The first RNA-based medicinal products to attract widespread attention were, of course, the RNA-based vaccines against SARS-CoV-2. But now other RNA-based medicinal products are emerging. RNA-based therapeutics, for example, represent a burgeoning market. In the estimation of Allied Market Research, the RNA-based therapeutics industry is expected to reach a valuation of $25.12 billion by 2030. Given that the corresponding figure for 2021 was just $4.93, the industry is registering a compound annual growth rate of 17.6%.

Leslie J. Williams
Leslie J. Williams
hC Bioscience

This kind of growth is possible because the RNA therapeutics industry is employing a range of new and powerful technologies. Also, the industry is developing much-needed products. “RNA-based therapeutics have immense potential to address many diseases that currently have suboptimal or no therapeutic option,” says Leslie J. Williams, co-founder, president, and CEO of hC Bioscience. “[These therapeutics rely on] a new modality by directly targeting the proteome.”

To explore some of the therapy-related research underway, GEN reached out to experts who are scheduled to deliver presentations at the 2nd Next Generation RNA Therapeutics Summit, which will take place May 16–18 in Boston. Here’s what we learned.

Programming the process

Jacob Becraft, PhD
Jacob Becraft, PhD
Strand Therapeutics

Despite the success with mRNA-based vaccines, Jacob Becraft, PhD, co-founder and CEO at Strand Therapeutics, indicates that therapeutics based on mRNA pose several challenges: getting mRNA to the desired tissue, getting enough drug to that tissue, and limiting toxic effects. He adds that we might be better able to meet these challenges if we look at delivery in a new way.

“Everyone’s been thinking about this problem as a delivery challenge, but mRNA programming allows us to take a broader view of delivery,” he explains. “By programming it, we simply nullify any message that goes to the wrong tissue.”

Instead of just creating a therapeutic mRNA, Becraft and his colleagues also program context into it. Doing that depends on understanding the genetic circuits involved in a disease. “With synthetic biology, we can program a genetic circuit that regulates the output of the protein therapeutic,” he details. Then, when the mRNA reaches a cell that includes certain markers—say, markers indicative of a cancer cell—the therapeutic protein is produced to destroy the tumor. If the mRNA enters a cell that lacks the specified markers, it gets degraded instead of activated.

According to Becraft, “cracking the nut” of cell-type-specific mRNA means being able to open up a plethora of potential targets and drugs.

Strand’s first drug will be aimed at solid tumors, and it’s going through the Investigational New Drug (IND) process. “In the latter half of this year, it will enter patients,” Becraft predicts. “That will be the world’s first programmed mRNA with inherent cell-type specificity to enter the clinic.”

Becraft adds that this therapeutic relies on self-amplifying mRNA. “It amplifies itself to increase the level of protein, provided it’s in the correct cell, and creates a cytokine,” he says. As a result, this therapy would solve the key limitations of existing approaches based on mRNA.

Going round in circles

Most biologists think of RNA as linear, and that’s the structure used in existing therapies, but RNA also forms circles. “The primary benefit of circular RNAs is that they are inherently more stable than linear RNAs,” says Brian Pickering, PhD, co-founder and CEO at Chimerna Therapeutics. That’s because nucleases inside cells attack RNAs from the ends. “Since a circular RNA does not have a beginning or an end, these nucleases are rendered ineffective,” Pickering explains. “This allows the therapeutic to perform its function over a longer time span.”

Scientists can use circular RNAs in many ways, depending on how they’re made. Chimerna’s technology adds self-cleaving ribozymes around the desired RNA. “This method of production allows us to produce circular RNAs both in vivo and in vitro,” Pickering asserts. “In the case of in vivo production, we deliver a DNA vector that expresses our RNA inside of the cell [where it is circularized].” For in vitro applications, Chimerna developed a process to make circular RNAs in bacteria. “The circular RNAs,” Pickering points out, “are then purified from bacteria using a proprietary affinity tag we engineer into our RNA sequences.”

Pickering insists that Chimerna’s biggest advance is its manufacturing platform. “Our platform,” he says, “does not suffer from shortages in raw materials, such as nucleotides, since the bacteria synthesize them for us.” In addition to other advances, Pickering emphasizes the importance of an affinity tag for purification: “Without this, it would be far too difficult to purify the RNA of interest from the other bacterial RNA.”

Endless opportunities

Scientists at Laronde also develop circular RNA, which the company calls endless RNA (eRNA). Avak Kahvejian, PhD, Laronde’s co-founder and chief scientific officer, says that the name for this circular RNA is “a little tongue in cheek, given that it has a double meaning. Besides being circular, eRNA lasts longer.”

Avak Kahvejian, PhD
Avak Kahvejian, PhD

Although circular RNA outlasts linear RNA, the specific duration of RNA hangs on several factors. “It depends on the design of the RNA, the cell type the RNA is in, and the conditions inside the cell,” Kahvejian specifies. “So, it’s contextual, but it does last significantly longer.”

Laronde developed a comprehensive eRNA platform. “It involves everything from putting together the sequence of nucleotides on a computer, to synthesizing and purifying it,” Kahvejian says. In addition, the Laronde platform can work at various scales. “We can make enough material so that you can do animal studies and human studies,” he asserts. “That lets us prototype many, many different examples.”

After generating prototypes, Kahvejian and his colleagues can try out many eRNAs to explore the application landscape. “When you change the open reading frame, you can get a wildly different protein, which in turn means a wildly different indication,” he explains. “The other reason you want to do this is so that you can understand how changing the nucleotides and their order impacts translation efficiency, stability, and so on.” By quickly generating eRNAs, he says, “we can both explore and learn rapidly.”

Although Laronde is still working at a preclinical stage, Kahvejian emphasizes that the company is “exploring a variety of applications and a wide range of indications.”

Improving performance

Nathaniel Wang, PhD
Nathaniel Wang, PhD
Replicate Bioscience

Other companies are exploring the potential of self-replicating RNA (srRNA). “With srRNA, you’re actually providing an instruction manual for the cell to produce the mRNA itself,” says Nathaniel Wang, PhD, founder and CEO of Replicate Bioscience. “You’re essentially putting a Xerox machine into the cell.” Consequently, with this approach, a cell produces high levels of mRNA for a longer period of time than it would with existing approaches. “That leads to more protein and a better therapeutic effect,” Wang insists.

Using srRNAs offers other benefits as well. One of them is lower dosing, which arises from amplification and durability. “It’s already been shown that you get better durability, which means less frequent dosing clinically,” Wang points out. “You can actually use a single dose and get a very good durable response right off the bat.”

By studying various types of srRNAs, Wang and his colleagues intend to create therapies that are capable of fine-tuning outcomes. “We can use the way that the RNAs interact with the cells to remodel them so that they become the type of protein-production factory that you need,” he explains. “Some proteins need to be secreted into the blood in order to have their effect, some need to be decorated with sugars, and some need to be chopped up into fragments and shown to the immune system.” Using a library of srRNAs, Wang and his colleagues can create such specializations.

Beyond adding desired features, Replicate’s library can improve performance. “When we do these engineered optimizations, the result is actually 100-fold better in terms of performance compared to conventional self-replicating RNAs,” Wang asserts. “The performance with our srRNA is actually over 1,000 times better [than that of linear RNA].”

Self Replicating RNA illustration
Replicate Bioscience, a developer of synthetic self-replicating RNA (srRNA) technology, says that srRNA vectors may be used to drive the in situ production of fully natural mRNAs, which may then result in the production of proteins that serve as vaccines and therapeutics. Replicate Bioscience adds that srRNA has several advantages over traditional viral vectors. These include greater safety, simplified manufacturing, larger payloads, and lower doses. This last advantage is illustrated here. Notice that an amplification mechanism allows srRNA to outperform mRNA in terms of therapeutic output.

Turning tRNA into therapies

Of all the RNA therapeutic modalities, mRNA probably receives the most attention. But therapeutics based on tRNA shouldn’t be overlooked. Like mRNA therapeutics, tRNA therapeutics are capable of addressing protein dysfunction at the level of translation.

“Each tRNA molecule reads the codon on mRNA that contains the instructions for a specific amino acid to form a specific protein, and mutations in the codon can lead to dysfunctional proteins—for example, prematurely truncated proteins,” hc Bioscience’s Williams explains. “tRNA-based therapeutics edit proteins in a codon-specific manner to restore protein function and allow the cell machinery to function normally, restoring homeostasis.”

Repair Defective Proteins
Engineered tRNA molecules from hC Bioscience are designed to repair or eliminate defective proteins. These molecules are generated with the company’s PTCX (“Patch”) and SWTX (“Switch”) platforms. (A) PTCX generates tRNAs that can recognize nonsense mutations in mRNA transcripts and suppress them by inserting an amino acid where it is needed, restoring the protein to full length and function. (B) SWTX generates tRNAs that can mark disease-causing proteins (that is, proteins resulting from a missense mutation) for destruction.

Williams adds that tRNA offers advantages in gene editing. These include context-free targeting and modulation of mutations anywhere in a single gene or across the genome. “Multiple mutations of the same kind anywhere in the genome can be addressed with a single engineered tRNA without having to develop gene- and location-specific guides,” she says. “Multiple genes with the same mutations driving distinct diseases can be treated with the same therapeutic.” Williams notes that there is interest in using tRNA to treat inherited rare genetic disorders and cancers.

Williams maintains that if tRNA is to advance as a modality, developers will need to employ a range of technologies, including tools for single-molecule analysis, animal models with varying mutational backgrounds, and multiple delivery platforms. By leveraging these technologies, developers will be able to rewrite proteins at the single-amino-acid level. These technologies, Williams emphasizes, represent an “unprecedented opportunity” to develop tRNA treatments for thousands of diseases.

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