New technology allows gene therapy dose regulation

Scientists have engineered a special molecular mechanism that can be integrated into gene therapies to allow clinicians to control dosing.

The achievement, published in the scientific journal Nature Biotechnology, gives gene therapy pioneers the first practical way to adjust therapeutic gene expression.

The failure to achieve such a basic safety function has contributed to the development of gene therapy that otherwise seeks to tackle genes. The approach of scientists tends to overcome a significant security challenge and can lead to greater use of the method.

Michael Farzan, who is a principal investigator from the Scripps Research team, demonstrated the power of their new switching technique by incorporating it into a gene therapy that produces the hormone erythropoietin, used as a treatment for anemia.

They showed that with a special embedded molecule, they were able to suppress the expression of their gene in very low concentrations and thus to improve gene expression across a wide dynamic range by using injected control molecules such as morpholinos. For other uses, the Food and Drug Administration has proven effective.

Michael said, I think that our approach offers the only practical way at present to regulate the dose of gene therapy in an animal or a human.

Gene therapies work by inserting copies of a therapeutic gene into the cells of a patient.

For instance, the patient was born without functional copies of the needed gene. The strategy has long been seen as having enormous potential to cure diseases caused by defective genes. 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, they cannot be switched off or modulated.

As a result, only a handful of gene therapies have been FDA-approved to date.

The investigator further mentioned that the simplicity of the technology and the approval of morpholines by the FDA may allow the new transgenic switching device to be used in a wide range of gene therapies.

A transgenic transformation from the family of ribonucleic acid (RNE) molecules, known as hammerhead ribozymes, has been developed by the Farzan team, including the studying co-authors Guocai Zhong, Ph.D. and Haimin Wang, respectively post-doctoral and Farzan lab research assistance.

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 the 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.

Farzan and his colleagues are now working on adapting their ribozyme exchange strategy to be used as a fail-safe gene therapy tool, which permanently disables errant transgenes.

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