Scientists at German biotech Curevac GmbH have reported they were able to raise erythropoietin (EPO) levels in pigs by treating them with sequence-engineered but chemically unmodified messenger RNA (mRNA) encapsulated in lipid nanoparticles.

The work is the first demonstration that using mRNA to raise protein levels can result in physiological responses in large animals.

It also shows the potential utility of mRNA whose bases have not been chemically modified.

"There is a dogma that modifications are necessary" both to boost translation levels and to avoid an immune response to the RNA, Curevac CEO Ingmar Hoerr told BioWorld Today.

Such an immune response is a problem in its own right for therapeutic proteins, and can, in a worst-case scenario, break tolerance to a protein that does not normally stimulate an immune response.

The Curevac team focused on the mRNA sequence itself, both within the protein-coding part of the mRNA and within regulatory sequences, rather than the chemical composition of the bases.

In their paper, which was published online in the June 8, 2015, issue of Molecular Therapy, the researchers directly compared the ability of chemically modified and sequence-engineered mRNA to boost the expression levels of EPO.

Recombinant EPO and follow-ons such as Thousand Oaks, Calif.-based Amgen's Epogen (epoetin alfa) and Aranesp (darbepoetin alfa) are used to treat anemia due to cancer treatments, chronic kidney disease and other causes. Proteins like EPO that are present in low levels in the blood are well suited to an mRNA-based approach.

In cell culture, the team found higher expression levels with the sequence-optimized mRNA than with base-modified mRNA.

When they tested their mRNA in mice, the authors wrote that "overall, protein levels were in the range of a previous study utilizing [chemically] pseudouridine-modified mRNA formulated and administered in the same manner as here. However, our sequence-engineered mRNA gave rise to longer lasting protein expression."

In monkeys, the authors also tested for telltale signs of an immune response in the form of either cytokine release or an antibody response to the EPO itself, but found neither.

When they treated pigs with the encapsulated mRNA, the animals showed an increase in immature red blood cells as well as increases in hematocrit, which measures the volume of red blood cells in the blood.

The authors also tested their encapsulated mRNA in cynomolgus monkeys, where some previous attempts to raise protein levels with DNA-based approaches had been unsuccessful. The monkeys, too, showed increases in immature red blood cells and hematocrit. Like the mice, they showed no evidence of cytokine release.

Chemical modification and sequence optimization, Hoerr noted, did not synergize in the experiments now published by the Curevac team. Combining base modification and sequence optimization resulted in a "worse product" with the advantages of neither.

MRNA ADVANTAGE

Using mRNA has several potential advantages over treating with the proteins themselves.

Some of those advantages are in the business realm – Hoerr noted that mRNA is one possible way to get patent extensions on protein therapeutics. But there are scientific advantages as well.

Despite its reputation for instability, "chemically, it's very stable – much more stable than proteins," he said.

As with other forms of RNA, delivery is "always the issue." The mRNA needs to be encapsulated into liposomes, and the liver is the best place for getting a high expression level.

Hoerr said Curevac is "very keen" to be the first company with an mRNA product to market. Other companies working on mRNA include Sarepta Therapeutics Inc., of Cambridge, Mass., with eteplirsen (AVI-4658) for Duchenne muscular dystrophy (DMD) in phase III. Moderna Therapeutics Inc. is still preclinical, albeit with both a rather large pipeline and some rather large deals. (See BioWorld Today, Jan. 14, 2015.)

To date, Curevac is furthest along in an area where the immune response is a silk purse, not a sow's ear: vaccines.

Curevac has two cancer vaccines in clinical trials. CV9104 for prostate cancer is in phase II, and CV9202 for non-small-cell lung cancer is in phase I trials in combination with various other agents and was licensed by Boehringer Ingelheim GmbH last year. (See BioWorld Today, Sept 18, 2014.)

Also in phase I is a prophylactic rabies vaccine. Earlier this year, the company received $52 million from the Bill & Melinda Gates Foundation to advance its prophylactic vaccine work.

The Gates Foundation's interest stems from mRNA's stability, which means that vaccines could be stored at room temperature – a very important cost savings for low- and middle-income countries.