A peptide conjugate enables systemic injection of the morpholino inducer and more durable induction of T3H38 ribozyme-controlled AAV transgene in mice

Genetic switches that allow for precise control over transgene expression timing or levels may improve the safety and expand the use of adeno-associated viral (AAV) vector-based gene therapy technologies. We previously engineered an efficient RNA switch system that comprises a novel self-cleaving ribozyme (T3H38) and an octaguanidine dendrimer-conjugated morpholino oligonucleotide (v-M8) complementary to the ribozyme. This switch system can be used to efficiently regulate AAV-delivered transgenes with an up to 200-fold regulatory range in mice. However, this switch system has a relatively short induction half-life and only works well when v-M8 was locally but not systemically administered, representing two key limitations of the system. To address these issues, here, we tested replacing the octa-guanidine dendrimer in the v-M8 morpholino oligo with a cell-penetrating peptide (CPP). Two CPP-conjugated morpholino oligos (B-M8 and B-MSP-M8) were synthesized and compared with v-M8 for the induction of T3H38-regulated AAV-luciferase in mice. One of the CPP-conjugated oligos (B-MSP-M8) not only showed significantly improved induction half-life over that of v-M8, but also enabled efficient induction of AAV transgene expression when the oligo was systemically administered. This study improves in vivo performance and broadens the utility of the T3H38 ribozyme-based RNA switch system in gene therapy applications.