Strategies to Minimize Innate Immune Stimulation to Maximize Messenger RNA Bioavailability

Craig Dobbs, Krist T. Azizian, Dongwon Shin, Sabrina Shore, TriLink BioTechnologies Jordana M. Henderson, Alexandre Lebedev, San Diego, CA 92121 Anton P. McCaffrey and Richard I. Hogrefe

Abstract Figure 2: CleanCap®, Enzymatic and ARCA, Figure 6: Sequence Engineering of FLuc mRNA Capping Comparison (Ψ or MOM1Ψ Substitution, Bioanalyzer) Messenger RNAs (mRNAs) are an exciting new class of nucleic acid therapeutics entering the clinic. mRNAs are useful in difficult to transfect, non-dividing cells. CleanCap Standard FLuc U depleted FLuc In contrast to plasmid or viral vectors, there is no risk of insertional mutagenesis [FU] [FU] 100 or oncogenesis. Transient mRNA expression is also desirable for cellular Enzymatic 100 Ψ Ψ reprogramming, genome editing (ZFNs, TALENs, CRISPR/Cas9) and vaccines. ARCA 80 80

For maximal expression in cells or target organs, transfected mRNAs must avoid 60 60 detection by pattern recognition receptors (PRRs) that evolved to sense Uncapped 40 pathogenic non-self RNAs. These include PRRs that recognize improperly capped Uncapped 40 Uncapped RNAs and double stranded RNA. PRR activation leads to cytokine production, 20 20 translational arrest and cell toxicity or death. Mammalian mRNAs are modified |post-transcriptionally to contain nucleotides with 2’-O-methyl residues, 0 0 m6 pseudouridine (Ψ) and N6-methyladenosine ( A). These modifications can reduce 25 200 500 1000 2000 4000nt 25 200 500 1000 2000 4000nt activation of PRRs and allow maximal translation of the transfected mRNA.

[FU] [FU] During mRNA capping, Cap 0 (m7GpppN) is formed as an intermediate. Methylation 70 25 MOM1Ψ MOM1Ψ of the 2’ position of the first and sometimes second nucleotide forms Cap 1 and Cap 60 2, respectively. Recombinant enzymes used to generate Cap 1 Figure 3: Pseudouridine 5'-Triphosphate Derivatives 20 50 mRNA are expensive, do not always go to completion and the RNA must be purified 15 prior to capping. We developed a novel co-transcriptional capping method that 40 »»Incorporation of modified nucleosides in mRNA helps to evade an 30 yields Cap 1 with high efficiency and lower costs in a “one pot” reaction. CleanCap® 10 immune response also facilitates modification of the 5’ end of mRNAs with diverse functional groups, 20 m6 5 including A. We developed a capping assay that allows direct assessment of »»Ψ or m5C/Ψ modifications of mRNA are current industry standard 10 mRNA capping. Capping efficiencies as high as 95% were obtained. »»Several novel pseudouridine NTPs were synthesized and tested in firefly 0 0 luciferase mRNA transcriptions 25 200 500 1000 2000 4000nt 25 200 500 1000 2000 4000nt We synthesized several Ψ derivatives to explore the impact on innate immune stimulation: N1-ethyl-Ψ (Et1Ψ), N1-fluoroethyl-Ψ (FE1Ψ), N1-propyl-Ψ (Pr1Ψ), N1- isopropyl-Ψ (iPr1Ψ) and N1-methoxylmethyl-Ψ (MOM1Ψ) 5’-triphosphates. As a control, we tested 5-methoxy-uridine alongside the pseudouridine R = H, Me, Et, FCH2CH2, Pr derivatives because it previously gave good activity. Luciferase mRNAs were fully iPr, MOM, POM, BOM Figure 7: In Vitro Translation and Cell Activity of substituted with these modifications and translational potential was monitored in wheat germ extracts. Activity was also measured in the THP-1 monocyte cell Modified Luciferase mRNAs line, which is a sensitive model for innate immune activation. A recent report »U depleted mRNA sequences showed that minimizing uridine content in mRNAs reduced immune stimulation »»U depleted mRNA sequences » resulted in higher activity in by unmodified mRNAs. Here we show that incorporation of our modified uridine translated better in wheat THP-1 cells residues into uridine depleted luciferase resulted in equivalent activity relative to germ extracts Ψ in THP-1 cells. Interestingly, translation in extracts did not mirror activity in »»mRNA with bulkier pseudouridine »»We therefore continued our THP-1 cells. modifications did not translate well studies using the U depleted mRNA sequence

Pseudouridine 5’-triphosphate derivatives; H = pseudouridine, Me = N1- methyl, Et = N1-ethyl, FE = fluoroethyl, Pr = propyl, iPr = isopropyl, MOM )

) Standard FLuc U depleted FLuc 6 Standard FLuc U depleted FLuc 7 = methoxy methyl, POM = pivaloxy methyl, BOM = benzyloxy methyl 7 6 pseudouridine 4 Background: Why mRNA Therapeutics? 2 4 mRNA is a popular new tool for gene expression 0.6 2 0.4 »» Does not have a risk of insertional mutagenesis Figure 4: mRNA Synthesis by In Vitro Transcription »» Can transfect difficult cells such as non-dividing cells 0.2 Raw Light Units (x10 Units Light Raw »» Is transient (x10 Units Light Raw 0 0 Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ WT WT WT WT Template, promotor, T7 RNA polymerase m1 Et1 FE1 Pr1 m1 Et1 FE1 Pr1 m1 Et1 FE1 Pr1 m1 Et1 FE1 Pr1 5moU 5moU 5moU 5moU Applications MOM1 MOM1 MOM1 MOM1 *ΨTP, NTP, CleanCapTM »» Genome editing (Transposons, Cre, ZFNs, TALENs and CRISPR/Cas9) T7 Promotor Transcription Translation in wheat germ extracts Activity in THP-1 cells »» Gene replacement »» Vaccines T emplate * *Ψ * Ψ Ψ Figure 8: N1-Substituted Ψ Derivatives Resulted Limitations HO HO »» Innate immune response to unmodified mRNA *Ψ in Lower Toxicity Compared to WT and Ψ in OPPP Solutions O FLuc mRNAs

»» Proper capping N FLuc mRNA »» Chemical modification of mRNA can prevent innate immune N T7 RNA Polymerase H2N N Translation 4 0.80

stimulation ) HN 7 »» Removal of dsRNA O 3 0.60 Light Light Signal = Luciferase Expression

2 0.40 Innate immune sensors recognize mRNA Schematic representation of transcription and translation 1 0.20 Transfection of cells with unmodified RNAs can lead to cell death with N1-modified pseudouridine 5’-triphosphate derivatives

Raw Light Units (x10 Units Light Raw 0 0 in the presence of CleanCap®: *N1-modified-Ψ (560 nm, AU) Absorbance due to activation of innate immune pathways Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ Ψ WT WT Et m1 Et1 FE1 Pr1 m1 FE1 Pr1 5moU 5moU Toll-like receptors 3, 7 & 8 recognize different RNA forms MOM1 MOM1 »Found in endosomes where some viruses enter cells » Activity in THP-1 cells Toxicity (MTT assay*) in THP-1 cells

Cytosolic sensors Figure 5: U Depletion of Primary Luciferase *Lower absorbance reflects decreased cellular metabolism indicating toxicity »»Protein Kinase R (PKR): dsRNA mRNA Sequence Improves Incorporation of »»MDA5: long dsRNA N1-Substituted Ψ Derivatives by T7 Polymerase »»IFITs: unmethylated cap structures »»RIG-I: 5’-triphosphate Conclusions »»Some N1-substituted Ψ derivatives did not incorporate well in WT mRNA »»We decreased the number of uridine residues in the sequence by »»We have synthesized a number of 5’-triphosphates of N1-modified substituting synonymous codons pseudouridine derivatives »»These nucleoside 5’-triphosphates were used for the synthesis of modified »»U depletion resulted in good incorporation Figure 1: Cap 0, Cap 1 and Cap 2 Structures of 5’- mRNAs by in vitro transcription using WT and U-depleted templates Ends of mRNAs »»Efficiency of transcription using U-depleted templates greatly improved quality Eukaryotic mRNAs have a Cap 1 or Cap 2 structure. and yield 150 »»N1-substituted Ψ mRNAs show potential translational and immunological Sensing of proper cap structure is thought to be involved in self/non-self WT FLuc properties RNA recognition. U depleted FLuc 125 WT eGFP »»Translational activity of modified mRNAs in wheat germ extracts did not Cap structure influences activation of PRRs directly correlate with cell activity, which may indicate differences in immune stimulation by these mRNAs »»RIG-I is activated by Cap 0 RNAs but not Cap 1 mRNAs (PMID: 18426922 100 and 20457754)

»»IFIT1 binds Cap0 RNAs more tightly than Cap 1 mRNAs (PMID: 24371270) 75 Contact O B1 HO OH P

O O (%) mRNA O Craig Dobbs O P O O 50 P O O [email protected] O O O R1 H2N N O O

N Transcription Relative B2 O P O 25 HN O N O 1 2 O Cap0: R and R =H 2 O OR 0 1 2 B3 Cap1: R =CH ; R =H H Et Pr 3 O P O WT Me FE iPr O POM BOM Cap2: R1=CH ; R2=CH O MOM 3 3 N1-ModificationΨ The Modified Nucleic 1-3 m6 1 O OH OTS B = A, C, G, U or A (B ) ® O P ORNA Acid Experts Seattle, WA O »»Relative efficiency of mRNA synthesis byin vitro transcription using Co-transcriptional capping with CleanCap® (Cap 1) helps evade an immune 09/18 T7 RNA polymerase with N1-modifiedΨ TPs; 7.5 mM triphosphates, www.trilinkbiotech.com response 37 °C for 3 h; 2100 Agilent Bioanalyzer with RNA nano chip