Development of an mRNA replacement therapy for phenylketonuria.

Phenylketonuria (PKU) is an inborn error caused by deficiencies in phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main cause of the disease whose signature hallmarks of toxically elevated levels of Phe accumulation in plasma and organs such as the brain, result in irreversible intellectual disability. Here, we present a unique approach to treating PKU deficiency by using an mRNA replacement therapy. A full-length mRNA encoding human PAH (hPAH) is encapsulated in our proprietary lipid nanoparticle LUNAR and delivered to a Pah enu2 mouse model that carries a missense mutation in the mouse PAH gene. Animals carrying this missense mutation develop hyperphenylalanemia and hypotyrosinemia in plasma, two clinical features commonly observed in the clinical presentation of PKU. We show that intravenous infusion of LUNAR-hPAH mRNA can generate high levels of hPAH protein in hepatocytes and restore the Phe metabolism in the Pah enu2 mouse model. Together, these data establish a proof of principle of a novel mRNA replacement therapy to treat PKU.

Synthesis and bioactivity of readily hydrolysable novel cationic lipids for potential lung delivery application of mRNAs.

Lipid nanoparticles (LNPs) mediated mRNA delivery has gained prominence due to the success of mRNA vaccines against Covid-19, without which it would not have been possible. However, there is little clinical validation of this technology for other mRNA-based therapeutic approaches. Systemic administration of LNPs predominantly targets the liver, but delivery to other organs remains a challenge. Local approaches remain a viable option for some disease indications, such as Cystic Fibrosis, where aerosolized delivery to airway epithelium is the preferred route of administration. With this in mind, novel cationic lipids (L1-L4) have been designed, synthesized and co-formulated with a proprietary ionizable lipid. These LNPs were further nebulized, along with baseline control DOTAP-based LNP (DOTAP+), and tested in vitro for mRNA integrity and encapsulation efficiency, as well as transfection efficiency and cytotoxicity in cell cultures. Improved biodegradability and potentially superior elimination profiles of L1-L4, in part due to physicochemical characteristics of putative metabolites, are thought to be advantageous for prospective therapeutic lung delivery applications using these lipids.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice.

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.

Effect of PEGylation on biodistribution and gene silencing of siRNA/lipid nanoparticle complexes.

PURPOSE: To determine the influence of physicochemical properties of lipid nanoparticles (LNPs) carrying siRNA on their gene silencing in vivo. Mechanistic understanding of how the architecture of the nanoparticle can alter gene expression has also been studied. METHODS: The effect of 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA) on hepatic distribution and FVII gene silencing was determined. FVII mRNA in hepatocytes and liver tissues was determined by Q-PCR. Hepatic distribution was quantified by FACS analysis using Cy5 labeled siRNA. RESULTS: Gene silencing was highly dependent on the amount of PEG-C-DMA present. FVII gene silencing inversely correlated to the amount of PEG-C-DMA in LNPs. High FVII gene silencing was obtained in vitro and in vivo when the molar ratio of PEG-C-DMA to lipid was 0.5 mol%. Surprisingly, PEGylation didn't alter the hepatic distribution of the LNPs at 5 h post administration. Instead the amount of PEG present in the LNPs has an effect on red blood cell disruption at low pH. CONCLUSION: Low but sufficient PEG-C-DMA amount in LNPs plays an important role for efficient FVII gene silencing in vivo. PEGylation did not alter the hepatic distribution of LNPs, but altered gene silencing efficacy by potentially reducing endosomal disruption.