform shows significant potential for gene therapy applications targeting resting CD4+ T cells.
• Bypassing intracellular barriers through mRNA delivery is an effective strategy to enhance gene expression in resting T cells.
• This approach provides a viable platform for resting T cell gene editing, potentially advancing HIV gene therapy efforts.