RNA Gene Therapy Seen to Effectively Treat Hemophilia A Mouse Model

A new type of gene therapy that uses RNA instead of DNA led to rapid and prolonged production of therapeutic levels of factor VIII (FVIII) in a mouse model of hemophilia A, a study reported.

The study, “Treatment of Hemophilia A Using Factor VIII Messenger RNA Lipid Nanoparticles,” was published in the journal of Molecular Therapy Nucleic Acids. 

Hemophilia A is caused by missing or defective FVIII, a clotting protein in the blood. Replacement therapy is a standard treatment that involves supplying the missing FVIII to patients, and can be given on-demand during acute bleeding episodes from trauma or surgery, and regularly to prevent bleeding episodes (prophylactic). 

However, as FVIII has a short duration in the body, patients must undergo frequent infusions, as common as three or four times a day.

Gene therapy is an alternative to replacement therapy, designed to deliver a working copy of the F8 gene to enable stable production of the FVIII protein. 

While most gene therapy approaches use DNA to carry genetic information, messenger RNA (mRNA) can also be used. mRNA is generated from DNA, and contains the information to make a protein. 

Researchers at Seattle Children’s Research Institute collaborated with scientists at Moderna Therapeutics — a company specializing in mRNA therapeutics, and that partly funded the study — to test whether a gene therapy using FVIII mRNA could be an effective way of treating hemophilia A.

The FVIII mRNA was encapsulated in a carrier made of biodegradable lipids (fats), called lipid nanoparticles (LNPs), which were then delivered by intravenous (IV, into the vein) injection to mice genetically altered to lack FVIII.

After showing efficient delivery to the liver and no liver toxicity in a control group of mice (given the LNPs but not treatment), lipid nanoparticles carrying different variants of FVIII mRNA were then injected into another mouse group.

Results in these treated animals showed blood clotting activity increased from six hours after injection, reaching a peak level of 5.5% after 24 hours. Control mice showed no factor VIII activity. 

A known issue with newly formed FVIII is delayed protein release from cells and elevated blood levels. To solve this problem, the researchers made two genetic changes to the mRNA that enhanced FVIII production. 

When modified FVIII mRNAs were injected in mice, one of the new variants — known as F8-N6delta2-F309S — led to the greatest increase in FVIII activity, more than 10 times (1,000%) higher than normal. Low levels of FVIII activity were already detected within 30 minutes of injection.

This new variant was used in all subsequent experiments, showing a half-life — the time for a compound’s amount in the body to be reduced by half — of about 22 hours (longer than the 8–12 hours typically seen with conventional replacement therapy).  

As high levels of FVIII can increase a risk of abnormal blood clot formation, different doses were tested. FVIII activity was dose-dependent, with the lowest dose resulting in full FVIII activity after the first day that then slowly declined over seven days. Mice treated with a moderate dose (0.2 mg/kg) continued to have significantly better blood clotting after one week, and the scientists thought this dose suitable for prophylactic treatment.

Repeated injections of the moderate dose — two to four times within two weeks — were then given mice to test the effects repeat dosing. Results showed that animals given the most injections (three or four) had reduced production of FVIII, becoming undetectable at day 11. This was associated with the formation of FVIII inhibitors — antibodies generated against the protein that lower its effectiveness — which are common in these mice following repeated dosing, the researchers said.

Finally, to mimic routine replacement therapy in patients at high risk of spontaneous bleeding, and to avoid the development of inhibitors in mice, the team used mice with a reduced immune response. Routine injections every five days for two months consistently produced FVIII with peak levels as high as 220% of normal levels. 

“In conclusion, F8 [FVIII] LNP treatment produced rapid and prolonged duration of FVIII expression that could be applied to prophylactic treatment and potentially various other treatment options,” the investigators wrote. “Our study showed potential for a safe and effective platform of new mRNA therapies for HemA [hemophilia A].”