Systemic delivery of factor IX messenger RNA for protein replacement therapy

Abstract

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.lipid nanoparticles | nonviral mRNA delivery | hemophilia B therapy | systemic delivery | hepatic diseases A berrant gene expression is the underlying cause for many pathologies and restoring the normal state by targeting genes through expression or knockdown is conceptually a simple solution (1). RNA-based therapeutics have some inherent advantages over DNA and viral vectors but their therapeutic use has been plagued by problems of poor translatability, lack of stability, inefficient delivery, and adverse immune reactions. Incremental improvements (5′ caps, codon optimization, use of optimized 5′ and 3′ UTRs, poly(A) modifications, modified nucleosides like 5-methyl cytosine (5MC), pseudouridine and 2 thio-UTP, etc.) have substantially improved the stability and translatability of RNAs while also making them immunologically silent. Furthermore, lipid nanoparticles (LNPs) have been developed as a nonviral option to encapsulate and deliver nucleic acids in vivo.Efficient in vivo delivery, however, has long been a major challenge because currently available LNPs can induce liver damage and stimulate an immune response (2). Lipid nanoparticles typically comprise four different lipids-an ionizable lipid, a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG) lipid. When formulated into LNPs, these amine-containing ionizable lipids electrostatically complex with the negatively charged RNA to facilitate cellular uptake. These improvements have resulted in increasing use of small interfering RNA (siRNAs) as a potential therapeutic for systemic in vivo delivery to treat diseases like transthyretin amyloidosis, hepatitis B virus, hypercholesterolemia, cancer, and so forth (Arbutus, Alnylam Pharmaceuticals, Quark Pharmaceuticals, Allergan, Calando Pharmaceuticals, and others) (3). However, obvious differences between mRNAs and siRNAs in terms of length, stability, charge density, and so forth, make the synthesis, packa...

mRNA mediates passive vaccination against infectious agents, toxins, and tumors

“…There was a transient weak increase of some cytokines in circulation (), which obviously did not hamper high protein expression. Of note, equal or even higher levels were considered unproblematic in a recent study on modified mRNA encoding factor IX (Ramaswamy et al , ). Histopathology of liver, the target organ of mRNA‐LNPs, did not reveal any signs of abnormality or inflammation ().…”

mRNA as novel technology for passive immunotherapy

“…Thereafter, it took almost two decades until further studies started to demonstrate the broad potential of mRNA-based protein therapies. Meanwhile, there is a plethora of publications on a huge variety of indications comprising anemia [188,218], hemophilia [223,224], myocardial infarction [155,225], cancer [226,227], lung disease such as surfactant B deficiency and asthma [228][229][230], metabolic disorders [231][232][233][234][235], fibrosis [195], skeletal degeneration [236], tendon impairment [237], and neurological disorders such as sensory nerve dysfunction, Friedreich's ataxia and Alzheimer's disease [238][239][240]. Whereas evidence for the therapeutic potential of mRNA is mostly restricted to mouse models, first data in swine indicate that mRNA-based protein therapies are feasible also in large animals [218,225].…”

“…Only very few studies looking at local administration used uncomplexed and thus unprotected mRNA [225,228,230,237]. The majority of investigations built on lipid-based formulations with a clear tendency to the application of LNPs [223,224,[231][232][233]239]. Most if not all groups purified their IVT mRNA before in vivo administration.…”

“…Likewise, there is a clear prevalence to chemically modified mRNA, although various examples suggest that this is not mandatory [218,223,239,240]. While most mRNAs harbored 5-methyl-cytosine and/or pseudouridine initially [155,188,226], there appears to be a trend towards the use of N1-methyl-pseudouridine at present [224,225]. Regarding the cap structure, almost all early studies cotranscriptionally generated cap0 mRNAs using ARCA [226,228,229].…”

Biomedical applications of mRNA nanomedicine

“…LNPs were used to deliver human FIX (hFIX) mRNA to the liver to treat hemophilia B in a FIX knockout mouse model. The hFIX mRNA was effectively translated into functional FIX protein by hepatocytes and secreted into the circulation where it alleviated the clotting defect of the mice [176]. …”