Applying CleanCap® mRNA: 4 Example Studies

Applying CleanCap® mRNA: 4 Example Studies
Posted in: Therapeutics

  1. Airway Tissue Remodeling in Chronic Obstructive Pulmonary Disease
  2. Local Gene Delivery to Dermal Fibroblasts for Wound Healing 
  3. mRNA Replacement Therapies for Cystic Fibrosis
  4. Secreted Truncated ACE2 Variants as Decoys for SARS‐CoV‐2


Historically, reporter genes have been delivered as DNA plasmids for transcription into mRNA before translation into a reporter protein. In biological systems, ideal reporter proteins generate highly sensitive signals that are easily measurable. Thus, they are often photoluminescent (e.g., GFP) or chemiluminescent (e.g., luciferase). Photoluminescence (aka fluorescence) occurs when a photon of light is absorbed and then emitted, whereas chemiluminescence occurs when photon emission follows a chemical or enzyme-catalyzed reaction. More recently, direct delivery of mRNA-encoded reporters has become more feasible, providing several advantages over plasmid DNA. In addition, the utilization of TriLink’s readymade mRNAs capped with CleanCap® reagent in reporter gene studies complements and supplements the advantages of mRNA reporters. This Zon blog post will briefly discuss general concepts for mRNA reporter genes, before describing four recent examples of mRNA therapies investigated using off-the-shelf CleanCap mRNA reporters.  

FIGURE 1. Plasmid-based delivery of a reporter protein. Taken from and free to use.

Widely Used Protein Reporters

One of the most widely used protein reporters is the photoluminescent green fluorescent protein (GFP). Originally isolated from jellyfish, scientists Roger Y. Tsien, Osamu Shimomura, and Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry for their discovery and development of GFP as a research tool. Chalfie et al.’s 1994 publication in Science, titled Green Fluorescent Protein as a Marker for Gene Expression, has received nearly 10,000 citations in Google Scholar. Cinelli et al. found that a double-mutant termed enhanced GFP (EGFP) offered higher-intensity emission compared to wild-type GFP. Subsequently, dozens of variations of fluorescent reporter proteins have been developed across the light spectra for distinct reporter assays. 

Luciferase enzymes are commonly used chemiluminescent reporters. As implied by its name, firefly luciferase (FLuc) is based on the light-emitting enzyme responsible for the glow of fireflies. FLuc catalyzes the ATP/O2-dependent conversion of luciferin to light-emitting oxyluciferin. Renilla luciferase (RLuc) is based on an enzyme found in sea pansies, and its biochemical and optical properties are distinct from those of FLuc (Lorenz et al.). Importantly, these differences allow for RLuc to be used to normalize the results of FLuc measurements ratiometrically. This approach is referred to as the FLuc/RLuc dual-luciferase reporter system, and it accounts for biochemical variability in cells.

β-Galactosidase (β-gal or beta-gal) is another common chemiluminescent protein. β-gal is the product of the bacterial LacZ gene, and it catalyzes the conversion of β-galactosides into monosaccharides, which can be detected by the artificial chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, termed X-gal. The enzyme cleaves the glycosidic bond in X-gal, forming galactose and 5-bromo-4-chloro-3-hydroxyindole, which dimerizes and oxidizes to an intense blue-colored product that is both easily detectable and quantifiable. 

Advantages of mRNA-Encoded Reporters

As in vitro transcribed (IVT) mRNA technology advances, mRNA reporter systems are increasingly being used over plasmid DNA. At least in part, this is likely due to the advantages that mRNA systems offer over plasmid DNA systems:

1. Encoding reporters in synthetic IVT mRNA bypasses the need to transport plasmid DNA from the cytoplasm into the nucleus for transcription into mRNA. This type of intracellular transport requires time, and it can be inefficient. Concern for complex kinetics and possible inefficiencies also applies to the need for subsequent export of the resultant mRNA into the cytoplasm, where translation into the reporter protein occurs.

2. IVT enables the synthesis of base-modified mRNA, providing increased stability and enhancing translation efficiency (as discussed in a Zon blog). 

3. IVT mRNA permits co-transcriptional capping to obtain naturally occurring 5’ cap structures, such as Cap 1. Biologically, Cap 1 structures decrease innate immune activation and improve translational efficiency (Vaidyanathan et al.). Co-transcriptional capping with TriLink’s CleanCap® reagents results in higher yields and purer mRNA during synthesis (Henderson et al.).

FIGURE 2. Structure of a 5-methoxyuridine moiety in mRNA. Drawn by Jerry Zon.

TriLink offers all of the above-mentioned mRNA reporters off-the-shelf in microgram to milligram scales, and can also accommodate bulk order requests. These CleanCap® mRNA reporters are available with either natural bases or in uridine-depleted forms. All uridines are replaced with 5-methoxyuridine (Figure 2) to further reduce innate inflammatory responses and increase translational efficiency. The following sections summarize recent (2020-2021) publications that employ one or more of TriLink’s off-the-shelf CleanCap® mRNA reporters.

Four Examples of mRNA Therapies Studied Using CleanCap® mRNA Reporters 
Space limitations necessitate brevity. Readers are encouraged to use the links provided to access the full article for each example.

Modulating Airway Tissue Remodeling in Chronic Obstructive Pulmonary Disease

Encompassing a group of diseases that cause airflow blockage and breathing-related problems, chronic obstructive pulmonary disease (COPD) affects sixteen million Americans.

A 2021 publication by Tam et al. investigated the underlying mechanisms of airway remodeling in COPD. Genome-wide association studies have shown that a gene variant in the Family with sequence similarity 13, member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with COPD. A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor-β1 (TGF-β1, Figure 3). To determine the role of FAM13A in TGF-β1 signaling, FAM13A−/− airway epithelial cells were generated using CRISPR-Cas9, while overexpression of FAM13A was achieved using lipid nanoparticles (LNPs) loaded with a custom Clean Cap® FAM13A mRNA. Uptake efficiency was tested by incorporating a non-exchangeable lipid dye into the LNP. CleanCap® EGFP served as an excellent control for testing transfection efficiency of the developed LNP system in their human airway epithelial cell line.

FIGURE 3. Structure of transforming growth factor-β1 (TGF-β1).

Briefly, wild-type (WT) and FAM13A−/− cells were treated with TGF-β1, followed by gene and/or protein expression analyses. Compared to WT cells, FAM13A−/− cells augmented TGF-β1-induced increase in collagen type 1 (COL1A1) and matrix metalloproteinase 2 (MMP2) expression. This effect was mediated by increased expression of β-catenin encoded by the CTNNB1 gene in FAM13A−/− cells, compared to WT cells after TGF-β1 treatment. FAM13A overexpression (via its LNP- CleanCap® mRNA) offered partial protection from TGF-β1-induced COL1A1 expression. Finally, airway epithelial-specific FAM13A protein expression was shown to be significantly increased in patients with severe COPD compared to control nonsmokers, as well as negatively correlated with lung function. 

In contrast, β-catenin (CTNNB1), previously linked to regulation by FAM13A, is decreased in the airway epithelium of smokers with COPD compared to non-COPD subjects. Together, these data demonstrate that FAM13A may protect from TGF-β1-induced fibrotic response in the airway epithelium due to its ability to sequester CTNNB1 from regulating downstream targets. Importantly, therapeutically increasing FAM13A expression in the airway epithelium of smokers at risk for COPD and those with mild COPD may reduce the extent of airway tissue remodeling.

Local Gene Delivery to Dermal Fibroblasts for Wound Healing

Over 6 million people in the US suffer from chronic wounds, typically due to underlying conditions like obesity, diabetes, or ischemia (Duran-Mota et al.). In 2014, wound care products accounted for $2.8 billion of the global healthcare budget, and by 2024, the advanced wound care market for surgical wounds and chronic ulcers is expected to exceed $22 billion. 

Current treatments involving antibiotic dressings and removal of damaged tissue are not only often ineffective, but also cause severe pain, emotional distress, and social isolation in patients for years or even decades, ultimately resulting in limb amputation. Additionally, drug release is not sustained to match the needed therapeutic window. An mRNA-based therapy is a viable alternative, as it promotes wound healing through modulation of local gene expression. However, protecting the mRNA therapy from degradation and maintaining efficient mRNA transfection into primary cells pose significant challenges to clinical translation. 

In their 2021 publication, Duran-Mota et al. describe a potential delivery method for mRNA therapies to address chronic wounds. In this paper, the authors developed an injectable, biodegradable, and biocompatible hydrogel-based wound dressing that delivers poly (β-amino ester) (pBAE) nanoparticles in a sustained manner over a range of therapeutic windows (Figure 4). They demonstrated that pBAE nanoparticles, successfully used as carriers in previous in vivo studies, protect mRNA cargo and permit efficient transfection into human dermal fibroblasts upon sustained release from the hydrogel wound dressing. 

FIGURE 4. Schematic diagram of a composite hydrogel wound dressing.  Designed for applications involving human dermal fibroblasts transfection, based on a pBAE-PEG injectable hydrogel doped with mRNA-loaded polyplexes. Taken from Duran-Mota et al. in and free to use.

In this investigation, CleanCap® EGFP mRNA (5moU) and CleanCap® FLuc mRNA (5moU) were used to evaluate the performance of pBAE nanoparticles. Briefly, human dermal fibroblasts were seeded and incubated to 80-90 % confluence prior to performing the transfection experiments using three different compositions of pBAE nanoparticles loaded with these 5moU-modified mRNA reporters. After 24 hours of incubation, cells were imaged for EGFP using a fluorescence microscope. Only one of the three pBAE formulations, termed C6RH, led to qualitatively brighter green fluorescence compared to jetMESSENGER®, a commercially available mRNA transfection agent that served as a positive control and performance benchmark. This improved performance was confirmed and quantified by measurements using flow cytometry, which gave transfection efficiencies of ~80% for C6RH and ~50% for the positive control. Importantly, cell viability after 24 hours was ~100% for C6RH vs. ~0% for the positive control. To determine luciferase protein expression after 24 hours, FLuc activity was measured with a luminometer as Relative Light Units (RLU), which yielded ~1 x 107 RLU for C6RH and ~5 x 105 RLU for the positive control. It was therefore concluded that this prototype wound dressing technology could enable the development of novel gene therapies to treat chronic wounds.

Development of mRNA Therapeutics for Cystic Fibrosis

Cystic fibrosis (CF) is a frequently occurring genetic disorder that primarily affects the lungs, pancreas, liver, kidneys, and intestine. Long-term issues include loss of lung function and shortened life expectancy. More than 30,000 people live with CF in the US, and more than 70,000 live with it worldwide (CF Foundation). 

CF is caused by the loss of epithelial cell chloride transport due to mutations in the CF transmembrane conductance regulator (CFTR) gene that encodes the CFTR protein. In people with CF, the loss of chloride transport due to defects in the CFTR protein results in the accumulation of thick, sticky mucus in the bronchi of the lungs, as well as loss of exocrine pancreatic function, impaired intestinal absorption, reproductive dysfunction, and elevated sweat-chloride concentration. Although tremendous advances have been made with small-molecule treatments for certain CF mutations, there are still no targeted therapies for mutations that cause little or no protein expression. 

A poster presentation in 2021 investigated the co-delivery of CFTR mRNA and lumacaftor (Maeshima et al., Figure 5). This small molecule acts as a protein-folding chaperone to correct folding of the most common CFTR mutant (ΔF508), thereby increasing the number of functional CFTR proteins trafficked to the cell surface (reviewed elsewhere). 

FIGURE 5. Structure of lumacaftor. Drawn by Jerry Zon.

Obtained from TriLink as a custom product, CleanCap CFTR IVT mRNA was delivered into human CF bronchial epithelial BMI-1 cells (ΔF508 mutation) at the air-liquid interface. Three different cationic liposomal formulations, each with five different cationic receptor-targeting peptides, were compared. The best performing formulation was then used to deliver CleanCap® FLuc mRNA (5moU) to mouse lungs and found to be 2.5-times more effective than a control. Further work to assess the delivery, expression, and function of CFTR IVT mRNA in mouse lungs using this lead formulation is in progress. These experiments form the basis of mRNA replacement therapies that could be revolutionary for CF patients with little or no CFTR protein expression.

Secreted Truncated ACE2 Variants as Decoys for SARS‐CoV‐2

Prophylactic mRNA COVID-19 vaccines, notably those developed by Pfizer-BioNTech and Moderna, utilize lipid nanoparticle formulations (LNPs) for delivery. Despite the successful utilization of LNPs to deliver mRNA, some of the challenges and potential improvements to be addressed in new formulations include (i.) finding less complex/cheaper lipids or lipid-like (aka lipoid) compounds, (ii.) finding a safer replacement for polyethylene glycol (PEG), which can be immunogenic, (iii.) developing formulations compatible with freeze-drying for long-term storage at room temperature or in a refrigerator, and (iv.) developing formulations to target specific cells or tissues. 

To investigate improving delivery, a 2021 publication by Li et al. reported the construction and multi-round optimization of a new combinatorial library of symmetric lipid-like compounds. The lead compound (Figure 6) was prepared into “lipid-like nano-assemblies” (LLNs) for intracellular delivery of mRNA. The authors utilized CleanCap mRNAs to test their novel LLN system.

FIGURE 6. Structure of tB-UC18. This tri-substituted benzene derivative has symmetrically positioned amino groups (X) bearing a cis-C18 lipid. Drawn by Jerry Zon.

Briefly, 293T or HeLa cells were transfected with various compositions of LLNs, each containing 200 ng or other doses of CleanCap® β-gal mRNA. The same doses of mRNA were complexed with Lipofectamine 2000 as a comparison for normalization of β-gal activity-% measured at various time points up to 48 hours, following cell lysis and colorimetric assay using O-nitrophenyl-β-D-galactopyranoside as a substrate. In addition, cells were fixed and rinsed before adding X-Gal for in situ staining and imaging, the results of which qualitatively matched those of the colorimetric assays. CleanCap® β-gal mRNA in LLNs exhibited more than three orders of magnitude higher resistance to degradation by serum than the resistance demonstrated by unprotected mRNA. Additionally, a single intravenous injection of LLNs into mice achieved efficient luciferase mRNA translation into the spleen 4 hours post-injection, without causing significant hematological and histological changes. 

FIGURE 7. 3D illustration of SARS-CoV-2 viruses binding to ACE2 receptors on a human cell. This binding is the initial stage of COVID-19 infection.

The researchers then applied their LLN system to study a potential COVID-19 therapeutic strategy. ACE2 is the receptor through which the SARS-CoV-2 virus enters human cells. ACE2 decoys can bind to the virus and block the binding of SARS-CoV-2 to the human ACE2 receptor (Figure 7), thus preventing viral entry and subsequent infection. Li et al. showed that LLN-mediated delivery of IVT mRNA of high-affinity truncated ACE2 variants (termed tACE2v) induced elevated expression and secretion of tACE2v decoys in 293T cells. This robust neutralization activity in vitro suggests that intracellular delivery of IVT mRNA encoding ACE2 receptor-mimics via LLNs may represent a novel intervention strategy for COVID-19.

Concluding Comments

Every application of IVT mRNAs requires intracellular delivery and translation into the encoded protein of interest. The examples discussed here demonstrate the utility of mRNA reporters to confirm delivery and translation in various model systems. Readily available, highly effective CleanCap® mRNA reporters greatly facilitate the design, implementation, and assessment of mRNA in your system. Initially optimizing protein expression through reporter mRNAs can save tremendous time, energy, and money. Additionally, when you want to test your mRNA of interest, TriLink fulfills your needs by producing a custom CleanCap mRNA. At the risk of restating this experimental strategy too simply, the familiar adage “don’t be penny wise and pound foolish” comes to mind.

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