Creating stable RNAs through circularization
What is RNA and what does it do?
RNAs are polymers of nucleic acids that are produced by every cell in your body. RNA is an extraordinarily adaptable molecule that can perform many different functions. One of RNA's primary functions is to provide instructions for making proteins in our cells. But RNAs can do so much more. RNAs can also fold into shapes that act like antibodies and recognize targets. These RNAs are called aptamers. Aptamers can be highly specific—they can recognize mutated proteins that occur in disease and not recognize the normal proteins. However, unlike antibodies, RNAs can function inside of our cells where many disease-driving proteins exist. RNA can also act like a molecular sponge for other RNAs in cells. By sequestering or degrading disease-causing RNAs we can inhibit their pathogenic roles and restore a cell's normal function.
Why aren't RNA therapeutics widely used?
The major roadblock that has previously prevented the use of RNAs as a therapeutic is that most RNAs are exceptionally unstable. They are rapidly removed by RNA-degrading cellular enzymes, which prevents them from accumulating to the concentrations needed for therapeutic purposes. For RNA aptamers to sequester a target protein, they need to accumulate to levels such that they have at least a one-to-one stoichiometry with the target protein. The majority of these enzymes recognize the beginning or end of RNA polymers.
Chimerna scientists have overcome this problem by developing a technology for making RNA as a circle, rather than the normal linear form that predominates in nature. RNA circles resist degradation, allowing them to accumulate to levels that have never previously been possible for RNA-based therapies. Our technology allows therapeutic RNAs to be expressed as stable RNA circles in any cell in the body using the cell's own machinery.
Genetic encodability: turning cells into drug-making factories
If any therapeutic is going to work in a patient, it must get to the correct tissue, correct cell, and to the correct part of the cell where it needs to function. This has always been challenging for RNA-based therapies because RNAs that are administered to a patient usually degrade once they enter a cell.
The first (and most common) method is to synthesize RNAs in a laboratory and then to deliver the circularized RNAs directly to cells. The RNAs are sometimes encased in lipid particles to help them enter into cells. The major drawback is that the RNAs are unstable and degrade quickly. As a result, the effect of the RNA is short-lived and the concentrations of the RNA are always low. RNAs have to be continually re-dosed in order to maintain RNA levels in the cell.
The second method is to have the cells make the RNAs. To do this, cells are provided with DNA that instructs the cell to make the RNA. The cell reads the DNA and then provides a steady supply of RNA that remains in the cell. This is because the cell itself makes the therapy. However, since these RNAs are still linear they last only a few minutes or hours and cannot accumulate to therapeutic levels.
Chimerna’s Tornado technology is a fundamentally new way to use DNA to make RNA in cells. The DNA directs the cell to synthesize an RNA of interest, but unlike in conventional DNA approaches, the RNA is directed to enzymes normally found in the cell that causes the RNA to be converted from a normal linear RNA into a circle. The front and back end of the RNA are “ligated” to each other. These circles are stable and accumulate to high levels in cells. The DNA can be delivered directly to tissues using lipid nanoparticles or packaged in adeno-associated viral particles to maximize cell-specific delivery. Chimerna scientists have turned our own cells into drug-making factories.
Self-processing: using the cell's own machinery to make circular RNAs
Tornado RNAs are expressed from a single transcript and require no other supplemental enzymes or factors. Within the transcript, the RNA aptamer or RNA of interest is flanked by two self-cleaving “ribozymes.” Each ribozyme is a sequence from nature that autocatalytically cleaves, leaving a unique arrangement of phosphates on the ends of the RNA. These ends are a substrate for a ubiquitous endogenous “RNA ligase” enzyme. This RNA ligase connects the two ends, resulting in a continuous circular RNA that contains the aptamer or RNA of interest.
Highly stable and abundant: Tornado-generated circular RNAs are more stable and abundant than linear RNAs.
Circular RNAs are stable because they lack the ends normally recognized by RNA-degrading enzymes in cells. This leads to high cellular accumulation. The Tornado expression systemis the most efficient circular RNA expression system for mammalian cells.
The difference in stability between linear and Tornado-generated circular RNAs is clear when compared side-by-side. This is exemplified by expressing linear or circular green fluorescent RNA aptamers in cells. The green signal is directly proportional to the amount of RNA in each cell.
Cells expressing Tornado circular RNA are visibly green while the same RNA expressed as a linear version barely has any detectable green signal.
For a therapeutic RNA, the RNAs should reach levels of at least 1:1 of RNA to target. Proteins are far more abundant than the levels of RNA aptamers that could be achieved using previous expression systems. This has prevented the use of RNA aptamer therapies. Using Tornado, circular RNA reach levels that are comparable with the most abundant cellular RNAs, such as 5.8S, 5S, and tRNA. Without Tornado-based circularization, linear aptamers are too unstable and low abundance to be detected. It is only because of the Tornado expression system that RNA therapies, for the first time, can achieve the expression levels they need to be effective.