Enzymatic Synthesis of RNA with Chemical Modifications
Richard Pomerantz, Ph.D. Associate Professor, Biochemistry and Molecular Biology, Thomas Jefferson University
Modified Enzymes Could Cut the Cost of RNA Manufacturing, Say Researchers
Making RNA oligonucleotides using traditional chemical methods is effective but expensive, according to researchers at Thomas Jefferson University, who say modified enzymes could help the industry reduce costs.
Project leader professor Richard Pomerantz, who is also the co-founder and CSO at Recombination Therapeutics, spoke about the potential of enzymatic oligo manufacturing at TIDES USA, telling delegates reduced cost is the major advantage.
“The cost of synthesizing RNA using phosphoramidite chemistry is about 10 to 20 times higher than synthesizing DNA oligos. The expected delivery time for RNA is approximately five to 15 times longer than equivalent length DNA oligos. And chemical synthesis of intermediate length or long length RNA with heavily modified nucleotides is extremely inefficient and costly.
“So new and efficient, cost-effective RNA synthesis technologies are needed for reducing the costs and increasing the yields of these products,” he said, citing aptamers, antisense RNAs and vaccines as examples.
Modified enzymes
Modified enzymes are a potential low-cost manufacturing alternative to phosphoramidite chemistry, according to Pomerantz, citing work at his laboratory.
“So we’ve been involved in bioengineering DNA dependent RNA polymerases by converting somewhat promiscuous DNA polymerases found in our human cells into promoter independent DNA dependent RNA polymerases. They’re essentially using a DNA template to synthesize an RNA product.
Pomerantz used a version of RNA polymerase 1 developed by his team as an example, explaining it is able to “synthesize the 95 nucleotide RNA product with three different base modifications that are typically used.
“For example, pseudo urethane, which is used for mRNA vaccines, is incorporated at a very similar rate as natural UTP. We’re also able to incorporate other five prime modifications such as methyl CTP.”
The enzyme can also help improve the stability of the RNA molecules it produces, which would be of significant benefit to developers using it for therapeutic applications.
Pomerantz said, “You need to really stabilize RNA for therapeutics and phosphorothioate-modified RNA is very important for therapeutic products. So we’re investigating the ability of the bioengineered RNA polymerase 1 to incorporate all four phosphorothioate ribonucleotides and make the same 95 nucleotide RNA product.”
Data so far suggest that bioengineered RNA polymerase I can make the same 95 base nucleotide that includes all four bases, although it took two hours to do so.
A second RNA polymerase the team is working on showed more promise, according to Pomerantz, who said, “This is a thermophilic, bacteriophage RNA polymerase from microorganisms and hot springs. And this has never been characterized before unpublished.
“This thermophilic DNA dependent RNA polymerase is able to now synthesize a 95 nucleotide RNA with 100% phosphorothioate modifications in just a couple of minutes.”
Advantages
There are other potential benefits of using enzymes to make oligonucleotides, according to Pomerantz, such as conjugation.
“Another interesting characteristic of primer dependent or promoter independent RNA synthesis using a DNA template is that because the primer could be any length, you could synthesize RNA with just about any conjugation at the five prime end.
“Many RNA-based therapeutics have conjugates to help their bioavailability and half-life. So now we’re demonstrating that we could have two MOA E or two methoxy, or two fluoro at the five prime end of the RNA primer, and that RNA primer can be extended to just about any length,” he said.
“We’re also working on a bioengineered RNA polymerase that’s a strong RNA terminal transferase. And this is an enzyme that’s been modified from a DNA polymerase present in human cells is very promiscuous. We have a patent on this. So we’re looking to partner or license this technology.”