DOI: 10.5061/dryad.q573n5tr8
title: Data from: Self-amplifying RNA generated with the modified nucleotides 5-methylcytidine and 5-methyluridine mediate strong expression and immunogenicity in vivo

description: When utilized in therapeutic applications, synthetic self-amplifying RNA
 can lead to higher and more sustained expression than standard messenger
 RNA. This feature is particularly important for gene replacement therapy
 applications where prolonged expression could reduce the dose and
 frequency of treatments. The inclusion of modified nucleotides in
 synthetic non-amplifying mRNA has been shown to increase RNA stability,
 reduce immune activation and enhance gene expression. Preclinical and
 clinical studies with self-amplifying RNA (saRNA) have so far exclusively
 relied on RNA containing the canonical nucleotides adenosine, cytidine,
 guanosine and uridine. For the first time, we show that non-canonical
 nucleotides, such as m5C and m5U, are sufficiently compatible with a
 replicon derived from Venezuelan equine encephalitis alphavirus mediating
 protein translation in vitro, while those containing m1ψ in place of
 uridine show no detectable expression. When administered in vivo, saRNA
 generated with m5C or m5U mediate sustained gene expression of the
 luciferase reporter gene with those incorporating m5U appearing to lead to
 more prolonged expression. Finally, distinct antigen-specific humoral and
 cellular immune responses were induced by modified saRNA encoding the
 model antigen ovalbumin. The use of modified nucleotides with saRNA-based
 platforms could enhance their potential to be used effectively in a
 variety of applications.
description: # Data from: Self-amplifying RNA generated with the modified nucleotides
 5-methylcytidine and 5-methyluridine mediate strong expression and
 immunogenicity in vivo This README.md file was generated on 2024-04-05 by
 Bassel Akache & Tyler Renner. GENERAL INFORMATION 1. Title of Dataset:
 Self-Amplifying RNA Generated with the Modified Nucleotides
 5-MethylCytidine and 5-MethylUridine Mediate Strong Expression &
 Immunogenicity in vivo Related Article of the same title submitted to
 Nucleic Acids Research Molecular Medicine. 2. Author information: Hiva
 Azizi1, Tyler M. Renner1, Gerard Agbayani1, Bryan Simard1, Renu Dudani1,
 Blair A. Harrison1, Umar Iqbal1, Yimei Jia1, Michael J. McCluskie1 and
 Bassel Akache1* 1National Research Council Canada, Human Health
 Therapeutics, Ottawa, Ontario, Canada, K1A 0R6 * To whom correspondence
 should be addressed: Bassel Akache, Email:
 [Bassel.Akache@nrc-cnrc.gc.ca](mailto:Bassel.Akache@nrc-cnrc.gc.ca) 3.
 Date of data collection: 2023 - 2024 4. Geographic location of data
 collection: Ottawa, Canada. 5. Funding sources that supported the
 collection of data: National Research Council of Canada. 6. Recommended
 citation for this data set: Azizi, Hiva et al. (2024), Data from:
 Self-Amplifying RNA Generated with the Modified Nucleotides
 5-MethylCytidine and 5-MethylUridine Mediate Strong Expression &
 Immunogenicity in vivo, Dryad, Dataset,
 [https://doi.org/10.5061/dryad.q573n5tr8](https://doi.org/10.5061/dryad.q573n5tr8) MATERIALS & METHODS In vitro RNA transcription The plasmid DNA templates used to generate the tested saRNAs were derived from SCR725 plasmid (Sigma-Aldrich, St. Louis, MO, USA), which encode the non-structural genes of VEEV and tagGFP2 downstream of its subgenomic promoter. The IRES-E3L-IRES-puromycin element was removed and the encoded fluorescent protein gene was replaced by genes encoding for a codon-optimized red Clickbeetle luciferase (CBR2opt) or Ovalbumin (derived from plasmids #108712 and #64599, respectively) (22, 23). ATG-1929 (Addgene plasmid # 108712; [http://n2t.net/addgene:108712](http://n2t.net/addgene:108712); RRID:Addgene_108712) was a gift from Keith Wood, while pcDNA3-OVA (Addgene plasmid # 64599; [http://n2t.net/addgene:64599](http://n2t.net/addgene:64599); RRID:Addgene_64599) was a gift from Sandra Diebold & Martin Zenke (22, 23). In addition, an adenine residue was inserted immediately downstream of the T7 promoter to enable the use of the co-transcriptional CleanCap Reagent AG (TriLink Bio Technologies, San Diego, CA) to generate RNA with stabilized Cap 1 structure. DNA templates were linearized with XbaI and purified by phenol chloroform extraction. RNA was generated using Megascript T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) as previously described (24). The modified nucleotides m1ψ (Jena Bioscience, Jena, Germany), m5C and m5U (Tri Link Bio Technologies) were included instead of the corresponding Cytidine and/or Uridine nucleotide to generate the saRNAs with modified nucleotide chemistry. Following in vitro transcription (IVT) of RNA, plasmid DNA was digested using Turbo DNase (Invitrogen, Waltham, MA, USA) as per manufacturer’s instructions. RNA was purified by acid phenol chloroform extraction, precipitated with ethanol and resuspended in RNase-free water. To assess RNA size and integrity, all saRNA samples were resolved on formaldehyde (2.6% v/v) denaturing agarose (1% w/v) gel. The concentration of saRNA samples were measured using Invitrogen’s Ribogreen assay reagent (Thermo Fisher Scientific), as per manufacturer’s recommendations. Detection of dsRNA by dot blot analysis Double-stranded (ds) RNA controls were generated to enable proper quantification of the dsRNA within the IVT samples. The template for generation of dsRNA controls consisted of linear DNA with T7 promoters at both ends to enable simultaneous transcription of the gene and its reverse complement from the 5’ and 3’ ends, respectively. The linear DNA was generated by polymerase chain reaction from a plasmid template encoding click beetle Luciferase (derived from same plasmid above) flanked by an alpha globin 5’ untranslated region (UTR) and mtRNR1-AES 3’UTR (see Supplementary Data for nucleotide sequence of template). The dsRNA was generated with either canonical or modified nucleotides as described above by with a T7 transcription kit. Annealing of the complementary RNA strands to form dsRNA was achieved by heating each sample at 95°C for 1 min in a hybridization buffer containing 10 mM Tris-HCl [pH 7.0] and 50 mM NaCl (Sigma-Aldrich) and then cooling gradually to room temperature over a period of 2 hours. Detection of dsRNA species within the IVT saRNA samples and the dsRNA controls was performed by dot blot as previously described (25). Briefly, 2-fold serial dilutions of saRNA containing 1 to 0.016 µg were prepared and spotted onto to a positively charged nylon membrane (Whatman Nytran SuPerCharge, Sigma-Aldrich), utilizing a Bio-Dot apparatus (Bio-Rad Laboratories, Hercules, CA, USA). As for the dsRNA controls, 4-fold serial dilutions of RNA containing 1 to 0.0003 µg were prepared and also loaded on the membrane. Once samples were loaded, the membrane was then allowed to dry and then blocked for 1 hour at room temperature, followed by overnight incubation with J2 anti-dsRNA antibody (Scicons, Budapest, Hungary) at 4°C. The blots were washed 3 times for a total of 15 minutes, before being hybridized with a goat anti-mouse HRP IgG secondary antibody (Southern Biotech, Birmingham, AL, USA). Following a 1-hour incubation, blots were washed as before and chemiluminescent signal was detected using Pierce ECL Western Blotting Substrate (Thermo Fisher, USA) on a ChemiDoc imaging system (Bio-Rad Laboratories). In vitro expression in HEK-293T cells HEK-293T cells were maintained in Dulbecco’s minimal essential medium (DMEM) media supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM glutamine (all from Thermo Fisher Scientific). For transfection of RNA, 7 x 105 cells were placed in a 6-well plate and incubated overnight. On the following day, 2 µg of GFP-encoding RNA were transfected using lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s instructions. Images of the cells at 24 hours post transfection were taken with a EVOS FLoid Cell Imaging System (Thermo Fisher Scientific) under both brightfield and fluorescent (GFP) modes. Cells were then washed and detached with PBS with 1% bovine serum albumin and 5 mM EDTA, prior to washing and resuspension in IC fixation buffer (Thermo Fisher Scientific). Cells were acquired on an LSR Fortessa (Becton Dickinson, Franklin Lakes, New Jersey, USA) to measure the number of cells positive for GFP expression. Analysis of flow cytometry data was conducted using FlowJo version 10.9.0 (Becton Dickinson). Preparation of saRNA/LNPs In vitro transcribed saRNA encoding luciferase or ovalbumin (OVA) was encapsulated within Genvoy-ILM ionisable lipid mixture (Precision NanoSystems, Vancouver, BC, Canada) at an N:P ratio of 8. Formation of LNPs was achieved by microfluidic mixing of RNA (aqueous) and ionisable lipids (organic) using the NanoAssemblr Ignite system (Precision NanoSystems) according to manufacturer’s recommendations with a flow rate ratio of 3 (aqueous: organic) and total flow rate of 12 (TFR; mL/min). Formulations were then diluted in Mg2+/Ca2+- free PBS, before undergoing buffer exchange and concentration using Amicon Ultra Centrifugal Filters (Millipore Sigma, St. Louis, MO, USA). Encapsulation efficiency of RNA was determined by ribogreen assay (Thermo Fisher Scientific) as per instructions accompanying the Genvoy-ILM ionisable lipid and found to be &gt;90%. In addition, particle size, polydispersity index (pdi) and zeta potential of the LNPs were verified using a Zetasizer NanoZS (Malvern Instruments, Malvern, UK) and found to be generally consistent within formulations (Supplementary Table 1). Institutional Review Board Statement: Mice were maintained at the small animal facility of the National Research Council (NRC) Canada in accordance with the guidelines of the Canadian Council on Animal Care. All procedures performed on animals in this study were approved by our Institutional Review Board (NRC Human Health Therapeutics Animal Care Committee) and covered under animal use protocol 2020.10. All experiments were carried out in accordance with the ARRIVE guidelines. In vivo Expression Analysis Female albino C57BL/6 mice (6-8 weeks old) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada). Mice (n=5-10 per group) were injected with 1 or 5 µg of LNP-encapsulated luciferase saRNA intramuscularly (i.m.) into the left tibialis anterior muscle. At the 1 µg dose, RNA containing m1ψ, m5C/m5U, m5C, m5U or all canonical nucleotides was administered, while only the latter three were administered at the higher 5 µg dose. Fifteen minutes prior to whole body imaging, D-luciferin (Perkin Elmer, Waltham, MA, USA) was injected subcutaneously into the scruff of the neck at a dose of 0.15 mg per gram of mouse body weight. An IVIS Lumina III small animal imager (Perkin Elmer) was used to measure the amount of luminescence emitted from the live mice using an open filter. Animals were imaged prior to treatment and at various time points after injection. Total flux (photons/second) was determined from a region of interest (ROI) encompassing the injection site using the Living Image 4.7.4 software (Perkin Elmer). Vaccination Studies Female C57BL/6 mice (6-8 weeks old) were obtained from Charles River Laboratories (Saint-Constant, QC, Canada). Mice (n=5-10 per group) were immunized by intramuscular (i.m.) injection (50 µL) into the left tibialis anterior (T.A.) muscle on Days 0 and 21 with 1 or 5 µg OVA-encoding saRNA containing m5C/m5U, m5C, m5U or all canonical nucleotides. On Day 14, 5 mice from the groups receiving 1 µg dose of RNA were anesthetized with isoflurane and bled via the submandibular vein with recovered serum used for quantification of antigen-specific antibody levels. They were then euthanized by cervical dislocation prior to collection of spleens for measurement of cellular immune responses by IFN-γ ELISpot. On Day 21 prior to immunization, the remaining 5 mice per group were bled as above. Finally on Day 28, terminal blood collections and spleen harvest were conducted on these mice. Samples were simultaneously collected from naïve animals for the assessment of background immune responses. Each of the samples from the individual mice was tested separately in the various readouts. Anti-OVA IgG ELISA Anti-OVA total IgG titers in serum were measured by indirect ELISA with OVA as previously described (26). Briefly, 96–well high-binding ELISA plates (Thermo Fisher Scientific) were coated with 10 µg/mL OVA protein diluted in PBS. Serum samples were serially diluted 3.162-fold and added to the plates to allow for binding of antibodies to the protein. Bound IgG was detected with goat anti-mouse IgG, IgG1 or IgG2c -horseradish peroxidase (HRP; 1:4,000, Southern Biotech, Birmingham, AL, USA) prior to the addition of the substrate o-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich). Bound IgG Abs were detected spectrophotometrically at 450 nm. Titers for IgG in serum were defined as the dilution that resulted in an absorbance value (OD450) of 0.2 and were calculated using XLfit software (ID Business Solutions, Guildford, UK). Samples that did not reach the target OD were assigned the value of the lowest tested dilution (i.e., 10) for analysis purposes. No detectable titers were measured in serum samples from naïve control animals. IFN- γ ELISpot IFN- γ ELISpot was also conducted as previously described (27). The levels of OVA-specific T cells were quantified by ELISpot using a mouse IFN-γ kit (Mabtech Inc., Cincinnati, OH, USA). Cells (2x105 per well) were stimulated in duplicate with 2 µg/mL of a peptide corresponding to the well-recognized CD8+ T cell epitope OVA257-264: SIINFEKL (JPT Peptide Technologies GmbH, Berlin, Germany). Cells were also incubated without any stimulants to measure background responses. Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD, Beachwood, OH, USA). For each animal, values obtained with media alone were subtracted from those obtained with each of the OVA peptide, and then converted to yield the number of antigen-specific IFN-γ+ spot forming cells (SFC)/106 splenocytes per animal. Statistical analysis Data were analyzed using GraphPad Prism® version 9 (GraphPad Software, Boston, MA, USA). 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Akache,B., Stark,F.C., Jia,Y., Deschatelets,L., Dudani,R., Harrison,B.A., Agbayani,G., Williams,D., Jamshidi,M.P., Krishnan,L., et al. (2018) Sulfated archaeol glycolipids: Comparison with other immunological adjuvants in mice. PloS One, 13, e0208067. DATA & FILE OVERVIEW 1. These data were collected to investigate the impacts of modified nucleotides on the expression and immunogenicity of proteins encoded within a self-amplifying RNA (saRNA) vector. 2. File list: File Name: Azizi et al Source Data File Sheet 1 Name: Figure 1B Sheet 1 Description: Raw image for formaldehyde agarose gel electrophoresis. Sheet 2 Name: Figure 1C Sheet 2 Description: Raw image for dot blot using the J2 anti-dsRNA specific antibody. Sheet 3 Name: Figure 2A Sheet 3 Description: HEK-293T cells were transfected in vitro with 1 µg saRNA encoding green fluorescent protein (GFP). After 24 hours, cells were detached with PBS + 1% bovine serum albumin and 5 mM EDTA, fixed and analyzed on an LSR II Fortessa. After gating on cells and singlets within FlowJo, the percentage of GFP-positive cells was determined. Sheet 4 Name: Figure 2B Sheet 4 Description: As above, except after gating on cells and singlets, the geometric mean fluorescence intensity was determined. Sheet 5 Name: Figure 3A Sheet 5 Description: Albino C57BL/6 mice were injected intramuscularly with 1 µg canonical, m5C, m5U or m1ψ-modified luciferase saRNA encapsulated within LNPs.The total luminescence signals obtained from a region of interest (ROI) of the injection site was quantified over a period of several weeks. Sheet 6 Name: Figure 3B Sheet 6 Description: Albino C57BL/6 mice were injected intramuscularly with 5 µg canonical, m5C or m5U-modified luciferase saRNA encapsulated within LNPs.The total luminescence signals obtained from a region of interest (ROI) of the injection site was quantified over a period of several weeks. Sheet 7 Name: Figure 3C Sheet 7 Description: Albino C57BL/6 mice were injected intramuscularly with 1 µg canonical, m5C, m5U or m5C/m5U-modified luciferase saRNA encapsulated within LNPs.The total luminescence signals obtained from a region of interest (ROI) of the injection site was quantified on Day 7 post-injection. Sheet 8 Name: Figure 3D Sheet 8 Description: Albino C57BL/6 mice were injected intramuscularly with 1 µg canonical, m5C, m5U or m5C/m5U-modified luciferase saRNA encapsulated within LNPs.The total luminescence signals obtained from a region of interest (ROI) of the injection site was quantified on Day 17 post-injection. Sheet 9 Name: Figure 5A Sheet 9 Description: Relative levels of OVA antigen specific titer on D14, D21 and D28 serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. Sheet 10 Name: Figure 5B Sheet 10 Description: Extrapolated levels of peptide reactive splenocytes for SIINFEKL on D14 and D28 splenocytes as determined by IFN-γ ELISpot, where the background of splenocytes in culture media is subtracted. Sheet 11 Name: Figure 5C Sheet 11 Description: Relative levels of OVA antigen specific titer of subclasses IgG1 and IgG2c on D28 serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. DATA-SPECIFIC INFORMATION FOR: Figure 1B 1. Number of variables: 2 2. Number of rows: 20 3. Variable list: Figure 1B Raw Gel Image 4. Missing data codes: None 5. Abbreviations used: saRNA = self-amplifying ribonucleic acid; IVT = in vitro transcription; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: This data sheet is used to illustrate qualitative data of a raw formaldehyde agarose gel. DATA-SPECIFIC INFORMATION FOR: Figure 1C 1. Number of variables: 2 2. Number of rows: 28 3. Variable list: Figure 1C Raw Membrane Image 4. Missing data codes: None 5. Abbreviations used: saRNA = self-amplifying ribonucleic acid; IVT = in vitro transcription; dsRNA = double stranded ribonucleic acid; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: This data sheet is used to illustrate qualitative data of a raw dot blot membrane. DATA-SPECIFIC INFORMATION FOR: Figure 2A 1. Number of variables: 3 2. Number of rows: 15 3. Variable list: Replicate Nucleotides % GFP 4. Missing data codes: None 5. Abbreviations used: GFP = green fluorescent protein; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Value obtained on FlowJo after gating on cells, singlets and the GFP positive population. DATA-SPECIFIC INFORMATION FOR: Figure 2B 1. Number of variables: 3 2. Number of rows: 15 3. Variable list: Replicate Nucleotides GFP gMFI 4. Missing data codes: None 5. Abbreviations used: GFP = green fluorescent protein; gMFI = geometric mean fluorescent intensity; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Value obtained on FlowJo after gating on cells, singlets and the GFP positive population. DATA-SPECIFIC INFORMATION FOR: Figure 3A 1. Number of variables: 5 2. Number of rows: 141 3. Variable list: Mouse ID Nucleotide Day Total Flux Dose 4. Missing data codes: None 5. Abbreviations used: m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Total flux (photons/second) was determined from a region of interest (ROI) encompassing the injection site using the Living Image 4.7.4 software (Perkin Elmer). DATA-SPECIFIC INFORMATION FOR: Figure 3B 1. Number of variables: 5 2. Number of rows: 106 3. Variable list: Mouse ID Nucleotide Day Total Flux Dose 4. Missing data codes: None 5. Abbreviations used: m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Total flux (photons/second) was determined from a region of interest (ROI) encompassing the injection site using the Living Image 4.7.4 software (Perkin Elmer). DATA-SPECIFIC INFORMATION FOR: Figure 3C 1. Number of variables: 5 2. Number of rows: 41 3. Variable list: Mouse ID Nucleotide Day Total Flux Dose 4. Missing data codes: None 5. Abbreviations used: m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Total flux (photons/second) was determined from a region of interest (ROI) encompassing the injection site using the Living Image 4.7.4 software (Perkin Elmer). DATA-SPECIFIC INFORMATION FOR: Figure 3D 1. Number of variables: 5 2. Number of rows: 41 3. Variable list: Mouse ID Nucleotide Day Total Flux Dose 4. Missing data codes: None 5. Abbreviations used: m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Total flux (photons/second) was determined from a region of interest (ROI) encompassing the injection site using the Living Image 4.7.4 software (Perkin Elmer). DATA-SPECIFIC INFORMATION FOR: Figure 5A 1. Number of variables: 5 2. Number of rows: 101 3. Variable list: Mouse ID Nucleotide Day Anti-Ova IgG (OD=0.2; GMT) Dose 4. Missing data codes: None 5. Abbreviations used: IgG = Immunoglobulin G; GMT = Geometric Mean Titer; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Relative levels of OVA antigen specific titer on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2. DATA-SPECIFIC INFORMATION FOR: Figure 5B 1. Number of variables: 5 2. Number of rows: 71 3. Variable list: Mouse ID Nucleotide Day Spot forming cells (SFCs) per million Dose 4. Missing data codes: None 5. Abbreviations used: SFCs = Spot forming cells; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD) and the background of splenocytes in culture media is subtracted. The value from the ELISpot was extrapolated from the experimental value obtained from 2x10^5 splenocytes. DATA-SPECIFIC INFORMATION FOR: Figure 5C 1. Number of variables: 6 2. Number of rows: 81 3. Variable list: Mouse ID Nucleotide Day Anti-Ova IgG (OD=0.2; GMT) Dose IgG Subclass 4. Missing data codes: None 5. Abbreviations used: IgG = Immunoglobulin G; GMT = Geometric Mean Titer; m5C = 5-MethylCytidine; m5U = 5-MethylUridine; m1ψ = N1-Methylpseudouridine 6. Other relevant information: Relative levels of OVA antigen specific titer on serum as determined by ELISA, defined as the dilution of serum where the optical density at 450nm reaches 0.2.