Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for...

Area of Science:

• Gene Therapy
• Molecular Biology
• Virology

Background:

• Efficient therapeutic protein expression is crucial for gene therapy success.
• Previous lentiviral gene transfer of alpha repeat protein (αRep4E3) showed anti-HIV-1 activity but lacked controllable integration.
• Uncontrollable integration sites pose challenges for clinical translation of gene therapies.

Purpose of the Study:

• To utilize CRISPR/Cas9 gene editing for precise integration of αRep4E3 into the AAVS1 safe harbor locus.
• To evaluate the expression efficiency of αRep4E3 under different promoters (CMV, EF1α, UbC) with and without codon optimization.
• To demonstrate the anti-HIV-1 efficacy of engineered cells in a proof-of-concept study.

Main Methods:

• CRISPR/Cas9 gene editing was employed to knock-in αRep4E3 genes into the AAVS1 locus.
• Expression levels of αRep4E3 were compared across CMV, EF1α, and UbC promoters in HEK293T cells.
• Codon optimization of αRep4E3mCherry was assessed for enhanced protein expression.
• Proof-of-concept study involved knocking in αRep4E3mCherry into Jurkat cells and challenging with HIV-1NL4-3.

Main Results:

• The EF1α promoter with codon-optimized αRep4E3mCherry demonstrated superior protein expression compared to other promoter/codon combinations.
• Engineered Jurkat cells expressing αRep4E3mCherry showed significant anti-HIV-1 activity.
• Reduced capsid (p24) protein levels and viral genome copies were observed in αRep4E3mCherry-expressing Jurkat cells post-HIV-1 challenge.

Conclusions:

• Precise gene editing using CRISPR/Cas9 enables controlled therapeutic protein expression at the AAVS1 locus.
• The αRep4E3 scaffold protein effectively interferes with viral RNA packaging, demonstrating potent anti-HIV-1 activity.
• αRep4E3 represents a promising antiviral molecule for developing a functional cure for HIV-1.