Gene Expression from Pseudouridine and 5-Methylcytidine Modified Messenger RNA

Jiehua Zhou1, Maggie L. Bobbin1, Julie R. Escamilla-Powers2, Anton P. McCaffrey2, and John J. Rossi1.

1Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010. 2TriLink BioTechnologies, Inc. San Diego, CA 92121.

Objectives Figure 2 Figure 6 Develop methodologies for gram scale synthesis of eGFP Stick mRNA Transcript Processing messenger RNA (mRNA) for gene therapy applications, as well as scalable purification methods that yield highly expressed, persistent and non-toxic mRNA. Transcription / DNase Treatment 5’ UTR eGFP ORF 3’ UTR Stick Cap A120 Nano- S-S carrier

Abstract Silica Membrane Purification / Phosphatase Treatment Recently, there has been significant interest in the use of mRNA based expression systems for gene therapy Stick allows hybridization to delivery applications and for the generation and manipulation of vehicles such as nanocarriers or aptamers stem cells. Several groups have shown that mRNAs are Phosphatase Treatment attractive vehicles for therapeutic gene expression in mammals (Kormann et. al. Nat. Biotechnol (2011) 29, 154; Kariko et. al. Molecular Therapy (2012) Epub ahead of Figure 3 Silica Membrane Purification HPLC Fractionation print). Additionally, Warren et al. demonstrated highly mRNA Transfection Results in Very Robust efficient induced pluripotent stem cell (iPSCs) generation Expression in HEK-293 Cells by transfection of mRNAs encoding reprogramming factors (Warren et al. Cell Stem Cell (2010) 7, 618). The authors suggested that iPSCs generated in this manner should be eGFP-Stick mRNA with Lipofectamine 2000, 48 hr safer than iPSCs derived by plasmid transfection or viral Figure 7 transduction as there is no risk of insertional mutagenesis and subsequent oncogenesis. Effect of HPLC Fractionation on eGFP Intensity A key insight for the development of an mRNA expression system was the recognition that mRNAs induce innate immune responses in transfected cells. Kariko et al. showed that substitution of uridine and cytidine residues GFP Plasmid with Lipofectamine 2000, 48 hr with pseudouridine (ΨU) and 5-methlycytidine dramatically reduced innate immune recognition of mRNA (Kariko et al. Molecular Therapy (2008) 16, 1833). In addition, pseudouridine modified RNA was translated more efficiently and had increased nuclease resistance.

These studies highlight the importance of development of Fraction # 1 2 3 4 5 6 7 8 stable, non immunogenic mRNA to support these applications. In this study, capped pseudouridine and 5-methlycytidine modified eGFP mRNAs were synthesized Figure 4 at milligram scales. Fluorescence activated cell sorting eGFP-Stick mRNA Expression in CEM Cells and CD34+ (FACS) demonstrated >95% transfection in HEK-293 cells. Hematopoietic Stem Cells is Long Lived Expression in human CEM T-cells and CD34+ hematopoietic μg loaded 1 0.6 1 1 1 1 0.5 0.9 stem cells was also examined. Transfected mRNAs showed RNASep™ Column (Transgenomic, Inc.) a surprisingly long duration of expression (8-10 days Glyoxal Treated RNA on Agarose Gel post-transfection). CEM Cells CD34+ cells 8 9 7 Gene therapy applications of mRNA will require scalable 8 6 7 5 purification methods that are able to produce mRNAs at 6 4 5 gram scales. Recently, it was shown that purification of 0.5 4 0.4 mRNA by high-performance liquid chromatography (HPLC) 3 0.3 2 0.2 Figure 8 dramatically reduced innate immune responses relative to eGFP % Positive Cells 1

% eGFP % Positive Cells 0.1 0 0.0 unpurified mRNA (Kariko et al. Nucleic Acids Research 39, 2 4 8 1 10 Mean eGFP Fluorescence Intensity e142). Here we compare mRNA purified by classic silica Days Days membrane chromatography to HPLC purified materials. TransIT®-mRNA Kit (Mirus Bio, LLC) 90000

80000 24 hrs 48 hours 70000 Transcriptions 72 hours 96 hours Transcriptions with T7 RNA polymerase were carried out in HPLC Purification of mRNA 60000 the presence of anti reverse cap analog (ARCA), 50000 Kariko, et. al. reported that relative to unpurified mRNA, pseudouridine and 5-methylcytosine. Transcription with 40000 HPLC purified mRNA was less immuno-stimulatory and traditional cap analog (mCAP) results in incorporation of 30000 expression was significantly higher in cultured cell and in the cap in the wrong orientation ~50% of the time. In 20000 vivo. However, a typical mRNA workup involves contrast, transcription with ARCA results in insertion of the 10000

purification of the mRNA on a silica membrane spin fluorescenceMean Intensity cap in proper orientation 100% of the time. After removal 0 column. Standard treatment was compared to HPLC of NTPs, RNAs were phosphatase treated to remove purification. residual 5' triphosphate. RNAiMax Fraction 1Fraction 2Fraction 3Fraction 4Fraction 5Fraction 6Fraction 7Fraction 8 No Treatment UnmodifiedSilica RNA Membrane CHO Cell Transfection Figure 5 100 ng mRNA RNAiMax™ (Life Technologies) Figure 1 mRNAs of Different Sizes Elute at Similar Times from HPLC Nucleotide Structures

O

N NH O O O PO N N NH2 + O O P O O Conclusion N NH PO O

+ NH N N O O Na • mRNA transfection results in high level gene 2 O 3 OH OH hLin28 (911 nt) hSox2 (1235 nt) expression without the risk of insertional OH O mutagenesis. ARCA - Anti Reverse Cap Analog (TriLink Cat. # N-7003) • As reported by Kariko, et. al., substitution with Modifications Reducing Immune Stimulation pseudo-U and 5-methyl-C dramatically increases NH O 2 mRNA expression. hOct4 (1364 nt) hMyc (1601 nt) NH NH N O O • HPLC purification isolates functionally distinct O P O O O P O N O O O O P O O O P O O mRNA populations. + + 4Li PO O 4Li PO O O O O O OH OH OH OH

Pseudouridine Triphosphate 5-Methylcytidine Triphosphate (TriLink Cat. # N-1019) (TriLink Cat. # N-1014) For further information, please contact Anton hKlf4 (1694 nt) McCaffrey, [email protected]

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