Safety, immunogenicity and efficacy of the self-amplifying mRNA ARCT-154 COVID-19 vaccine: pooled phase 1, 2, 3a and 3b randomized, controlled trials.

Combination of waning immunity and lower effectiveness against new SARS-CoV-2 variants of approved COVID-19 vaccines necessitates new vaccines. We evaluated two doses, 28 days apart, of ARCT-154, a self-amplifying mRNA COVID-19 vaccine, compared with saline placebo in an integrated phase 1/2/3a/3b controlled, observer-blind trial in Vietnamese adults (ClinicalTrial.gov identifier: NCT05012943). Primary safety and reactogenicity outcomes were unsolicited adverse events (AE) 28 days after each dose, solicited local and systemic AE 7 days after each dose, and serious AEs throughout the study. Primary immunogenicity outcome was the immune response as neutralizing antibodies 28 days after the second dose. Efficacy against COVID-19 was assessed as primary and secondary outcomes in phase 3b. ARCT-154 was well tolerated with generally mild-moderate transient AEs. Four weeks after the second dose 94.1% (95% CI: 92.1-95.8) of vaccinees seroconverted for neutralizing antibodies, with a geometric mean-fold rise from baseline of 14.5 (95% CI: 13.6-15.5). Of 640 cases of confirmed COVID-19 eligible for efficacy analysis most were due to the Delta (B.1.617.2) variant. Efficacy of ARCT-154 was 56.6% (95% CI: 48.7- 63.3) against any COVID-19, and 95.3% (80.5-98.9) against severe COVID-19. ARCT-154 vaccination is well tolerated, immunogenic and efficacious, particularly against severe COVID-19 disease.

Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases1. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation2-7. However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression.

Liver lipopolysaccharide binding protein prevents hepatic inflammation in physiological and pathological non-obesogenic conditions.

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels. As expected, the MCD increased hepatocyte vacuolation, liver inflammation and fibrosis markers, also increasing liver Lbp mRNA. In this model, liver Lbp gene knockdown resulted in a pronounced worsening of the markers of liver inflammation (also including CD68 and MPO activity), fibrosis, ER stress and protein carbonyl levels, all indicative of non-alcoholic steatohepatitis (NASH) progression. At cellular level, Lbp gene knockdown also increased expression of the proinflammatory mediators (Il6, Ccl2), and markers of fibrosis (Col1a1, Tgfb1) and protein carbonyl levels. In agreement with these findings, liver LBP mRNA in humans positively correlated with markers of liver damage (circulating hsCRP, ALT activity, liver CRP and KRT18 gene expression), and with a network of genes involved in liver inflammation, innate and adaptive immune system, endoplasmic reticulum stress and neutrophil degranulation (all with q-value<0.05). In conclusion, current findings suggest that a significant downregulation in liver LBP levels promotes liver oxidative stress and inflammation, aggravating NASH progression, in physiological and pathological non-obesogenic conditions.

A phase I/II randomized, double-blinded, placebo-controlled trial of a self-amplifying Covid-19 mRNA vaccine.

Coronavirus disease-19 (Covid-19) pandemic have demonstrated the importantance of vaccines in disease prevention. Self-amplifying mRNA vaccines could be another option for disease prevention if demonstrated to be safe and immunogenic. Phase 1 of this randomized, double-blinded, placebo-controlled trial (N = 42) assessed the safety, tolerability, and immunogenicity in healthy young and older adults of ascending levels of one-dose ARCT-021, a self-amplifying mRNA vaccine against Covid-19. Phase 2 (N = 64) tested two-doses of ARCT-021 given 28 days apart. During phase 1, ARCT-021 was well tolerated up to one 7.5 µg dose and two 5.0 µg doses. Local solicited AEs, namely injection-site pain and tenderness were more common in ARCT-021vaccinated, while systemic solicited AEs, mainly fatigue, headache and myalgia were reported in 62.8% and 46.4% of ARCT-021 and placebo recipients, respectively. Seroconversion rate for anti-S IgG was 100% in all cohorts, except for the 1 µg one-dose in younger adults and the 7.5 µg one-dose in older adults. Anti-S IgG and neutralizing antibody titers showed a general increase with increasing dose, and overlapped with titers in Covid-19 convalescent patients. T-cell responses were also observed in response to stimulation with S-protein peptides. Taken collectively, ARCT-021 is immunogenic and has favorable safety profile for further development.

Immune gene expression analysis indicates the potential of a self-amplifying Covid-19 mRNA vaccine.

Remarkable potency has been demonstrated for mRNA vaccines in reducing the global burden of the ongoing COVID-19 pandemic. An alternative form of the mRNA vaccine is the self-amplifying mRNA (sa-mRNA) vaccine, which encodes an alphavirus replicase that self-amplifies the full-length mRNA and SARS-CoV-2 spike (S) transgene. However, early-phase clinical trials of sa-mRNA COVID-19 vaccine candidates have questioned the potential of this platform to develop potent vaccines. We examined the immune gene response to a candidate sa-mRNA vaccine against COVID-19, ARCT-021, and compared our findings to the host response to other forms of vaccines. In blood samples from healthy volunteers that participated in a phase I/II clinical trial, greater induction of transcripts involved in Toll-like receptor (TLR) signalling, antigen presentation and complement activation at 1 day post-vaccination was associated with higher anti-S antibody titers. Conversely, transcripts involved in T-cell maturation at day 7 post-vaccination informed the magnitude of eventual S-specific T-cell responses. The transcriptomic signature for ARCT-021 vaccination strongly correlated with live viral vector vaccines, adjuvanted vaccines and BNT162b2 1 day post-vaccination. Moreover, the ARCT-021 signature correlated with day 7 YF17D live-attenuated vaccine transcriptomic responses. Altogether, our findings show that sa-mRNA vaccination induces innate immune responses that are associated with the development of adaptive immunity from other forms of vaccines, supporting further development of this vaccine platform for clinical application.

Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation.

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP-Lbp UNA-siRNA) on the development of fatty liver. Weekly LNP-Lbp UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD). In mice fed a high-fat and high-sucrose diet, which displayed induced liver lipogenesis, LBP downregulation led to reduced liver lipid accumulation, lipogenesis (mainly stearoyl-coenzyme A desaturase 1 [Scd1]) and lipid peroxidation-associated oxidative stress markers. LNP-Lbp UNA-siRNA also resulted in significantly decreased blood glucose levels during an insulin tolerance test. In mice fed a standard chow diet or an MCD, in which liver lipogenesis was not induced or was inhibited (especially Scd1 mRNA), liver LBP downregulation did not impact on liver steatosis. The link between hepatocyte LBP and lipogenesis was further confirmed in palmitate-treated Hepa1-6 cells, in primary human hepatocytes, and in subjects with morbid obesity. Altogether, these data indicate that siRNA against liver Lbp mRNA constitutes a potential target therapy for obesity-associated fatty liver through the modulation of hepatic Scd1.

Downregulation of peripheral lipopolysaccharide binding protein impacts on perigonadal adipose tissue only in female mice.

BACKGROUND AND AIMS: The sexual dimorphism in fat-mass distribution and circulating leptin and insulin levels is well known, influencing the progression of obesity-associated metabolic disease. Here, we aimed to investigate the possible role of lipopolysaccharide-binding protein (LBP) in this sexual dimorphism. METHODS: The relationship between plasma LBP and fat mass was evaluated in 145 subjects. The effects of Lbp downregulation, using lipid encapsulated unlocked nucleomonomer agent containing chemically modified-siRNA delivery system, were evaluated in mice. RESULTS: Plasma LBP levels were associated with fat mass and leptin levels in women with obesity, but not in men with obesity. In mice, plasma LBP downregulation led to reduced weight, fat mass and leptin gain after a high-fat and high-sucrose diet (HFHS) in females, in parallel to increased expression of adipogenic and thermogenic genes in visceral adipose tissue. This was not observed in males. Plasma LBP downregulation avoided the increase in serum LPS levels in HFHS-fed male and female mice. Serum LPS levels were positively correlated with body weight and fat mass gain, and negatively with markers of adipose tissue function only in female mice. The sexually dimorphic effects were replicated in mice with established obesity. Of note, LBP downregulation led to recovery of estrogen receptor alpha (Esr1) mRNA levels in females but not in males. CONCLUSION: LBP seems to exert a negative feedback on ERα-mediated estrogen action, impacting on genes involved in thermogenesis. The known decreased estrogen action and negative effects of metabolic endotoxemia may be targeted through LBP downregulation.

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.

Lipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines.

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.

Systemic delivery of factor IX messenger RNA for protein replacement therapy.

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.

Long Non-coding RNA BGas Regulates the Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic fibrosis (CF) is a life-shortening genetic disease. The root cause of CF is heritable recessive mutations that affect the cystic fibrosis transmembrance conductance regulator (CFTR) gene and the subsequent expression and activity of encoded ion channels at the cell surface. We show that CFTR is regulated transcriptionally by the actions of a novel long noncoding RNA (lncRNA), designated as BGas, that emanates from intron 11 of the CFTR gene and is expressed in the antisense orientation relative to the protein coding sense strand. We find that BGas functions in concert with several proteins including HMGA1, HMGB1, and WIBG to modulate the local chromatin and DNA architecture of intron 11 of the CFTR gene and thereby affects transcription. Suppression of BGas or its associated proteins results in a gain of both CFTR expression and chloride ion function. The observations described here highlight a previously underappreciated mechanism of transcriptional control and suggest that BGas may serve as a therapeutic target for specifically activating expression of CFTR.

Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth.

PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells.