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...

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8 + T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4 + T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

A self-transcribing and replicating RNA (STARR™) based vaccine (LUNAR®-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolong SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell mediated immunity produced a strong viral antigen specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for IFN-γ and IL-4 positive CD4+ T helper lymphocytes as well as anti-spike glycoprotein IgG2a/IgG1 ratios supported a strong Th1 dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of Lunar-COV19 as a single dose vaccine.

The use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine. Using a plasmid encoding an anti-poxvirus monoclonal antibody (as a reporter of protein expression), we showed that improved immunogenicity is likely due to increased in vivo DNA delivery, resulting in more target protein. Specifically, after four days, up to 30 ng/mL of functional monoclonal antibody were detected in the serum of rabbits injected with the LNP-formulated DNA. We pragmatically applied the technology to the production of human neutralizing antibodies in a transchromosomic (Tc) bovine for use as a passive immunoprophylactic. Production of neutralizing antibody was increased by >10-fold while utilizing 10 times less DNA in the Tc bovine. This work provides a proof-of-concept that LNP formulation of DNA vaccines can be used to produce more potent active vaccines, passive countermeasures (e.g., Tc bovine), and as a means to produce more potent DnA-launched immunotherapies. It is now possible to rapidly determine the genomic sequence of emerging infectious disease threats, even before the microbe has been isolated. That is, nonspecific amplification and sequencing of nucleic acid can be performed when virus is difficult to propagate and/or in samples where viable virus no longer exists. This genomic information can then be used to design and synthesize candidate nucleic acid-based vaccines. The vaccines can be used as active vaccines, or as passive vaccines to produce antibody-based medical countermeasures. In order to increase the potency of plasmid DNA vaccines, we have explored the possibility of formulating the DNA using lipid nanoparticles (LNPs). The microbes targeted in this research include Andes virus (ANDV) and Zika virus (ZIKV). ANDV is a South American, New World hantavirus and member of the Family Hantaviridae, Order Bunyavirales. The enveloped virion contains a single stranded, tripartite, negative sense RNA genome. The three segments, S, M, and L, encode the nucleocapsid protein (N), the envelope glycoproteins (G n /G c), and the RNA-dependent RNA polymerase, respectively. ANDV is rodent-borne and causes a severe disease referred to as hantavirus pulmonary syndrome (HPS) in humans. ANDV is the only hantavirus known to transmit person-to-person. The case fatality rate of this virus is >35% even in modern intensive care units. There are no vaccines or drugs approved to prevent or treat HPS 1 .

We explored an emerging technology to produce anti-Hantaan virus (HTNV) and anti-Puumala virus (PUUV) neutralizing antibodies for use as pre-or post-exposure prophylactics. The technology involves hyperimmunization of transchomosomic bovines (TcB) engineered to express human polyclonal IgG antibodies with HTNV and PUUV DNA vaccines encoding G n G c glycoproteins. For the anti-HTNV product, TcB was hyperimmunized with HTNV DNA plus adjuvant or HTNV DNA formulated using lipid nanoparticles (LNP). The LNP-formulated vaccine yielded fivefold higher neutralizing antibody titers using 10-fold less DNA. Human IgG purified from the LNP-formulated animal (SAB-159), had anti-HTNV neutralizing antibody titers >100,000. SAB-159 was capable of neutralizing pseudovirions with monoclonal antibody escape mutations in G n and G c demonstrating neutralization escape resistance. SAB-159 protected hamsters from HTNV infection when administered pre-or post-exposure, and limited HTNV infection in a marmoset model. An LNP-formulated PUUV DNA vaccine generated purified anti-PUUV IgG, SAB-159P, with a neutralizing antibody titer >600,000. As little as 0.33 mg/kg of SAB-159P protected hamsters against PUUV infection for pre-exposure and 10 mg/kg SAB-159P protected PUUV-infected hamsters post-exposure. These data demonstrate that DNA vaccines combined with the TcB-based manufacturing platform can be used to rapidly produce potent, human, polyclonal, escaperesistant anti-HTNV, and anti-PUUV neutralizing antibodies that are protective in animal models.