Streamlined mRNA Capping Strategies for Enhanced Therapeutic Potential

trilinkbiotech.com | 1 Technical note Impact of cap structures on the performance of in vitro– transcribed mRNAs Exploring strategies for optimal capping to maximize mRNA potency TriLink BioTechnologies, part of Maravai LifeSciences Introduction Capping is a crucial modification of in vitro–transcribed mRNAs, enhancing their stability, facilitating translation initiation, and enabling self/non–self-discrimination in therapeutic applications. At its most basic level, capping involves the addition of a 7-methylguanosine (m7G) to the 5′ end of the mRNA, generating a Cap-0 structure which is often recognized as non-self in higher eukaryotes. The first transcribed base can also be methylated at the 2′ ribose position to generate the more common Cap-1 structure found on endogneous mRNAs in higher eukaryotes. mRNA capping can occur post-transcriptionally through the action of specific enzymes (enzymatic capping) or co-transcriptionally, as exemplified by TriLink's CleanCap® technology. Enzymatic capping takes place after in vitro transcription (IVT) and typically uses Vaccinia Capping Enzyme (VCE) to add m7G to the 5′ end followed by a separate 2′-O-methyltransferase enzyme to modify the first transcribed base and convert Cap 0 to Cap 1. It often requires purification after IVT, increasing processing time and potential yield loss. Co-transcriptional capping, in contrast, is a one-step process that adds the m7G cap to the nascent mRNA transcript as it is being synthesized by RNA polymerase. Early versions of co-transctriptional capping such as antireverse cap analog (ARCA) utilize a dinucleotide cap with reduced capping efficiencies around 70% and generate a Cap 0, which can result in reduced potency and unwanted innate immunogenicity. This has been improved with CleanCap technology in which a trinucleotide cap analog such as m7GpppAmpG is added to initiate and cap mRNAs during synthesis. Compared to ARCA, CleanCap technology revolutionizes mRNA synthesis by creating a Cap-1 structure in a single-step reaction with ≥95% efficiency while improving mRNA translation in both in vitro and in vivo settings1,2. In this tech note, we evaluate the quality attributes and protein expression of mRNAs synthesized using CleanCap® AG (original analog), CleanCap® AG 3′ OMe (upgraded analog), and CleanCap® M6 (latest analog). Additionally, we compare the efficacy of CleanCap M6 with the enzymatic method. Our findings provide insights into the therapeutic potential of mRNAs capped with CleanCap M6. Evaluation of mRNA quality attributes with enzymatic capping and CleanCap technology Enzymatic capping has traditionally been used to modify synthetic RNA. However, CleanCap technology, introduced in 2017, addresses challenges associated with enzymatic capping, such as extended processing times, potential for low yield, and higher costs3. CleanCap technology enables co-transcriptional capping while allowing modifications to the cap's chemical structure for enhanced functionality. For instance, 3′-O-methylation of the m7G ribose facilitates proper cap incorporation4, while N6-methylated adenosine can increase protein expression at least in part by inhibiting decapping5. Following the development of CleanCap AG, we subsequently introduced CleanCap AG 3′ OMe and, more recently, CleanCap M6 (Figure 1). In our latest peer-reviewed publication6, we have shown that CleanCap M6 increases mRNA stability, at least in part by impairing Dcp2-mediated decapping. P O OH O O O HO OMe N HN N N O H2N P O O O O O P O OH O O P OH O O HO OH N NH N N O NH2 N N N N NH2 OMe P O OH O O O HO OMe N HN N N O H2N P O O O O O P O OH O O P OH O O HO OH N NH N N O NH2 N N N N HN OMe Me P O OH O O O HO OH N HN N N O H2N P O O O O O P O OH O O P OH O O HO OH N NH N N O NH2 N N N N NH2 OMe trilinkbiotech.com | 2 Figure 1. Structures of CleanCap cap analogs for mRNA. CleanCap AG has a 2′-O-methyl modification on the +1A which confers the Cap-1 structure. CleanCap AG 3′ OMe additionally features a 3′-O-methyl modification on the m7G. CleanCap M6 includes an m6A modification on the +1A of CleanCap AG 3′ OMe. CleanCap® AG CleanCap® AG 3′ OMe CleanCap® M6 A A A mRNA extension mRNA extension mRNA extension +1 2′ OMe +1 2′ OMe +1 2′ OMe m7G 3′ OMe m7G 3′ OMe m6A G G G m7G m7G m7G Before investigating their functionality, we first compared the quality attributes of mRNAs capped using different analogs. We examined full-length integrity, capping efficiency, and double-stranded RNA (dsRNA) levels, and found comparable mRNA quality across all three cap analogs (Table 1). Following their standard protocols, the enzymatic and CleanCap capping typically yield approximately 5 mg/mL of crude mRNA. However, with CleanCap analog’s optimized protocols — such as our proprietary CleanScript® IVT method using CleanCap AG or CleanCap AG 3′ OMe7 , or the pulse-feed protocol with CleanCap M68 — we can achieve mRNA yields as high as 10 mg/mL. Table 1. Comparing mRNA quality attributes across different capping methods. Firefly luciferase (FLuc) mRNAs were synthesized by IVT, capped using different analogs, and incorporated with N1-methylpseudouridines in place of uridines. Their mRNA integrity was analyzed by IP-RP-HPLC, capping efficiencies were determined by LC-MS, and dsRNA levels were assessed by J2 immunoblot. Capping method (Cap 1) Full-length integrity Capping efficiency dsRNA (J2 dotblot) Enzymatic 92.8% 99% <10 ng/µg CleanCap AG 93.3% 97% <5 ng/µg CleanCap AG 3′OMe 94% 96% <5 ng/µg CleanCap M6 97% 97% <5 ng/µg Assessment of protein expression from mRNAs with three CleanCap analogs To investigate the influence of 5′ cap structures on protein expression, we compared mWasabi mRNAs with CleanCap AG 3′ OMe or CleanCap M6 in vitro. We selected CleanCap AG 3′ OMe for the comparison with CleanCap M6 since we had previously shown CleanCap AG 3′ OMe performed better than ARCA and CleanCap AG2. We transfected the mRNAs into HeLa and HEK293T cell lines, measured fluorescence at 24 hours post-transfection, and found the M6 modification promoted higher protein expression in both cell lines (Figure 2). trilinkbiotech.com | 3 Next, to examine protein expression in vivo, we encapsulated N1-methylpseudouridine–modified FLuc mRNAs that were capped with CleanCap AG, CleanCap AG 3′ OMe, or CleanCap M6 into lipid nanoparticles (LNPs) and administered them through tail vein injection to female CD-1 mice (n = 5 per group). We then monitored luciferase activity for 48 hours using whole-body bioluminescence imaging at various time points. Consistent with the in vitro data, CleanCap M6–capped mRNAs exhibited significantly higher luminescence over time compared to those capped with CleanCap AG and CleanCap AG 3′ OMe (Figure 3). A B Figure 3. In vivo protein expression from mRNA with three CleanCap analogs. (A) N1-methylpseudouridine–modified, uridine-depleted FLuc mRNA was transcribed with the indicated cap analogs. 1 mg/kg dose of mRNA:LNP was delivered to mice by tail vein injection. Representative mouse per cohort by whole-body luciferase activity at 24 hrs post delivery is shown. (B) Measurement of the total luciferase activity from six timepoints (3-48 hrs post mRNA:LNP delivery) using area-under-the-curve calculations is shown. (*** p < 0.01, one-way ANOVA; error bars are standard error of mean.) Figure 2. Protein expression in cultured cells. mWasabi mRNA was capped with either CleanCap AG 3′ OMe or CleanCap M6, transfected into HeLa and HEK293T cells (n = 5 technical replicates per group), and measured for fluorescence at 24 hrs post transfection. (*** p < 0.001, t-tailed t-test. Error bars are standard error of mean.) Integrated flux (photons/s) ** ** ** CleanCap AG 0.0E+00 1.0E+12 2.0E+12 CleanCap AG 3′ OMe CleanCap M6 CleanCap AG CleanCap AG 3′ OMe 24h post-injection CleanCap M6 A.U. Fluorescence *** 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 CleanCap AG (3′ OMe) HeLa-mWasabi, 24hr CleanCap M6 A.U. Fluorescence *** 250000 200000 150000 100000 50000 0 CleanCap AG (3′ OMe) HEK293T-mWasabi, 24hr CleanCap M6 In vivo comparison of protein expression using CleanCap M6 and enzymatic capping We investigated the performance of CleanCap M6, our most potent analog, in vivo by comparing it to enzymatically capped mRNA. Notably, enzymatic production of the M6 modification is currently not possible due to enzyme limitations, and thus the enzymatically capped mRNA is a canonical Cap-1 cap. Similar to previous experiments, trilinkbiotech.com | 4 Reduced dosing with CleanCap M6 mRNA To explore potential dose-sparing benefits, we compared CleanCap M6 mRNA to CleanCap AG mRNA in mice. We administered equal doses (1 mg/kg) of both mRNA types and then introduced a significantly lower dose (0.3 mg/kg) of CleanCap M6 mRNA. We consistently observed increased protein expression with CleanCap M6 when equal doses of both mRNA types were administered at 1 mg/kg (Figure 5). Furthermore, our findings revealed that CleanCap M6 mRNA, at a dose of 0.3 mg/kg, achieved comparable in vivo expression levels to CleanCap AG mRNA at 1 mg/kg. This suggests the potential for reduced dosing requirements. Reducing the necessary dose while maintaining effectiveness can lead to cost savings and may minimize dose-related side effects. Standard dose (1 mg/kg) CleanCap® AG Standard dose (1 mg/kg) CleanCap® M6 Low dose (0.3 mg/kg) CleanCap® M6 Figure 4. In vivo protein expression of CleanCap M6 and enzymatically capped mRNAs. (A) Whole-body luciferase activity of mice after 12 hrs post injection of 1 mg/kg FLuc mRNA:LNP is shown. The only difference between the two group was mRNA capping strategy. (B) Time-course measurements of luciferase activity after FLuc mRNA:LNP delivery are plotted. (*** p < 0.001, two-tailed t-test. Error bars are standard error of mean. n = 9/group) (C) Measurement of the total luciferase activity from six timepoints (3-48 hrs post mRNA:LNP delivery) using area-under-the-curve calculations is shown. (*** p < 0.001, two-tailed t-test. Error bars are standard error of mean. n = 9/group) Figure 5. M6 modifications allow for lower mRNA dosing regimes to obtain similar protein expression levels. (*** p <0.001, one-way ANOVA. Error bars are standard error of mean. n = 7/group). we injected LNP-encapsulated FLuc mRNAs into mice (n = 9 per group) via tail vein and subsequently measured luciferase activity over 48 hours post-injection. The results indicate higher protein expression with CleanCap M6 (Figure 4), suggesting that the M6 modification strongly enhances the potency of mRNA compared to enzymatic capping. Integrated flux (photons/s) Matched dose Low dose 4.0E+12 3.0E+12 2.0E+12 1.0E+12 0.0E+12 CleanCap AG (1 mg/kg) CleanCap M6 (1 mg/kg) CleanCap M6 (0.3 mg/kg) *** *** Enzymatic 12 h post-injection CleanCap M6 9.0E+10 8.0E+10 7.0E+10 6.0E+10 5.0E+10 4.0E+10 3.0E+10 2.0E+10 1.0E+10 0.0E+10 0 6 12 18 24 30 36 42 48 54 Flux (photons/s) Hours post FLuc mRNA-LNP infusion CleanCap M6 Enzymatic capping 1.8E+12 1.6E+12 1.4E+12 1.2E+12 1.0E+12 8.0E+11 6.0E+11 4.0E+11 2.0E+11 0.0E+10 Integrated flux (photons/s) Enzymatic capping CleanCap M6 *** *** *** *** 10770 Wateridge Circle, Suite 200 San Diego, CA 92121 | 800.863.6801 | trilinkbiotech.com © 2025 TriLink BioTechnologies. All rights reserved. Comparing capping methods for protein replacement To evaluate the impact of capping on protein replacement therapy (a major application of mRNA therapeutics), we tested CleanCap AG 3′ OMe and CleanCap M6 with human erythropoietin (hEPO) mRNA9 . We encapsulated hEPO mRNA with either cap into LNPs and administered them to C57BL/6NCrl mice. We then measured serum hEPO levels by ELISA. The results indicated that CleanCap M6 mRNA consistently produced higher levels of hEPO protein compared to CleanCap AG 3′ OMe mRNA (Figure 6), suggesting its potential superiority for protein replacement applications. Figure 6. hEPO serum level in mice after hEPO mRNA delivery. LNPformulated mRNA was tail vein–delivered to the mice at 0.1 mg/kg (n = 5 per group). Serum was collected 24 hrs post delivery and measured for hEPO by ELISA. (** p < 0.005, student’s t-test. Error bars are standard error of mean.) hEPO level (ng/mL) 800 700 600 500 400 300 200 100 0 CleanCap AG (3′ OMe) CleanCap M6 ** Conclusion This technical note evaluates the performance of mRNAs with CleanCap cap analogs and enzymatic capping. CleanCap technology, which enables co-transcriptional capping and chemical modifications, demonstrates several advantages. • Improved mRNA quality: mRNAs capped with CleanCap technology demonstrate comparable or improved quality attributes to enzymatically capped mRNAs. Low dsRNA levels were also observed with CleanCap M6. • Enhanced protein expression: CleanCap M6 modification consistently leads to higher protein expression in vitro and in vivo, outperforming enzymatic capping and other CleanCap analogs. • Dose-saving potential: CleanCap M6 mRNA can achieve comparable in vivo expression at one-third mRNA per dose compared to CleanCap AG mRNA. • Promising for protein replacement: CleanCap M6 mRNA outperforms CleanCap AG 3′ OMe mRNA in protein replacement applications, producing higher levels of hEPO protein. Overall, CleanCap technology, particularly with the M6 modification, offers a promising approach for improving mRNA therapeutics through low dsRNA, enhanced protein expression, and potential for reduced dosing. References 1. Henderson JM et al. (2021) Cap 1 messenger RNA synthesis with co-transcriptional CleanCap® analog by in vitro transcription. Curr Protoc 1(2):e39. doi: 10.1002/cpz1.39 2. Henderson JM et al. (2022) Technical note: In vivo expression of exogenous mRNA synthesized with co-transcriptional capping. TriLink BioTechnologies. 3. White paper (2023): The economics of synthetic mRNA capping strategies. TriLink BioTechnologies https://www.trilinkbiotech.com/ cleancap-learning 4. Stepinski J et al. (2001) Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG. RNA 7(10):1486. doi: 10.1017.S1355838201014078 5. Mauer J et al. (2017) Reversible methylation of m6 Am in the 5′ cap controls mRNA stability. Nature 541(7637): 371. doi: 10.1038/ nature21022 6. Mandell ZF et al. (2025) CleanCap M6 inhibits decapping of exogenously delivered IVT mRNA. Mol Ther Nucleic Acids 36(1). doi: 10.1016/j.omtn.2025.102456 7. Technical note (2024) Optimized IVT process for better mRNA production. TriLink BioTechnologies https://go.trilinkbiotech.com/ cleanscript-technote 8. Henderson J et al. (2024) Technical note: Enhanced mRNA production by in vitro transcription and co-transcriptional capping. TriLink BioTechnologies https://www.trilinkbiotech.com/cleancap-m6-pulse-feed 9. Lippin Y et al. (2005) Human erythropoietin gene therapy for patients with chronic renal failure. Blood 106(7):2280. doi: 10.1182/ blood-2004-11-4174 041725