Summary:

Like most genetic disorders, Hemophilia B is caused by a mutation that leads to a dysfunctional protein. Hemophilia B patients are susceptible to life threatening bleeds when injured due to a clotting defect caused by a mutated coagulation factor IX (FIX). They also suffer from recurrent bleeding into joints and muscles which could result in pain, discomfort and ultimately loss of mobility. This debilitating condition currently has very few approved treatment options on the market. Patients are treated with either a recombinant or a purified serum FIX. Both types of treatments are relatively effective but highly costly, should be administered frequently and can lead to patients developing antibodies in the long term. Gene therapy and specifically RNA therapeutics therefore, can be a promising alternative for hemophilia B treatment. Messenger RNA (mRNA) delivery is anticipated to be safer and more efficacious than conventional gene delivery methods such as plasmids and viral vectors which could integrate into host genome and/ or cause severe immune responses. mRNA sequences are relatively easy to manufacture and can be customized to patients’ needs. They can be highly advantageous over the current treatments of hemophilia B because the translation and maturation of protein (FIX) in its native environment minimizes immunogenicity and adverse reactions. However, mRNA delivery is challenging due to its instability in biological systems. Nanoparticles provide an efficacious vehicle for nucleic acid delivery, including mRNA. In this article, the Verma lab at the Salk Institute in collaboration with Arcturus Therapeutics, describe an FIX mRNA lipid nanoparticle (LNP) system that efficaciously addresses the root cause of hemophilia B.

In their article featured in PNAS, 2017, the Verma lab describes an mRNA-LNP called LUNAR (lipid-enabled and unlocked nucleic acid modified RNA) that successfully alleviates hemophilia B clotting defects by expressing FIX in vivo. Using the NanoAssemblr^TM Benchtop instrument, they formulated a modified human FIX(hFIX) mRNA with a proprietary ionisable lipid (ATX) and the animal studies indicated blood FIX levels that significantly exceeded the acceptable clinical levels. Additionally, LUNAR was up to twofold superior to its counterpart formulation with the well-known ionisable lipid DLin-MC3-DMA. The authors then assessed the efficacy of LUNAR in a FIX knockout mouse model where they could restore the FIX levels back to normal physiological levels. Additionally, LUNAR kept hFIX at above the clinically acceptable level for up to 6 days. Furthermore, two catalytically enhanced variants of hFIX were incorporated in LUNAR and compared to the wild type hFIX. The results indicated the variants to be significantly more efficacious in enhancing clotting activity. This can allow for lower dose treatments, bringing down the costs and making the treatment more accessible to the public. The variant LUNAR was then tested against the current standard of care for hemophilia B: recombinant human FIX (rhFIX). LUNAR’s clotting effect lasted up to 3 days longer than rhFIX pointing to a greater therapeutic efficacy.  LUNAR was also repeatedly injected to mice over a 4-month period to monitor the safety and immunogenicity of the formulation. The mice showed no significant sign of distress and grew normally during the period. Additionally, the blood work indicated normal cytokine profiles (immune response markers).

This article highlights the significance of gene therapy and in particular mRNA therapeutics for treatment of disorders caused by loss-of-function mutations. The FIX mRNA-LNP described here -LUNAR- is highly effective in alleviating the clotting defect caused by hemophilia B and the authors have improved the therapeutic effect by optimizing the formulation in vivo. A side by side comparison indicated higher therapeutic efficacy for LUNAR than the current standard of care for hemophilia B. Thus, LUNAR represents a new generation of safe and efficient treatments for genetic disorders and indicates the great potential of gene therapy to satisfy as yet unmet medical needs.

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.