US20260218208
2026-07-30
Chemistry; metallurgy
C12N15/635
Mutant reverse tetracycline transactivator (rtTA) proteins and engineered nucleic acids are designed to regulate gene expression efficiently. These mutants are particularly useful in applications such as cellular reprogramming, tissue regeneration, and treatment of various diseases, including neurodegenerative, cardiovascular, and genetic disorders. The engineered nucleic acids can be delivered via viral and non-viral vectors, allowing for precise control of gene expression through tetracycline-responsive elements.
Inducible gene expression systems, like the tetracycline-on (Tet-On) system, are crucial for gene therapy, offering controlled expression of therapeutic genes. Traditional Tet-On systems, however, suffer from unintended activation, or "leakiness," which limits their effectiveness. The invention addresses this by introducing mutations in the rtTA protein that enhance sensitivity to tetracycline and reduce leakiness, thus improving the safety and efficacy of gene expression regulation.
The innovation is based on four specific mutations in the rtTA3 protein, resulting in a new variant, rtTA4. These mutations occur at positions G72, G12, F67, and R171, significantly enhancing the system's performance. The modifications allow for tighter control over gene expression, reducing unwanted activity and improving the system's overall toxicity profile, making it suitable for in vivo applications.
The mutant rtTAs, along with engineered nucleic acids and recombinant viruses, are incorporated into pharmaceutical compositions and kits. These are used to promote gene expression by administering the engineered nucleic acids and tetracycline to the target cells or tissues. The approach allows for the transient expression of transgenes, crucial for therapeutic interventions in various medical conditions.
The nucleic acids encoding the mutant rtTA can be linked to either constitutive or tissue-specific promoters, allowing for targeted gene expression. Additional elements, such as tetracycline repressors and posttranscriptional regulatory elements, are included to enhance control and expression levels. Viral vectors, including AAV and lentiviral vectors, are optimized for delivery, ensuring efficient gene transfer and expression in target tissues.