Synthetic RNA Switch-Based Temporal and Dose Control of in Vivo Gene Therapies

PROJECT SUMMARY One-time in vivo gene therapies, based on adeno-associated viral (AAV) vector delivery of genes encoding therapeutic proteins, noncoding RNAs, or genome/epigenome editors, may provide long-lasting (years of, or even lifelong) treatments or cures for many rare and common diseases. However, we cannot currently tune or inactivate transgene expression to reflect disease progression or the emergence of adverse events or contraindications over time, limiting the utility of AAV gene therapies. The constrained packaging capacity of AAV vectors (~4.7 kb) has thwarted development of genetic switches that can regulate transgene expression timing or levels—the solution to these limitations. To date, just a few AAV-compatible switches function in animals. Typically, these switches are activated or repressed by ligands with undesirable side effect, including rapamycin, a potent immune suppressant; tetracycline, an antibiotic not suitable for chronic use; and branaplam, a compound that causes peripheral neurotoxicity. We previously engineered an efficient synthetic RNA ON switch based on our novel self-cleaving ribozyme T3H38, which is regulated by a complementary morpholino oligonucleotide. At 63 bp long, the tiny T3H38 ribozyme can be inserted into the 3′ UTR of a transgene. Its regulator, a 25 nt morpholino oligo is part of a class of chemically modified RNA drugs that have proven safe for chronic use in humans. The T3H38 ribozyme showed a ~200-fold regulation in AAV transgene expression in mice upon administration of the complementary morpholino oligo to the animals. Optimizing the T3H38 ribozyme switch towards a leak-free system with no detectable baseline transgene expression in the absence of the morpholino oligo could transform AAV-based gene therapy. For example, it can enable safe use of AAV to express a variety of transgenes with narrow therapeutic windows (e.g., cytokines, hormones, genome editors), where an overdose or prolonged exposure can cause severe adverse events, or to conditionally express therapeutics with major contraindications (e.g., cytokines), specific disease conditions where a drug should not be used or should be discontinued. In preliminary studies, we have developed an enhanced RNA ON switch (regulatory range: up to 35,000-fold in mice) based on the T3H38 ribozyme. Here, we will (i) optimize the enhanced RNA switch to engineer an ultra-efficient switch with negligible leakiness; (ii) use the optimized switch for precise dose control of an AAV expressing erythropoietin—a paradigmatic biologic with a short half-life, a narrow therapeutic window, and major contraindications—for the treatment of chronic Epo-deficient anemia in a mouse model; and (iii) further optimize the switch system in mouse airways for temporal control of an AAV expressing a broadly anti-coronavirus immunoadhesin—a prototype of a broadly effective prophylaxis for immunocompromised individuals against a panel of coronaviruses of pandemic potential. The completion of this project will provide a broadly useful and potentially transformative regulatable gene therapy technology and proofs of concept showcasing the utility of this technology.