Cystic Fibrosis Publications

LUNAR® LNP delivery of CFTR mRNA restores channel function and improves mucociliary clearance in human and ferret cystic fibrosis airways

Lipid nanoparticle (LNP)-based delivery of CFTR-mRNA holds promise for treating pulmonary manifestations of cystic fibrosis (CF). LUNAR®-CFTR is a novel proprietary lipid nanoparticle (LNP) encapsulating codon-optimized human CFTR mRNA for CFTR replacement therapy. In this study, we examined the potential of LUNAR®-CFTR for delivering mRNA and restoring CFTR function in primary human and ferret airway epithelial cells and mucus-laden CF ferret airways. LUNAR® effectively delivered CFTR, Cre, and tdTomato mRNAs into multiple airway epithelial cell types of human and ferret in vitro and transgenic ferrets in vivo. The LUNAR®-CFTR was able to restore ion transport in polarized CF human bronchial epithelia to levels comparable to those achieved with elexacaftor/tezacaftor/ivacaftor. Mucociliary clearance (MCC) assays in CF ferrets further showed that a single dose of LUNAR®-mediated human CFTR mRNA delivery improved mean MCC values by 3-fold. The improved MCC was in line with an increase in CFTR-mediated transepithelial anion transport from the CF ferret trachea transfected with LUNAR®-CFTR in vivo. These findings underscore the potential of LUNAR® LNP as a robust vehicle for delivering CFTR-encoding therapeutic mRNA into airway epithelial cells and underscore the utility of CF ferrets as a reliable tool to evaluate the efficiency of gene therapy in mucus-laden airways.

The Safety and Tolerability of Inhaled LUNAR®-CFTR mRNA (ARCT-032) Demonstrated in a Phase 1 Study

LUNAR-CFTR was generally safe and well tolerated.
Post-dose mild respiratory symptoms and FEV1 decline was mitigated by albuterol pretreatment.
The observed fever (objective or subjective) and nonspecific symptoms within a few hours of dosing were also reported with higher doses in a previous. mRNA-LNP CF program (Rowe 2023). In those studies, most of those AEs were considered moderate in severity; in this Phase 1 study they were mild.
Encouraging Phase 1 safety results inform future studies (e.g., use doses below where most AEs were observed) and support clinical advancement of LUNAR-CFTR.
The FDA has issued a ‘study may proceed’ letter for a Phase 2, multicenter, multiple-ascending-dose study of LUNAR-CFTR targeting pwCF who are not on HEMT. The study is planned for Q4 2024.

LUNAR® -CFTR mRNA replacement therapy restores CFTR expression and function in human bronchial epithelial cells

Presentation:
LUNAR®-CFTR successfully transfected and expressed a functional CFTR protein in dF508 and G542X/R1162X null HBE cells in a dose dependent manner.
In presence of a Cl- gradient, LUNAR-CFTR treatment restored chloride currents in CF cells to levels approximating those in wild type cells.
In absence of a Cl- gradient, LUNAR-CFTR induced Cl- current at 60% of WT current levels but was comparable to those corrected by ETI, the clinically approved CFTR modulator for CF patients.
Repeated dosing of 5 mg of LUNAR-CFTR administered at half-life time intervals (3 days) achieved Cl- currents comparable to a single 20 mg dose and comparable to ETI induced currents, indicating beneficial effects of dose accumulation.
LUNAR®-CFTR is currently being studied in a Phase 1 clinical trial in healthy volunteers and adults with CF.

LUNAR® -CFTR mRNA replacement therapy restores CFTR expression and function in human bronchial epithelial cells

Poster:
LUNAR®-CFTR successfully transfected and expressed a functional CFTR protein in F508del and G542X/R1162X null HBE cells in a dose dependent manner.
In presence of a Cl- gradient, LUNAR-CFTR treatment restored chloride currents in CF cells to levels approximating those in wild type cells.
In absence of a Cl- gradient, LUNAR-CFTR induced Cl- current at ~60% of wild type current levels but was comparable to those corrected by ETI, the clinically approved CFTR modulator for CF patients.
Repeated dosing of 5 μg of LUNAR-CF administered at half-life time intervals (3 days) achieved Cl- currents comparable to a single 20 μg dose and comparable to ETI induced currents, indicating beneficial effects of dose accumulation.
LUNAR®-CFTR is currently being studied in a Phase 1 clinical study in healthy volunteers and adults with CF.

ARCT-032 (LUNAR®-CFTR) Improves Mucociliary Clearance in CF Ferrets

LUNAR® platform demonstrates efficient delivery of mRNA payloads and transfection in various cell types in ferret airways.
ARCT-032 (LUNAR®-CFTR) improved CFTR activity in CF ferret model as demonstrated by significantly improved MCC.
These in vivo results complement finings from in vitro studies in human bronchial epithelial cells (HBEs) derived from CF patients where ARCT-032 restored CFTR-mediated chloride conductance.
ARCT-032 is currently being studied in a Phase 1 trial in healthy volunteers and adults with CF.

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.

Self-amplifying COVID-19 mRNA vaccination induces longitudinally enhanced antibody function in a Phase 3 trial

Sustained and functional antibody responses to respiratory pathogens through vaccination is critical for global public health. The development and deployment of mRNA vaccines during the coronavirus disease 2019 (COVID-19) pandemic was a landmark achievement in modern medicine and ushered in a new age of vaccine innovation. The mRNA-based vaccines elicited strong antibody responses, both neutralizing and extra-neutralizing, against the viral Spike protein. The antibody levels waned with time since vaccination, and that coupled with the antigenic drift of the virus prompted updates to the mRNA vaccine composition and evaluation of other mRNA modalities. Self-amplifying mRNA (sa-mRNA) vaccines such as ARCT-154 can prolong antigen production and durability of humoral immune response post-immunization, and can thus be administered at a lower dose. How this translates into the overall humoral architecture compared to that shaped by conventional mRNA vaccinations, however, is unclear. Here, we analyze serum-based antibody responses in a subset of participants from a recent Phase III trial comparing neutralizing immune responses elicited by ARCT-154 and mRNA BNT162B2 as a post-hoc research. All participants had received three doses of mRNA COVID-19 vaccines and were randomized to receive a booster dose of ARCT-154 or BNT162B2. Primary outcomes of this research were to quantify waning responses against ancestral/wild type SARS-CoV-2 Spike (WT Spike) and a panel of diverse SARS-CoV-2 variant Spikes. Through a systems serology approach, we identified that the sa-mRNA vaccine ARCT-154 elicited a unique antibody response compared to BNT162B2 defined by a sustained, activating profile to the vaccine-encoded Spike protein and a broad spectrum of drifted Spikes. Notably, potently activating FcγRIIIA-binding antibodies showed a sustained stimulation in the ARCT-154-treatment arm, and this translated to an enhanced natural killer (NK) cell activation. The NK-activation through ARCT-154 was present for both target WT Spike and the antigenically drifted BA.5 Spike, which was the predominant form of SARS-CoV-2 during the observation period. Our results support a model whereby prolonged antigen expression and presentation moves immune profiles towards activating phenotypes with broad antigenic coverage.

Immunogenicity and safety of self-amplifying mRNA COVID-19 vaccine (ARCT-2303), with or without co-administration of seasonal inactivated influenza vaccine in adults: a phase 3, randomised, controlled, observer-blind, multicentre study

A recently licenced self-amplifying mRNA (sa-mRNA) COVID-19 vaccine induces a robust, broad, and long-lasting immune response, extending the arsenal of efficacious COVID-19 countermeasures. We ran a clinical study to assess the benefits of vaccine strain update and the feasibility of co-administration with influenza vaccines.

Immunogenicity of ARCT-154, a self-amplifying mRNA COVID-19 vaccine, in different booster settings

A self-amplifying mRNA (sa-mRNA) vaccine against COVID-19 (ARCT-154) was evaluated for immunogenicity in three clinical studies with four booster scenarios: first homologous booster, first heterologous booster, second heterologous booster, and a two-dose series in previously infected participants. Neutralizing antibody responses were measured four weeks after vaccination using pseudovirus microneutralization assays against the Wuhan-Hu-1 and SARS-CoV-2 variants. ARCT-154, administered as a homologous booster, resulted in a geometric mean fold-rise (GMFR) of 6.5 (95 % confidence interval, 5.6–7.5) and seroconversion rate (SCR) of 77 % (69.8–83.2), compared with 1.4 (0.9–2.2) and 17.3 % (8.2–30.3) with placebo. A first heterologous booster of ARCT-154 achieved a GMFR of 36.7 (17.4–77.5) and SCR of 91.7 % (61.5–99.8) against Wuhan-Hu-1, with
GMFRs ranging from 20.0 to 29.4 for Beta, Delta, and Omicron BA.1 variants. When comparing ARCT-154 and BNT162b2 mRNA vaccine as a second booster dose, GMFRs against Wuhan-Hu-1 were 6.8 (6.0–7.6) and 4.4 (4.0–4.8), and SCRs were 66.1 % (61.1–70.9) and 51.2 % (46.0–56.4), respectively. Similar inter-group differences were shown for the Omicron BA.4/5 and persisted for ≥12 months. Following natural infection, one dose and two doses of ARCT-154 resulted in GMFR of 4.4 (2.2–7.0) and 6.2 (3.8–10.0), and SCR of 58.8 % (32.9–81.6) and 73.3 % (44.9–92.2) against Wuhan-Hu-1, respectively. Neutralizing antibodies remained elevated for at least 6 months. These results confirm that ARCT-154, administered as a homologous or heterologous booster after previous COVID-19 vaccination or natural exposure, provides robust, broad, and durable immune responses against SARS-CoV-2 viruses.

A second-generation, self-amplifying COVID-19 Vaccine: World’s first approval and distribution in the Japanese market with vaccine hesitancy

The mRNA vaccine is a milestone in immunotherapeutics, as symbolized by the 2023 Nobel Prize for Physiology or Medicine awarded to Drs. Karikó and Weissman. Whereas the conventional, “first-generation” mRNA vaccine was globally distributed to hundreds of millions of people to decrease COVID-19 prevalence, further advanced constructs have been pursued by researchers and pharmaceutical manufacturers. The key feature of the “second-generation” mRNA vaccine is a self-amplifying replicon that may allow a low dose to ensure durable immunogenicity. In clinical trials, ARCT-154 indeed showed effectiveness (magnitude, persistence, and breadth) superior to conventional mRNA vaccines, with similar or less frequent adverse responses, and acquired its world’s first approval in November 2023 in Japan (brand name: KOSTAIVE manufactured by Meiji Seika Pharma, Tokyo, Japan) to prevent COVID-19 infection. Real-world distribution of KOSTAIVE was started in October 2024, and researchers are collecting data on its effectiveness and safety despite nonscientific, but persistent, antivaccine skepticism.

Safety profile of self-amplifying mRNA SARS-CoV-2 vaccine ARCT-154 in adults: a pooled phase 1/2/3 randomized clinical study

Public health concerns due to ongoing emergence of SARS-CoV-2 variants necessitates further development of improved COVID-19 vaccines. One major innovation are self-amplifying mRNA vaccines such as ARCT-154 (Arcturus Therapeutics Inc.) which induces superior immunogenicity compared with conventional mRNA in terms of magnitude, breadth and persistence of neutralizing antibodies

Differential clearance rate of proteins encoded on a self-amplifying mRNA COVID-19 vaccine in muscle and lymph nodes

ARCT-154, a recently approved self-amplifying mRNA (saRNA) vaccine for SARS-CoV-2, has shown superior induction and prolonged maintenance of neutralizing antibodies compared to the conventional mRNA vaccine BNT162b2. However, the scientific evidence explaining this superiority remained elusive. Hence, we explored the temporal changes in spike protein and replicase components following a single dose of ARCT-154 vaccination in mice. The encoded spike protein reached its highest level approximately 3 days after vaccination and quickly disappeared from the rectus femoris muscle, the injection site. Although the spike protein levels also peaked at an early time point in the lymph nodes, it remained detectable 28 days after the vaccination and then disappeared by 44 days after the vaccination. Expression of nsP1, nsP2 and nsP4 was observed in the injected muscle and/or the lymph nodes for up to 15 days post-vaccination. Data were analyzed using unpaired two-tailed Mann–Whitney U-tests. These data suggest that prolonged expression of spike proteins in lymph nodes may, if not entirely, be responsible for the induction of higher and prolonged levels of neutralizing antibodies by the saRNA vaccine.

A randomized trial comparing safety, immunogenicity and efficacy of self-amplifying mRNA and adenovirus-vector COVID-19 vaccines

This phase 3 trial compared safety, tolerability, immunogenicity and efficacy of the self-amplifying mRNA COVID-19 vaccine, ARCT-154, with ChAdOx1-S adenovirus-vector vaccine. In four centers in Vietnam adult participants aged 18‒85 years were randomly assigned to receive two doses, 28 days apart, of either ARCT-154 (n = 1186) or ChAdOx1-S (n = 1180). Both vaccines were well tolerated with similar safety and reactogenicity profiles consisting of mainly mild-to-moderate solicited adverse events and few related serious adverse events. Higher neutralizing antibody responses persisting to one-year post-vaccination after ARCT-154 compared with ChAdOx1-S were associated with a generally higher efficacy against COVID-19. In an exploratory analysis relative vaccine efficacy of ARCT-154 vs. ChAdOx1-S against any COVID-19 from Day 36 to Day 394 was 19.8% (95% CI: 4.0–33.0). Self-amplifying mRNA vaccine offers potential immunological advantages in terms of immunogenicity and efficacy over adenovirus-vector vaccine without compromising safety.

Immunogenicity of a booster dose of a bivalent (Asp614Glyand omicron BA.4/5 variant) self-amplifying mRNAnSARS-CoV-2 booster vaccine versus the BNT162b2 omicronBA.4/5 mRNA vaccine: a randomised phase 3 trial

We previously showed that ARCT-154, a self-amplifying mRNA COVID-19 vaccine, had improved immunogenicity and antibody persistence compared with conventional mRNA or adenovirus vector vaccines. In this study, we compared ARCT-2301, a bivalent self-amplifying mRNA vaccine (Asp614Gly and omicron BA.4/5 variant), with the bivalent Comirnaty omicron BA.4-5 vaccine, to determine whether this improved response persisted in bivalent formulations against different SARS-CoV-2 variants.

Self-amplifying mRNA COVID-19 vaccine induces a superior immune response compared with mRNA vaccine that persists to 12 months postvaccination.

In fully immunized adults the superiority of neutralizing antibody responses against ancestor SARS-CoV-2 following a booster dose of ARCT-154 compared with BNT162b2 persists through one year and is also observed against all tested variants, supporting further development of sa-mRNA vaccines.

Comparison of the immunogenicity of bivalent self-amplifying mRNA and mRNA vaccines against various SARS-CoV-2 variants up to six months postvaccination.

As with monovalent vaccine, the bivalent sa-mRNA formulation demonstrates superior immunogenicity over conventional bivalent mRNA vaccine, a higher immune response persisting up to six months after a booster dose, and improved breadth, supporting the robustness of the sa-mRNA vaccine platform for future vaccine strain updates.

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.

Persistence of immune responses of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2

New COVID-19 sa-mRNA Results from CSL and Arcturus Therapeutics Demonstrate Longer Duration of Immunity Compared to Conventional COVID-19 mRNA Vaccine Booster

Immunogenicity and safety of a booster dose of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2 mRNA COVID-19 vaccine: a double-blind, multicentre, randomised, controlled, phase 3, noninferiority trial

Licensed mRNA COVID-19 vaccines require booster doses to sustain SARS-CoV-2-specific responses, creating the need for novel, broadly immunogenic vaccines. We aimed to compare the immunogenicity, safety, and tolerability of ARCT-154—a self-amplifying mRNA vaccine against SARS-CoV-2 D614G variant—with the BNT162b2 (Comirnaty; Pfizer–BioNTech) mRNA vaccine when administered as a fourth-dose booster.

Self-Amplifying mRNA SARS-COV-2 vaccines elicit robust, cross-reactive and durable neutralizing booster responses in previously mRNA vaccinated adults

• This study provides evidence that a booster dose of sa-mRNA vaccine induces a robust, broadly cross-reactive, and durable neutralizing immune response, that persists through 12 months post-vaccination.
• Favorable safety and reactogenicity was observed for all three sa-mRNA vaccines.

Booster dose of self-amplifying SARS-CoV-2 RNA vaccine vs. mRNA vaccine: a phase 3

Licensed mRNA vaccines demonstrated initial effectiveness against COVID-19 but require booster doses to broaden the anti-SARS-CoV-2 response. There is an unmet need for novel highly immunogenic and broadly protective vaccines. We compared immunogenicity and tolerability of ARCT-154, a novel self-amplifying mRNA vaccine with the mRNA vaccine, Comirnaty. Methods We compared immune responses to ARCT-154 and Comirnaty booster doses in healthy 18-77-year-old Japanese adults initially immunised with two doses of mRNA COVID-19 vaccine (Comirnaty or Spikevax) then a third dose of Comirnaty at least 3 months previously. Neutralising antibodies were measured before and 28 days after booster vaccination. The primary objective was to demonstrate non-inferiority of the immune response against Wuhan-Hu-1 SARS-CoV-2 virus as geometric mean titre (GMT) ratios and seroresponse rates (SRR) of neutralising antibodies; key secondary endpoints included the immune response against the Omicron BA.4/5 variant and vaccine tolerability assessed using participant-completed electronic diaries.

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.

Impact of ARCT-810 mRNA Therapy on Dietary Intake and Biochemical Data in Adolescents and Adults with Ornithine Transcarbamylase Deficiency

• This poster presents preliminary data. Evaluation of the safety and efficacy of ARCT-810 is ongoing.
• ARCT-810 was generally safe and well tolerated throughout the study period.
• After ARCT-810 administration, all participants exhibited weight gain with increases in protein intake which may suggest improved dietary protein tolerance.
• The observed decreases in mean levels of first morning fasting plasma ammonia and glutamine despite increased dietary protein intake suggests possible improvements in urea cycle flux following ARCT-810 administration.

Interim Results of Phase 2 Trial of ARCT-810 mRNA Therapy in Patients with Ornithine Transcarbamylase Deficiency

ARCT-810 is an investigational mRNA treatment intended to treat ornithine
transcarbamylase deficiency (OTCD).
In the spf/ash mouse model of OTCD following challenge with a high protein
diet, single and multiple doses of ARCT-810 demonstrated improvement in
key ureagenesis biomarkers and increased overall survival1.
Single doses of ARCT-810 in healthy volunteers and OTCD patients were
safe and well tolerated.
Rapid plasma clearance of lipid (approximately 48 hours) with extended
plasma mRNA exposure (approximately 2 to 4 weeks)
A Phase 2 EU study (NCT05526066) was previously conducted in 8
patients randomized 3:1 (ARCT-810: placebo) at 0.3 mg/kg IV q2w dosing
for up to 6 doses.
Stable adult participants with mild OTC deficiency were enrolled and
required to maintain stable dietary protein intake. While biomarker
assessments were limited, encouraging trends were observed in ammonia
and glutamine levels following study drug administration.
An additional dose ranging study was initiated to further characterize
biomarker response and establish therapeutic effect.

Restoration of Urea Cycle Function in OTC Deficiency ARCT-810 mRNA Therapy Across Two Clinical Studies

Treatment with the mRNA therapy resulted in a mean reduction from baseline in glutamine over the treatment period across two independent Phase 2 studies.
Exploratory (post hoc) analyses indicate that the observed decreasing trend in glutamine is not random.
Based on inspection of available data, the degree of glutamine reduction appears to be proportional to baseline control and severity of the individual patient.
These data represent preliminary biomarker evidence of mRNA-based enzyme replacement following ARCT-810 administration in a monogenic metabolic disease.

Interim Results Following Multiple Doses of ARCT-810

ARCT-810 is an investigational mRNA treatment for ornithine transcarbamylase deficiency (OTCD) formulated with a proprietary lipid nanoparticle (LNP), including an ionizable lipid to target mRNA to the liver. The mRNA is then translated into functional OTC enzyme.
• In the spf/ash mouse model of OTCD following challenge with a high protein diet, single and multiple doses of ARCT-810 demonstrated improvement in biomarkers of ureagenesis and increased survival1.
• Single doses of ARCT-810 in healthy volunteers and OTCD patients were safe and well tolerated with rapid clearance of lipid
(approximately 48 hours) and longer plasma mRNA exposure (approximately 2 to 4 weeks).
• A Phase 2 program including EU and US studies was initiated to evaluate safety, pharmacodynamic, and pharmacokinetics of multiple doses (up to 6) of ARCT-810.

Characterization and treatment monitoring of ureagenesis disorders using stable isotope

Urea cycle disorders (UCDs) are a group of rare conditions, possibly life-threatening and without definitive cure besides liver transplantation. Traditional biochemical analyses/biomarkers cannot reliably determine changes in the UC-function from baseline to post-intervention. We describe a UHPLC-HRMS method to assess ureagenesis in plasma and dried blood spots for [15N]urea and [15N]amino acids, using [15N]ammonium chloride as tracer. [15N]enrichment of urea and amino acids was studied in controls (n = 22) and patients (n = 59), the latter showing characteristic ureagenesis variations according to their underlying metabolic defect. Follow-up of therapies was successful, as we observed restoration of [15N]urea production and lowering of [15N]glutamine. There were no adverse events, and only minimal amounts of tracer and samples required with a short sample preparation time and analysis. Thus, the method proved to be safe and efficient to monitor UCD patients of variable severity pre- and post-therapy, being suitable as physiological endpoint for development of therapies.

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.

Lipid Nanoparticle Delivery of TALEN mRNA Targeting LPA Causes Gene Disruption and Plasma Lipoprotein(a) Reduction in Transgenic Mice

Lipoprotein(a), or Lp(a), is encoded by the LPA gene and is a causal genetic risk factor for cardiovascular disease. Individuals with high Lp(a) are at risk for cardiovascular morbidity and are refractory to standard lipid-lowering agents. Lp(a)-lowering therapies currently in clinical development require repetitive dosing, while a gene editing approach presents an opportunity for a single-dose treatment. In this study, mRNAs encoding Transcription Activator-Like Effector Nucleases (TALENs) were designed to target human LPA for gene disruption and permanent Lp(a) reduction. TALEN mRNAs were screened in vitro and found to cause on-target gene editing and target protein reduction with minimal off-target editing. TALEN mRNAs were then encapsulated with LUNAR®, a proprietary lipid nanoparticle (LNP), and administered to transgenic mice that expressed a human LPA transgene. A single dose of TALEN mRNA-LNPs reduced plasma Lp(a) levels in mice by over 80%, which was sustained for at least 5 weeks. Moreover, both standard and long-read next generation sequencing confirmed the presence of gene-inactivating deletions at LPA transgene loci. Overall, this study serves as a proof-of-concept for using TALEN-mediated gene editing to disrupt LPA in vivo, paving the way for the development of a feasible gene editing therapy for patients with high Lp(a).

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing TatmRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with down regulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells’ transcriptome and proteome profiles.

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.