Scientists at Scripps Research in Jupiter have developed a special molecular switch that could be embedded into gene therapies to allow doctors to control dosing.
The new transgene switching system to be used in a wide variety of envisioned gene therapies. The lab made a new transgene switch from a family of ribonucleic acid (RNA) molecules called hammerhead ribozymes. These ribozymes cut themselves in two as soon as they are copied out into RNA from the DNA that encodes them.
A therapeutic transgene containing the DNA of such a ribozyme will thus be copied out in cells into strands of RNA, called transcripts, that will tend to separate into two pieces before they can be translated into proteins. However, this self-cleaving action of the ribozyme can be blocked by RNA-like morpholinos that latch onto the ribozyme’s active site; if this happens, the transgene transcript will remain intact and will be more likely to be translated into the therapeutic protein.
The ribozyme thus effectively acts as an “off switch” for the transgene, whereas the matching morpholinos, injected into the tissue where the transgene resides, can effectively turn the transgene back “on” again–to a degree that depends on the morpholino dose.
The scientists started with a hammerhead ribozyme called N107 that had been used as an RNA switch in prior studies, but they found that the difference in production of a transgene-encoded test protein between the “off” and “on” state was too modest for this ribozyme to be useful in gene therapies. However, over months of experimentation they were able to modify the ribozyme until it had a dynamic range that was dozens of times wider.
The team then demonstrated the ribozyme-based switch in a mouse model of an EPO gene therapy, which isn’t yet FDA-approved but is considered potentially better than current methods for treating anemia associated with severe kidney disease. They injected an EPO transgene into muscle tissue in live mice, and showed that the embedded ribozyme suppressed EPO production to a very low level.
Injection of a small dose of the morpholino molecules into affected tissue strongly reversed that suppression, allowing EPO production to rise by a factor of more than 200–and stay there for more than a week, compared to a half-life of a few hours for EPO delivered by a standard injection. Those properties make the ribozyme-based switch potentially suitable for real clinical applications.
“We got what I would have said before was an impossible range of in vivo regulation from this system,” Farzan says.
Farzan and his colleagues are now working to adapt their ribozyme switch technique so that it can be used as a gene therapy failsafe mechanism, deactivating errant transgenes permanently.
Widespread use of gene therapy technologies is limited in part by the lack of small genetic switches with wide dynamic ranges that control transgene expression without the requirement of additional protein components. In this study, we engineered a class of type III hammerhead ribozymes to develop RNA switches that are highly efficient at cis-cleaving mammalian mRNAs and showed that they can be tightly regulated by a steric-blocking antisense oligonucleotide. Our variant ribozymes enabled in vivo regulation of adeno-associated virus (AAV)-delivered transgenes, allowing dose-dependent and up to 223-fold regulation of protein expression over at least 43 weeks. To test the potential of these reversible on-switches in gene therapy for anemia of chronic kidney disease6, we demonstrated regulated expression of physiological levels of erythropoietin with a well-tolerated dose of the inducer oligonucleotide. These small, modular and efficient RNA switches may improve the safety and efficacy of gene therapies and broaden their use.
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