Platform Publications

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 translation2,3,4,5,6,7. 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.

TALENs delivered as mRNA cargo in LNPs cause LPA gene editing and Lp(a) reduction in vivo

● Achieved proof-of-concept for TALEN-mediated Apo(a) reduction in vitro using TALEN mRNAs
● Demonstrated in vivo efficacy by LNP delivery of TALEN mRNA that led to Lp(a) reduction in a transgenic mouse model
● TALEN-mediated gene editing was dose responsive
● TALEN mRNA-LNP formulations were well-tolerated in vivo
● No detectable off-target activity in vitro

Lipid nanoparticle delivers phenylalanine ammonia lyase mRNA to the liver leading to catabolism and clearance of phenylalanine in a phenylketonuria mouse model

Phenylketonuria (PKU) is a genetic disorder affecting around 1 in 12,000 live births (1), caused by a mutation in the phenylalanine hydroxylase (PAH) gene in the liver which facilitates the catabolism of phenylalanine (Phe). Without a functional copy of PAH, levels of Phe in the blood and tissues rise, resulting in potentially life- threatening damage to the central nervous system. (2) Treatment options for PKU are limited, and center around adherence to a strict PKU diet that suffers from poor patient compliance. There are two approved drugs available, one of which must be used in conjunction with the PKU diet and another that has serious immuno- logical side effects. Here we demonstrate that the LUNAR® delivery technology is capable of delivering mRNA for a replacement enzyme, the bacterial phenylalanine ammonia lyase (avPAL), into the hepatic tissue of a PKU mouse, and that the enzyme is capable of metabolizing Phe and reducing serum levels of Phe for more than five days post-transfection. We further demonstrate the ability of LUNAR to deliver a plant-derived PAL protein with a similar impact on the level of serum Phe. Taken together these results demonstrate both the capability of LUNAR for the targeted delivery of PAL mRNA into hepatic tissue in vivo, replacing the defective PAH protein and successfully reducing serum Phe levels, thereby addressing the underlying cause of PKU symptoms. Secondly, that plant-based PAL proteins are a viable alternative to bacterial avPAL to reduce the immunogenic response.

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.

tRNA therapeutics burst onto startup scene

Companies advance tRNA therapeutics to overcome mutant stoppages in protein synthesis shared by thousands of genetic diseases and cancers.

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 Pahenu2 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 Pahenu2 mouse model. Together, these data establish a proof of principle of a novel mRNA replacement therapy to treat PKU.

Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG)-trinucleotide repeats in causative genes. These diseases include spinal and bulbar muscular atrophy (SBMA), Huntington’s disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias. Targeting expanded CAG repeats is a common therapeutic approach to polyQ diseases, but concomitant silencing of genes with normal CAG repeats may lead to toxicity. Previous studies have shown that CAG repeat-targeting small interfering RNA duplexes (CAG-siRNAs) have the potential to selectively suppress mutant proteins in in vitro cell models of polyQ diseases. However, in vivo application of these siRNAs has not yet been investigated. In this study, we demonstrate that an unlocked nucleic acid (UNA)- modified CAG-siRNA shows high selectivity for polyQ expanded androgen receptor (AR) inhibition in in vitro cell models and that lipid nanoparticle (LNP)-mediated delivery of the CAG-siRNA selectively suppresses mutant AR in the central nervous system of an SBMA mouse model. In addition, a subcutaneous injection of the LNP-delivered CAG-siRNA efficiently suppresses mutant AR in the skeletal muscle of the SBMA mouse model. These results support the therapeutic potential of LNP-delivered UNA-modified CAG-siRNAs for selective suppression of mutant proteins in SBMA and other polyQ diseases.

CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.

Property Driven Design and Development of Lipids for Efficient Delivery of siRNA

Ionizable cationic lipids are critical components involved in nanoparticle formulations, which are utilized in delivery platforms for RNA therapeutics. While general criteria regarding lipophilicity and measured pKa in formulation are understood to have impacts on utility in vivo, greater granularity with respect to the impacts of the structure on calculated and measured physicochemical parameters and the subsequent performance of those ionizable cationic lipids in in vivo studies would be beneficial.

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.

Anti-HFRS Human IgG Produced in Transchromosomic Bovines Has Potent Hantavirus Neutralizing Activity and Is Protective in Animal Models

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.

Challenges and Potential Solutions for Development of Successful Potency Assay in mRNA Therapeutics

mRNA Therapeutics require development of potency assays early during the pre-clinical stage. Conventional potency methods for protein biologic may not apply directly to mRNA therapeutic drug substance. Cell-based potency assays have to be carefully evaluated to make sure that the read-outs correspond to the actual potency of the drug substance and not an artifactual value. mRNAs can be evaluated for potency at level of protein expression (cell-free), cellular protein expression/stability and enzyme activity in vitro.

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.

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 samRNA 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.

Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset

RNA vaccines have shown efficacy in preventing coronavirus disease 2019 (COVID-19) as early as 12 days after the first dose. Vaccine efficacy onset presents a unique opportunity to define the necessary elements of immunity against COVID-19. Kalimuddin et al. tracked the serological and T cell responses longitudinally in 20 healthcare workers after the first Pfizer/BioNTech BNT162b2 vaccine dose. Anti-spike immunoglobulin G (IgG) and IgA antibodies and spike-specific T cells were detectable at day 10 after the first dose; neutralizing and receptor-blocking antibodies remained mostly undetectable at this time point. These results suggest that binding antibodies and T cell responses are responsible for early protection against COVID-19 and call for circumspection on the prevailing notion that neutralizing antibodies are absolutely required for protection.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces 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)g 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.

Restoring ornithine transcarbamylase (OTC) activity in an OTC-deficient mouse model using LUNAR-OTC mRNA

Patients of ornithine transcarbamylase (OTC) deficiency lack the OTC activity in the liver. Using an OTC deficiency mouse model, we demonstrated that LUNAR OTC, an OTC mRNA medicine, enables full restoration of OTC activity in mouse liver. Moreover, the OTC activity in the plasma is a surrogate assay for OTC activity in the liver, which can serve as a pharmacodynamic endpoint without the necessity of liver biopsies.

mRNA Therapy For Ornithine Transcarbamylase Deficiency

LUNAR®-OTC treats patients suffering from ornithine transcarbamylse deficiency (OTCD) using mRNA to replace the wild-type human enzyme. OTCD is a rare metabolic disease in which the urea cycle cannot efficiently convert ammonia into urea. The efficacy of LUNAR®-OTC was evaluated in an animal model of OTCD ameliorating certain OTCD disease phenotypes in mice.

NACFC Arcturus Therapeutics Plenary 1 Hope for All Addressing the Needs of Those with Untreated CF Mutations

LUNAR®-hCFTR mRNA (ARCT-032) Restores CFTR Expression & Function in vitro

LUNAR®-mRNA Transduces Epithelium in Presence of CF Mucus

LUNAR®-CF: An mRNA Replacement Approach for Cystic Fibrosis Lung Disease

Arcturus Technology targeting cystic fibrosis lung disease

LUNAR®-CF: An mRNA Replacement Approach for Cystic Fibrosis Lung Disease

Mutation agnostic treatment for CF lung disease

LUNAR®-CF, A Novel mRNA Therapeutic Approach to Correct the Root Cause of Cystic Fibrosis Lung Disease

LUNAR®-CF is an aerosolized mRNA replacement therapy to treat the root cause of Cystic Fibrosis (CF) Lung Disease (CFLD) and is agnostic to genotype. We present
robust preclinical data to support the advancement of LUNAR®-CF as a therapeutic approach for CFLD. A codon-optimized human CFTR mRNA (hCFTR) encapsulated in
LUNAR®, a proprietary lipid nanoparticle, was developed to deliver hCFTR mRNA into airways epithelium. Physicochemical properties of LUNAR® were stable after
aerosolization.

LUNAR®-CF, an aerosolized mRNA replacement Therapy for Cystic Fibrosis Lung Disease

LUNAR®-CF is an aerosolized mRNA replacement therapy to treat Cystic Fibrosis (CF) Lung Disease, a therapeutic approach agnostic to a patient’s genotype. A healthy copy of the human CFTR mRNA is encapsulated into lipid nanoparticles (LUNAR®-hCFTR), aerosolized to patient’s airways using a vibrating mesh nebulizer to directly deliver a de novo human CFTR mRNA into epithelial cells. This human CFTR mRNA encodes a fully functional human CFTR protein that will be beneficial to facilitate mucociliary clearance and improve CF lung disease.

LUNAR® selective delivery of nebulized mRNA into murine lung epithelial cells

LUNAR® lipid nanoparticles carrying the mRNA payload reaches the target cell, where it fuses with the plasma membrane forming an intracellular endosome. This endosomic particle undergoes a pH-mediated disruption that causes the breakdown of the biodegradable nanoparticle and the delivery of the mRNA into the cytoplasm.

LUNAR-CF a mRNA Replacement Therapy for Cystic Fibrosis

Arcturus Therapeutics is a nucleic acid medicines company focused on developing RNA therapeutics to treat rare diseases. Our proprietary LUNAR® lipid-mediated delivery technology enables the efficient delivery of any mRNA into a variety of cell types and tissues, and can be optimized for multiple routes of administration.