New technology allows control of gene therapy doses

Technique appears to solve a major safety issue and could lead to much more use of gene therapies

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New Delhi: Scientists at Scripps Research have developed special switch-like molecules that can be embedded in gene therapies to allow doctors to control the dosing of those therapies over wide ranges in individual patients.

The feat, reported in Nature Biotechnology, offers gene therapy designers what may be the first viable technique for adjusting the activity levels of therapeutic genes in patients. The lack of such a basic safety feature has helped limit the development of gene therapies, which otherwise have held great promise.

The scientists demonstrated the power of their new switching technique by incorporating it into a gene therapy that produces the hormone erythropoietin (EPO), used as a treatment for anemia. They showed that they could suppress the expression of the EPO gene to very low levels with a special embedded molecule, and could then increase the gene’s expression, over a wide dynamic range, using injected control molecules called morpholinos that the U.S. Food and Drug Administration (FDA) has found to be safe for other applications.

“I think that our approach offers the only practical way at present to regulate the dose of a gene therapy in an animal or a human,” says principal investigator Michael Farzan, PhD, Professor and Co-Chair of the Department of Immunology and Microbiology at Scripps Research in Florida.

Gene therapies work by inserting copies of a therapeutic gene into the cells of a patient, for example because the patient was born without functional copies of the gene. With the added gene, called a transgene, the cells can now produce copies of the therapeutic protein that the transgene encodes.

The strategy has long been seen as having enormous potential. It could cure genetic diseases caused by defective genes, for example. It also could enable the steady, long-term delivery to patients of therapeutic molecules that are impractical to deliver in pills or injections because they don’t survive for long in the body. However, gene therapies have been viewed as inherently risky because once they are delivered to a patient’s cells, the added transgene copies can’t be switched off or modulated, for example if their activity becomes harmfully excessive. So far only a handful of gene therapies have been FDA-approved.

Farzan’s team, including study co-first authors Guocai Zhong, PhD and Haimin Wang, respectively a postdoctoral researcher and a research assistant in the Farzan lab, crafted a transgene switch from a family of ribonucleic acid (RNA) molecules called hammerhead ribozymes. These ribozymes have the remarkable property that they 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.

The small size of the ribozyme, the simplicity of the technique, and the fact that morpholinos like the ones used in the study are already FDA-approved, could allow the new transgene switching system to be used in a wide variety of envisioned gene therapies, Farzan adds.

The scientist and his colleagues are now working to adapt their ribozyme switch technique so that it can be used as a gene therapy fail safe mechanism, deactivating errant transgenes permanently.


The research paper, “A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo” was written by Guocai Zhong, Haimin Wang, Wenhui He, Yujun Li, Huihui Mou, Zachary Tickner, Mai Tran, Tianling Ou, Yiming Yin, Huitian Diao, and Michael Farzan, all of Scripps Research at the time of the study.
Funding for the research was provided by the National Institutes of Health (R37 AI091476, AI1129868, DP1 DA043912 199).