The ability to control the expression of therapeutic genes within a “therapeutic window” is crucial to ensure the development of safe and effective gene therapies, but to date, there has been no control strategy that itself doesn’t have potential safety issues, limiting applications in the clinic. Researchers at Baylor College of Medicine have now described an RNA-based switch, the pA regulator, that they have shown can control mammalian gene expression without generating the unwanted immune responses that are associated with other gene control approaches that use foreign regulatory transactivator proteins. Reporting in Nature Biotechnology on tests with the system in human cells, and in live mice, the researchers say the new technology offers a promising solution that could be applied for clinical gene therapy applications.

Laising Yen, PhD, associate professor of pathology and immunology and of molecular and cellular biology at Baylor, and colleagues, described their work in a paper titled “Control of mammalian gene expression by modulation of polyA signal cleavage at 5′ UTR.”

Just like a doctor adjusts the dose of a medication to the patient’s needs, the expression of therapeutic genes, those modified in a person to treat or cure a disease via gene therapy, also needs to be maintained within a therapeutic window. Staying within the therapeutic window is important as too much of the protein could be toxic, and too little could result in a small or no therapeutic effect.

Although the principle of therapeutic window has been known for a long time, developing a safe method has proven problematic. As the authors noted in their paper, “Current gene transfer methods used in gene and cell therapy, such as adeno-associated virus (AAV), have intrinsic difficulties in executing the ‘conditional’ and ‘reversible’ gene control, which often require further calibrations after delivery.”

And while the commonly used tet-on system is a powerful gene regulation tool in biological studies, its use of foreign regulatory ‘transactivator’ protein can trigger immune responses in nonhuman primates, and so there are no approved clinical applications based on this system. “In fact, immune response against foreign protein epitopes remains a key hurdle to successful gene therapy as it eliminates gene expression and damages transduced host cells,” the team further wrote. Corresponding author, Yen, added, “Although there are several gene regulation systems used in mammalian cells, none has been approved by the U.S. Food and Drug Administration for clinical applications, mainly because those systems use a regulatory protein that is foreign to the human body, which triggers an immune response against it. This means that the cells that are expressing the therapeutic protein would be attacked, eliminated or neutralized by the patient’s immune system, making the therapy ineffective.”

Having worked on this technology for more than a decade Yen and colleagues have now reported on a new solution. The pA regulator mammalian gene regulation system, they claim, overcomes key challenges associated with existing gene expression control systems without using foreign, immunogenic regulatory proteins. “… the system allows any intact protein to be expressed as a transgene product without modifying its coding sequence, circumventing the transgene-specific immune responses observed in other systems,” the investigators further wrote.

“The solution we found does not involve a foreign regulatory protein that will evoke an immune response in patients,” Yen further commented. “Instead, we use small molecules to interact with RNA, which typically do not trigger an immune response. Other groups also have made attempts to resolve this critical issue, but the drug concentrations they used are beyond what the FDA has approved for patients. We were able to engineer our system in such a way that it works at the FDA-approved dosage.”

Yen and his colleagues developed a system that turns genes on to different levels on cue using small molecules at FDA-approved doses. The switch is placed in the RNA, the copy of genetic material that is translated into a protein. This approach allows the researchers to control the protein’s production a step back by controlling its RNA.

The RNA of interest is first engineered to contain an extra polyA signal, akin to a “stop sign” that genes naturally use to mark the end of a gene. When the machinery of the cell detects a polyA signal in the RNA, it automatically makes a cut and defines the cut point as the end of the RNA. “In our system, we use the added polyA signal, not at the end, but at the beginning of the RNA, so the cut destroys the RNA and therefore the default is no protein production. It is turned off until we turn it on with the small molecule,” Yen said.

To turn on the gene at the desired level, the team engineered a switch on the RNA. They modified a section of the RNA near the polyA signal such that it can now bind to a small molecule, FDA-approved tetracycline in this case.

“When tetracycline binds to that section that functions as a sensor on the RNA, it masks off the polyA signal, and the RNA will now be translated into protein,” Yen said. Moreover, the authors stated, “Unlike the tet-on system that requires one promoter to generate the regulatory transactivator protein and a second promoter to express the transgene, the pA regulator is a compact single promoter system that can be implemented in a single plasmid or viral vector.”

Importantly, and in contrast to other systems that might generate additional amino acid sequences to the transgene coding sequence, which could promote transgene-specific immune responses, “… the pA regulator introduces no change to the coding sequence of target genes,” the team added. Moreover, they noted, “In contrast to the ‘irreversible’ and ‘on or off two-stage only’ gene control generated by CRISPR/Cas9 DNA editing, the pA regulator controls gene expression in a ‘dose-dependent’ and ‘reversible’ manner, both features crucial for effective therapeutics as well as the biological study of gene function.”

The team tested their pA regulator approach in different contexts, both to control expression of transgenes in human cells and in live mice, and also to  control endogenous gene expression in the human genome, “all at the FDA-approved dose,” they noted. “The pA regulator effectively controls the luciferase transgene in live mice and the endogenous CD133 gene in human cells, in a dose-dependent and reversible manner with long-term stability.”

The authors outlined how the technology would fit as part of a clinical gene therapy that provides a gene to compensate for a malfunctioning gene underlying a patient’s disorder. In this situation the gene the patient received would have the switch incorporated, allowing the physician to control production of the therapeutic protein. So, if the patient only required a small amount of the therapeutic protein, they would take only a small dose of tetracycline, so turning on the therapeutic gene by just a small amount. If the patient needed more therapeutic protein, then they would take more tetracycline to boost protein production. To stop production of the therapeutic protein the patient would just stop taking tetracycline, as, in the absence of tetracycline, the switch will be back to its default off position.

Some diseases may benefit from the presence of constant low levels of therapeutic protein, and in that case, there is flexibility in the technology to pre-adjust the default level of gene expression to specified levels of protein production, while retaining the option of dialing up  expression further by administering tetracycline.

“This strategy allows us to be more precise in the control of gene expression of a therapeutic protein. It enables us to adjust its production according to disease’s stages or tune to the patients’ specific needs, all using the FDA-approved tetracycline dose,” Yen said. “Our approach is not disease-specific, it can theoretically be used for regulating the expression of any protein, and potentially has many therapeutic applications. In addition, this system is more compact and easier to implement than the existing technologies. Therefore, it also can be very useful in the lab to turn a gene of interest on or off to study its function.”

The authors further concluded, “We envision that it could open new windows of opportunity for biological studies as well as clinical applications such as ex vivo and in vivo gene and cell therapy.

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