Tunable Switch Via Oral Small Molecule Controls Gene Therapy Expression Levels

The Xon switch system allows scientists to fine-tune levels of gene expression. Photomicrograph courtesy of Luis Tecedor

limjr [at] chop.edu (By Jillian Rose Lim)

Today's gene therapies allow scientists to revise the script of a patient's DNA to correct defective or deficient gene expression responsible for disease development. But such breakthroughs have their challenges. Once a therapy is delivered into a patient's tissue, it's difficult to regulate levels of expression: Too much can have detrimental effects, while too little can fail to yield a result.

Now, Children's Hospital of Philadelphia researchers have engineered a delivery system — called the X^on system — to fine-tune levels of gene therapy expression, a "dimmer switch" that advances precision medicine for common, rare and complex conditions.

"We're taking the field of gene therapy to an entirely new level, where we can approach disorders where fine control of gene therapy products are required for safety, utility, and success," said senior study author Beverly Davidson, PhD, Director of the Raymond G. Perelman Center for Cellular and Molecular Therapeutics and Chief Scientific Strategy Officer at CHOP.

Dr. Davidson and her team, together with collaborators from the Novartis Institutes for BioMedical Research (NIBR), published the first research describing this innovation in Nature. They showed that by using a small molecule drug in combination with gene therapy vectors, they could control dosing of protein expressed to achieve the maximum therapeutic benefit.

"We developed a tunable switch, in order to control the level of gene therapy product that occurs in cells," said Dr. Davidson, who is also on faculty at the Perelman School of Medicine of the University of Pennsylvania. "This switch is controlled with an oral drug that can be used in children for an inherited disease. It's the hand, if you will, that controls the amount of a gene therapy product. The dose of a drug that you take controls how high that switch is turned up."

Developing a 'Dimmer Switch'

In order to develop the switch system, the researchers leveraged a small molecule discovered at NIBR that has the ability to harness the system by which scientists direct the metaphorical switch: alternative RNA splicing. Alternative splicing enables a single gene to code for multiple proteins depending on where the RNA is cut.

The Davidson Lab engineered the X^on system to control this process by regulating which protein-coding exons are spliced in or out, and repurposed the small molecule to suit their goal of creating a splicing switch that can be applied to any genetic elements they target. Once taken orally, the small molecule initiates the gene therapy by splicing the desired corrective gene into its active form. The X^on system works effectively in the brain, as well as peripheral tissues throughout the body.

"Even at lower doses, this small molecule splicing modulator could change the splicing of different things that we then engineered for our gene therapy approach," Dr. Davidson said.

Balancing Expression

X^on allows scientists to manage the robustness of the gene they seek to express by adjusting drug doses or redosing to "turn up" the switch as needed. Furthermore, oral drugs have a calculable lifespan — scientists can predict when that switch will automatically "dim," meaning the gene therapy will cease to stay active inside the cell. As such a drug leaves the cell, it automatically dims back down, almost like a timer. That timer is dependent on the half-life of the protein that you turn on. If that protein lives weeks, then the switch comes back to zero very slowly. If the protein has a half-life of a day, the switch returns to zero very quickly.

"The newly developed switch not only controls protein levels, but if needed, those proteins can be induced again and again by the simple ingestion of this oral drug," said Alex Mas Monteys, PhD, a research assistant professor in Davidson's Lab at CHOP and co-lead of the study.

In the paper, Dr. Davidson, Dr. Mas Monteys and colleagues showed proof of principle studies for Xon's use in dosing a blood product, erythropoietin (EPO), in the liver. A hormone used to treat anemia associated with chronic kidney disease, EPO plays a key role in the production of the red blood cells that carry oxygen from the lungs to the rest of our bodies.

"Right now, patients receive doses of the recombinant protein through injections," Dr. Davidson said. "Our data show that you can use an oral small molecule splicing modulator in a pulsatile manner as frequently as your body needs to be exposed to that recombinant product."

A patient would take the oral drug and get a dose of EPO, helping to boost levels of hematocrit — the volume percentage of red blood cells in blood. As those hematocrit levels wane over weeks, the patient might take another dose to increase hematocrit levels.

"You would only take as much drug as you needed to get the boost in EPO that would induce the hematocrit," Dr. Davidson said.

The researchers also showed proof of principle for the Xon delivery system's ability to provide what Dr. Davidson describes as a "burst of expression" for gene editing to happen — a noted advantage over current gene therapies. Scientists have successfully engineered viral vectors to deliver genetic material in cases where they cannot use lipid nanoparticle technology, but manipulating viruses' editing machinery faces certain challenges, such as our bodies' immune response. Viral vectors also sometimes have "off-target" effects on other genes.

"The problem with viruses is that they're there for the long haul," Dr. Davidson said. "And in something like the brain or the eye, you're going to have the editing machinery expressed for the life of that cell, which could be the life of an individual."

With the switch system, the vector delivers the adeno-associated virus expressing the editing machinery, and once the cell is exposed to a splicing modulator, the editing machinery turns on and remains for a short period of time, after which it lays silent in the cell. This results in lower immune response and fewer off-targets from editing due to prolonged exposure.

Although the current paper focuses on the use of X^on with gene therapy delivered via viruses, Dr. Davidson and Dr. Mas Monteys anticipate the technology also could be engineered for CAR-T cell therapy, where the switch system could pause therapy if needed.

Next Steps: On to Off

By enabling the careful balance of protein expression, X^on revolutionizes gene therapy as we know it.

"Where the field needs to go now is to address this fine line where too little expression is a problem and patients have disease, but too much is just as much of an issue— it creates a new problem," Dr. Davidson said. "This solves that issue of allowing expression of the correct amount."

CHOP and NIBR are collaborating to develop next-generation small molecule splicing modulators and the X^on system to achieve fine-tuned gene regulation across multiple clinical applications. Novartis is testing the Xon system in a handful of potential gene therapies, while Dr. Davidson, Dr. Mas Monteys and colleagues are optimizing the idea of a switch system even further.

"What we present in this paper is Xon, wherein we turn things on, but what we're working on now is an 'X off,'" Dr. Davidson said. "With X off, you would deliver the gene therapy and it would be on, and then you would take a drug to turn it off." Such a drug would help clinicians moderate gene expression in an even more precise manner.

The work was supported by NIBR, the Hereditary Disease Foundation and National Institutes of Health grant 5T32HG009495-04, and Children's Hospital of Philadelphia Research Institute.

Editor's Note: Dr. Davidson is an inventor of the X^on system. CHOP has licensed this technology to Novartis. CHOP, and by extension Dr. Davidson, have received compensation from Novartis in exchange for the licensing of the X^on technology. CHOP's and Dr. Davidson's participation in this research was reviewed and approved by CHOP's Conflict of Interest Committee.