Revolutionize mRNA Manufacturing Using a Novel RNA Polymerase

©2024 Maravai LifeSciences. This document and its contents are confidential and proprietary and are not to be shared or redistributed without express consent. RNA synthesis & analysis • Uridine depleted Firefly luciferase (Fluc), eGFP, Cas9, mCherry mRNA and/or saRNA sequences • CleanCap® analogs, rNTPs & RNA polymerase reagents from TriLink BioTechnologies • DNaseI treatment / LiCl (mRNA) or RNeasy (saRNA) purification • Yield: UV spectrometry • Purity: Fragment Analyzer analysis • Capping efficiency: LC-MS analysis • dsRNA content: J2 Immunoblot or ELISA (Vazyme) Innate immune response • A549-dual NFkB-SEAP/IRF ISG54-Lucia luciferase reporter cells (InvivoGen) • MessengerMAX transfection reagent (Thermo Fisher) • Poly (I:C) & 3p-hpRNA positive controls (InvivoGen) • Analysis: QUANTI-Luc 4 (InvivoGen) Protein Expression • A549 cells (ATCC) • MessengerMAX transfection reagent (Thermo Fisher) • M6-capped GFP mRNAs • Analysis: GFP fluorescence over time by Incucyte (Sartorius) A novel RNA polymerase variant for reduced dsRNA byproducts in mRNA manufacturing Stephanie Ramos1 , Benjamin Hudson1 , John Davidson2 , Andrew Ujita1 , Coleen Vo1 , Farinaz Rezvani 1 , Anthony Truong 1 , Deidra Broxterman 1 , Nona Albohassani 1 , Robin Lee 1 , Kate Broderick 3 1TriLink BioTechnologies, Part of Maravai LifeSciences, San Diego, California 92121, USA; 2Alphazyme, Part of Maravai LifeSciences, Jupiter, Florida 33458, USA; 3Maravai LifeSciences, San Diego, California 92121, USA Abstract Results Conclusion The success of mRNA vaccines during the COVID pandemic has fueled interest in leveraging the mRNA platform beyond infectious disease vaccines and into cancer vaccines, cell and gene therapies, and protein replacement therapies. With this comes increased need for high-yielding manufacturing processes that generate quality mRNA with minimal unwanted byproducts. Double-stranded RNA (dsRNA) is a critical byproduct with immunostimulatory properties that can impair the tolerability, safety, and potency of mRNA vaccines and therapeutics. Here we present data for an RNA polymerase variant, CleanScribe RNA Polymerase, that consistently reduces dsRNA formation as compared to wildtype T7 across a variety of IVT conditions and mRNA templates. CleanScribe RNA Polymerase efficiently incorporates cap analogs and modified NTPs with mRNA integrities comparable to wildtype T7. CleanScribe RNA Polymerase: • Provides simple in vitro transcription (IVT) integration by directly substituting wildtype (WT) T7 RNA polymerase • Reduces formation of dsRNAs by up to 85%, generating higher purity of in vitro– transcribed mRNAs and saRNAs • Produces comparable IVT yields, capping efficiency, and RNA integrity Methods & Materials Acknowledgements We thank members of the TriLink and Alphazyme Research & Development and Product Management groups for helpful discussions and critical evaluation. References • Karikó et al. (2011) Nucleic Acids Res 39(21):e142 • Ravichandran et al. (2023) Nat Biotechnol 41, 560–568. Figure 1. CleanScribe RNA Polymerase significantly reduces dsRNA in IVT reactions. eGFP, Fluc and Cas9 mRNAs were synthesized with the indicated co-transcriptional cap analog according to manufacturer instructions using either wildtype (WT) or CleanScribe RNA polymerase, then purified by LiCl precipitation. dsRNA levels of the IVT reactions were measured by A. J2 dot blot, and B. dsRNA ELISA. dsRNA dot blot dsRNA ELISA (2 µg of mRNA loadzed per sample) eGFP FLuc eGFP FLuc eGFP FLuc 0 20 40 60 80 100 C a p p ni g E f icie n c y %( ) WT T7 v8 Standard AG 3'OMe CleanScript AG 3'OMe IVT: M6 WT T7 RNA pol CleanScribe RNA Pol CleanCap® AG 3′ OMe % dsRNA relative to WT T7 RNA pol eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 CleanCap® M6 WT T7 RNA pol CleanScribe RNA pol EGFP FLuc 20 ng dsRNA std 16 ng 8 ng 4 ng 2 ng 1 ng WT T7 RNA pol CleanScribe RNA pol CleanCap® AG 3′ OMe CleanCap® M6 Figure 2. CleanScribe RNA Polymerase does not compromise mRNA yield or quality. mRNAs from Figure 1 were analyzed for A. yield by UV spectrometry, B. capping efficiency by LC-MS, and C. purity by Fragment Analyzer (FA; representative eGFP-mRNAs shown; similar results obtained for all mRNAs). eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 2 4 6 8 10 mRNA Yield (mg/mL) eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 2 4 6 8 10 mRNA Yield m( g/mL) mRNA yield (mg/mL) CleanCap® AG 3′ OMe CleanCap® M6 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 Capping efficiency (%) CleanCap® AG 3′ OMe CleanCap® M6 WT T7 RNA pol CleanScribe RNA Pol 0 2000 4000 6000 8000 10000 12000 14000 16000 1700 1800 1900 2000 2100 RFU Time (sec) CleanCap® AG 3′ OMe CleanCap® M6 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 1700 1800 1900 2000 2100 RFU Time (sec) A. B. A. B. C. WT T7 RNA pol CleanScribe RNA pol EGFP FLuc 20 ng dsRNA std 16 ng 8 ng 4 ng 2 ng 1 ng WT T7 RNA pol CleanScribe RNA pol eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 % dsRNA relative to WT T7 RNA pol eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 2 4 6 8 10 mRNA Yield (mg/mL) eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 2 4 6 8 10 mRNA Yield (mg/mL) mRNA yield (mg/mL) eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 Capping Ef iciency (%) eGFP FLucCas9 eGFP FLucCas9 eGFP FLucCas9 0 20 40 60 80 100 Capping Ef iciency (%) Capping efficiency (%) 0 4000 8000 12000 16000 20000 1700 1800 1900 2000 2100 RFU Time (sec) Figure 3. CleanScribe RNA Polymerase further reduces dsRNA in IVTs already optimized for low dsRNA. eGFP, Fluc and Cas9 mRNAs were synthesized with AG 3’OMe cap analog with TriLink’s optimized CleanScript® IVT conditions using either wildtype (WT) or CleanScribe RNA pol, then purified by LiCl precipitation. dsRNA levels of the IVT reactions were measured by A. J2 dot blot, and B. dsRNA ELISA. mRNAs were analyzed for C. yield by UV spectrometry, D. capping efficiency by LC-MS, and E. purity by FA (representative eGFP-mRNAs shown; similar results obtained for all mRNAs). A. B. C. D. E. eGFP FLuc Cas9 0 2 4 6 8 10 mRNA Yield (mg/mL) mRNA yield (mg/mL) eGFP FLuc Cas9 0 20 40 60 80 100 Capping Ef iciency (%) Capping efficiency (%) eGFP FLuc Cas9 0 20 40 60 80 100 % dsRNA relative to WT T7 RNA pol % dsRNA relative to WT T7 RNA pol Figure 4. CleanScribe RNA Polymerase incorporates N1-methylpseudouridine. eGFP, Fluc and Cas9 mRNAs were synthesized with CleanCap® M6 cap analog and replacing wildtype Uridine with N1- methylpseudoruidine using either wildtype (WT) or CleanScribe RNA polymerase, then purified by LiCl precipitation. mRNAs were analyzed for A. dsRNA levels by dsRNA ELISA, B. yield by UV spectrometry, and C. capping efficiency by LC-MS. A. B. C. WT T7 RNA pol CleanScribe RNA Pol WT T7 RNA pol CleanScribe RNA Pol Fluc mCher y Fluc-5mC mCher y-5mC 0 20 40 60 80 100 % dsRNA relative to WT T7 RNA pol Fluc mCher y Fluc-5mC mCher y-5mC 0 2 4 6 8 10 saRNA Yield (mg/mL) 0 10000 20000 30000 40000 50000 3500 3750 4000 4250 4500 4750 5000 RFU Time (sec) FLuc 0 2500 5000 7500 10000 12500 15000 17500 20000 3500 3750 4000 4250 4500 4750 5000 RFU Time (sec) mCherry Figure 5. CleanScribe RNA Polymerase reduces dsRNA for long self-amplifying RNAs. Fluc and mCherry self-amplifying RNAs (saRNAs) were synthesized with CleanCap® AU analog, and either all wildtpe rNTPs or wildtype cytidine replaced with 5-methylcytidine (5mC), using either wildtype (WT) or CleanScribe RNA pol, then purified by RNeasy column filtration. saRNAs were analyzed for A. dsRNA levels by dsRNA ELISA, B. yield by UV spectrometry, and C. purity by FA (representative unmodified saRNAs shown; similar results obtained for all saRNAs). 0 24 48 72 96 0 1×10 7 2×10 7 3×10 7 4×10 7 5×10 7 Time (hours) GFP Expression (Mean ± SD Total Integrated Intensity) WT T7 RNA Pol CleanScribe TM RNAPol Figure 6. dsRNA reductions by CleanScribe polymerase translate to increased mRNA potency. eGFP mRNAs were synthesized with CleanCap® M6 analog described in Fig 1 and formulated for 5 ng/well, 96-well plate transfections in MessengerMAX. A. A549-dual reporter cell supernatants were collected 24 hours post transfection and analyzed for ISG54-Lucia luciferase expression as a readout for innate interferon responses. B. A549 cells were analyzed for mRNA-encoded protein expression by GFP fluorescence over time. All conditions performed in triplicate. Innate inflammatory response Cell-based protein expression A. B. C. A. B. Media Poly (I:C) 3p-hpRNA WT T7 RNA Pol CleanScribe TM RNA Pol 0 20000 40000 60000 ISG54 Induction (Mean ± SD Luciferase RLUs) Media Poly (I:C) 3p-hpRNA WT T7 RNA Pol CleanScribe TM RNA Pol Experimental conditions