Modified mRNA Production for HIV-1 Vaccine

Modified mRNAs were produced by in vitro transcription using T7 RNA polymerase (Megascript, Ambion) on linearized plasmids encoding codon-optimized CH848 10.17DT gp160s, CH848 10.17DT SOSIP trimers, or CH848 10.17DT trimer-ferritin NPs. All the HIV-1 modified mRNA constructs used in this study and their corresponding plasmids were listed in Table S1. One-methylpseudouridine (m1Ψ)-5’-triphosphate (TriLink, Cat# N-1081), instead of UTP was used to produce nucleoside-modified mRNAs. Modified mRNAs contain 101 nucleotide-long polyadenylation tails for optimized expression. Modified CH848 10.17DT SOSIPv4.1 trimer and CH848 10.17DT SOSIPv5.2.8 trimer mRNAs were capped using ScriptCap m7G capping system and ScriptCap 2′-O-methyl-transferase kit (ScriptCap, CellScript) (Pardi et al., 2013 (link)). Capping of all other in vitro transcribed mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog, CleanCap (TriLink, Cat# N-7413). All mRNAs were purified by cellulose purification, as described (Baiersdorfer et al., 2019 (link)). All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at −20 °C.

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Mu Z., Wiehe K., Saunders K.O., Henderson R., Cain D.W., Parks R., Martik D., Mansouri K., Edwards R.J., Newman A., Lu X., Xia S.M., Bonsignori M., Montefiori D., Han Q., Venkatayogi S., Evangelous T., Wang Y., Rountree W., Tam Y., Barbosa C., Alam S.M., Williams W.B., Pardi N., Weissman D, & Haynes B.F. (2021). Ability of nucleoside-modified mRNA to encode HIV-1 envelope trimer nanoparticles. bioRxiv.

Publication Preprint 2021

Megascript ScriptCap 2′-O-methyl-transferase kit

Agarose gel electrophoresis Cellulose Codon Ferritin Frozen Hiv 1 Mrnas Nucleoside Nucleotide Plasmids Polyadenylation T7 rna polymerase Tails Transcription Transferase Triphosphate

Corresponding Organization : University of Pennsylvania

Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Acuitas Therapeutics (Canada)

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Variable analysis

independent variables

Transcription using T7 RNA polymerase on linearized plasmids encoding codon-optimized CH848 10.17DT gp160s, CH848 10.17DT SOSIP trimers, or CH848 10.17DT trimer-ferritin NPs
Use of one-methylpseudouridine (m1Ψ)-5'-triphosphate instead of UTP to produce nucleoside-modified mRNAs
Capping method (ScriptCap m7G capping system and ScriptCap 2′-O-methyl-transferase kit for CH848 10.17DT SOSIPv4.1 trimer and CH848 10.17DT SOSIPv5.2.8 trimer mRNAs, and CleanCap trinucleotide cap1 analog for all other in vitro transcribed mRNAs)

dependent variables

Not explicitly mentioned

control variables

Purification of all mRNAs by cellulose purification
Storage of all mRNAs frozen at -20 °C

positive controls

Not mentioned

negative controls

Not mentioned

Annotations

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