Nucleic Acids Research Advance Access published September 2, 2011
`Nucleic Acids Research, 2011, 1–10
`doi:10.1093/nar/gkr695
`
`Generating the optimal mRNA for therapy:
`HPLC purification eliminates immune activation
`and improves translation of nucleoside-modified,
`protein-encoding mRNA
`Katalin Kariko´ 1, Hiromi Muramatsu1, Ja´ nos Ludwig2 and Drew Weissman3,*
`
`1Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA, 2Institute of Clinical Chemistry
`and Pharmacology, University of Bonn, Bonn, Germany and 3Department of Medicine, University of
`Pennsylvania, Philadelphia, PA, USA
`
`Received May 26, 2011; Revised August 9, 2011; Accepted August 10, 2011
`
`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`ABSTRACT
`In vitro-transcribed mRNA has great therapeutic po-
`tential to transiently express the encoded protein
`without the adverse effects of viral and DNA-based
`constructs. Mammalian cells, however, contain RNA
`sensors of the innate immune system that must
`be considered in the generation of
`therapeutic
`RNA. Incorporation of modified nucleosides both
`reduces innate immune activation and increases
`translation of mRNA, but
`residual
`induction of
`type I interferons (IFNs) and proinflammatory cyto-
`kines remains. We identify that contaminants,
`including double-stranded RNA,
`in nucleoside-
`modified in vitro-transcribed RNA are responsible
`for innate immune activation and their removal by
`high performance liquid chromatography (HPLC)
`results in mRNA that does not induce IFNs and in-
`flammatory cytokines and is translated at 10- to
`1000-fold greater levels in primary cells. Although
`unmodified mRNAs were translated significantly
`better
`following purification,
`they still
`induced
`high levels of cytokine secretion. HPLC purified
`nucleoside-modified mRNA is a powerful vector for
`applications ranging from ex vivo stem cell gener-
`ation to in vivo gene therapy.
`
`INTRODUCTION
`
`Our understanding of the importance of RNA in biologic-
`al processes and the therapeutic potential has substantially
`increased with the discovery of non-coding regulatory
`RNAs. The use of mRNA has also expanded, including
`the delivery of mRNA to generate induced pluripotent
`stem (iPS) cells (1–3) and in vivo administration to
`
`express therapeutic proteins (4). The recognition that the
`immunogenicity of RNA could be reduced by the incorp-
`oration of modified nucleosides with a concomitant
`increase in translation (5), potentially allows efficient ex-
`pression of intra and extracellular proteins in vivo and ex
`vivo without activation of
`innate immune pathways.
`Unfortunately, modified nucleoside-containing RNA
`transcribed by phage RNA polymerase transcription still
`retains a low level of activation of such pathways (3,5–7).
`The remaining activation of RNA sensors by nucleoside
`modified RNA could be because the modifications do not
`completely suppress the RNAs ability to activate sensors
`or due to contaminants with structures that activate in the
`presence of nucleoside modification. It is well established
`that RNA transcribed in vitro by phage polymerase
`contains multiple contaminants,
`including short RNAs
`produced by abortive initiation events (8) and double-
`0
`ex-
`stranded (ds)RNAs generated by self-complementary 3
`tension (9), RNA-primed transcription from RNA tem-
`plates
`(10) and RNA-dependent RNA polymerase
`activity (11).
`Large quantities of RNA can be easily prepared by
`in vitro transcription from DNA templates using phage
`RNA polymerase or solid-phase chemical synthesis. For
`uses that require further purification, such as NMR (12),
`crystallography (13) and therapeutic applications (14), a
`number of techniques have been developed. Preparative
`denaturing
`polyacrylamide
`gel
`electrophoresis
`is
`commonly used to purify in vitro-transcribed RNA,
`however, this method is suitable only for short RNAs
`[reviewed in (15)]. Long RNAs can be separated on
`denaturing agarose gels, but they are not translatable
`due to covalent modifications introduced by the denatur-
`ants glyoxal and formaldehyde (16). Chromatography
`based on size exclusion can efficiently remove unincorpor-
`ated nucleoside triphosphates, small abortive transcripts
`and plasmid template from the desired RNA product
`
`*To whom correspondence should be addressed. Tel: +1 215 573 8491; Fax: +215 349 5111; Email: dreww@mail.med.upenn.edu
`
`ß The Author(s) 2011. Published by Oxford University Press.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
`by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`CUREVAC EX2031
`Page 1
`
`

`

`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`polystyrene-divinylbenzene
`copolymer microspheres
`(21 100 mm column). Buffer A contained
`(2.1 mm)
`0.1 M triethylammonium acetate (TEAA), pH = 7.0 and
`Buffer B contained 0.1 M TEAA, pH = 7.0 and 25%
`acetonitrile (Transgenomics). Columns were equilibrated
`with 38% Buffer B, loaded with RNA and run with a
`single or 2 linear gradients to 55 or 65% Buffer B over
`20–30 min at 5 ml/min. RNA analyses were performed
`with the same column matrix and buffer system using a
`7.8 mm 50 mm column at 1.0 ml/min.
`
`RNA isolation from column fractions
`
`RNA content from desired fractions was concentrated and
`desalted using Amicon Ultra-15 centrifugal filter units
`(30K membrane) (Millipore) by successive centrifugation
`
`at 4000g for 10 min (4
`C) in a Sorvall ST16R centrifuge
`(Thermo Scientific) and dilution with nuclease free water.
`The RNA was recovered by overnight precipitation at
`20
`
`C in NaOAc
`(0.3 M, pH 5.5),
`isopropanol
`(1 volume) (Fisher) and glycogen (3 ml) (Roche).
`
`Dot blot
`
`RNA (200 ng) was blotted onto super charged Nytran,
`dried, blocked with 5% non-fat dried milk in TBS-T
`buffer
`(50 mM Tris–HCl,
`150 mM NaCl,
`0.05%
`Tween-20, pH 7.4), and incubated with dsRNA-specific
`mAb J2 or K1 (English & Scientific Consulting) for
`60 min. Membranes were washed six times with TBS-T
`and reacted with HRP-conjugated donkey anti-mouse Ig
`(Jackson Immunology), washed six times and detected
`with ECL Plus Western
`blot
`detection
`reagent
`(Amersham).
`Images were
`captured on a Fujifilm
`LAS1000 digital imaging system. dsRNA (25 ng) used as
`a positive control was derived from sense and antisense
`strands of T7TS UTR sequence (328 bp). Blots were
`reprobed with 32P-labeled DNA complementary to the
`0
`3
`-UTR of the RNA to document the presence of RNA.
`
`Complexing of RNA
`
`Lipofectin (Invitrogen) complexing was performed as
`described previously (5) using 0.8 ml of Lipofectin and
`0.1 mg of RNA per well of a 96-well plate. Complexing
`of RNA to TransIT mRNA (Mirus Bio) was performed
`according to the manufacturer combining RNA (0.1 mg)
`with TransIT mRNA (0.3 ml) and boost (0.2 ml) reagents.
`
`Cell transfections
`
`For Lipofectin complexed RNA, medium was removed
`and 50 ml of complexed RNA was added to 5 x 104 293T
`or DCs per well. Cells were incubated for 1 h and the
`replaced with 200 ml
`Lipofectin-RNA mixture was
`complete medium. For TransIT complexed RNA, 17 ml
`of complex was added to cells, 293T, DCs, or 2  105 kera-
`tinocytes cultured in 183 ml complete medium. Cells were
`lysed in firefly or Renilla specific lysis reagents (Promega)
`at 24 h post RNA addition. Aliquots were assayed for
`enzyme activities using firefly and Renilla luciferase
`assay systems
`(Promega) and a LUMAT LB 950
`luminometer (Berthold/EG&G; Wallac). Expression of
`
`CUREVAC EX2031
`Page 2
`
`2 Nucleic Acids Research, 2011
`
`under native conditions (17,18), but is limited in its ability
`to remove contaminants with similar sizes and contamin-
`ants complementary to the RNA selected to purify. No
`technique has been reported for purification and prepara-
`tive isolation of long in vitro-transcribed mRNA that
`removes contaminating complementary strands and pre-
`serves its translatability.
`The development of mRNA to use as a tool to replace
`in
`vivo
`intra-
`and extracellular proteins
`and to
`transdifferentiate, reprogram and differentiate cells ex
`vivo requires the RNA to have high translatability and
`no RNA sensor activation. In this report, we identify
`that contaminants from in vitro-transcribed RNA are a
`source of innate immune activation and their removal in-
`creases RNA translation and eliminates type I interferon
`and inflammatory cytokine secretion.
`
`MATERIALS AND METHODS
`
`Cells
`
`Human embryonic kidney 293T cells (American Type
`Culture Collection) were cultured in Dulbecco’s modified
`Eagle’s medium (DMEM) supplemented with 2 mM L-glu-
`tamine (Life Technologies) and 10% fetal calf serum
`(FCS)
`(HyClone)
`(complete medium). Human and
`murine dendritic cells (DCs) were generated as described
`(5). Human keratinocytes were obtained from the Skin
`Disease Research Core (Penn) and grown in MCDB
`with bovine pituitary extract (140 mg/ml) (Sigma) and
`70 mM Ca++ on collagen (0.01 mg/ml)
`(Invitrogen)
`coated plates.
`
`mRNA synthesis
`
`mRNAs were transcribed as previously described (5),
`using linearized plasmids
`encoding firefly luciferase
`(pT7TSLuc and pTEVLuc), codon-optimized murine
`erythropoietin (pTEVmEPO), enhanced green fluorescent
`protein (pTEVeGFP), Metridia luciferase (pT7TSMetluc)
`or Renilla luciferase (pT7TSRen and pTEVRen) and T7
`RNA polymerase (Megascript, Ambion). All mRNAs were
`transcribed to contain 30 or 51-nt long poly(A) tails.
`Additional poly(A) tail was added with yeast poly(A) poly-
`merase (USB) and noted as An. Triphosphate-derivatives of
`pseudouridine ( ) and 5-methylcytidine (m5C) (TriLink)
`were used to generate modified nucleoside containing
`RNA. All RNAs were capped using the m7G capping
`0
`-O-methyltransferase (ScriptCap,
`kit with or without 2
`CellScript) to obtain cap1 or cap0. We did not observed
`differences in the immunogenicity of cap0- and cap1-
`containing nucleoside-modified RNAs. All RNAs were
`analyzed by denaturing or native agarose gel electrophor-
`esis. Pseudouridine-modified mRNAs encoding KLF4,
`LIN28, cMYC, NANOG, OCT4 and SOX2 were a kind
`gift of CellScript, Inc.
`
`HPLC purification of RNA
`
`liquid
`RNA was purified by High performance
`chromatography (HPLC) (Akta Purifier, GE Healthcare)
`using
`a
`column matrix of
`alkylated non-porous
`
`

`

`Nucleic Acids Research, 2011
`
`3
`
`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`Figure 1. In vitro-transcribed RNA is
`immunogenic and contains
`in vitro transcripts encoding
`dsRNA contaminants.
`(A) 200 ng of
`mEPO and containing the
`indicated modified nucleosides were
`blotted and analyzed with K1 and J2 dsRNA-specific mAbs. The
`dsRNA positive control contained a 328 bp long dsRNA (25 ng).
`(B) DCs were treated with Lipofectin-complexed Renilla luciferase
`firefly
`and Metridia
`luciferases
`(T7TSRenA30),
`(T7TSLucA30,
`T7TSMetlucA30), and mEPO (TEVmEPOA51) mRNAs. TNF-a levels
`were measured in the supernatants at 24 h. (C) DCs were treated with
`TransIT-complexed in vitro transcripts encoding Renilla and firefly
`luciferases (T7TSRenA30, T7TSLucA30), eGFP (TEVeGFPA51) and
`mEPO (TEVmEPOA51). IFN-a levels were measured in the super-
`natants at 24 h. Error bars are standard error of the mean. Data
`shown is from one experiment that is representative of 3–6.
`
`of RNAs with coding sequences for mammalian and
`0
`0
`reporter proteins flanked by different 5
`- and 3
`-UTRs
`were analyzed. The RNAs were cell-delivered following
`complexing with Lipofectin, a cationic lipid, or TransIT,
`a membrane active polymer and lipid mixture. RNA com-
`plexed with Lipofectin induced high levels of TNF-a and
`moderate levels of IFN-a, while RNA complexed with
`
`CUREVAC EX2031
`Page 3
`
`eGFP in DCs was documented using an inverted
`epifluorescent Nikon microscope mounted with a Nikon
`D40 digital camera. Murine EPO protein was measured
`with a specific ELISA assay (R&D Systems).
`
`RNA immunogenicity analyses
`DCs (murine or human) (5 104 cells/well) in 96-well
`plates were treated with medium, R-848 (Invivogen), or
`Lipofectin- or TransIT-complexed RNA or poly(I:C)
`(Sigma). Supernatant was harvested after 24 h and the
`IFN-a,
`IFN-b (PBL InterferonSource), or
`levels of
`TNF-a (Biosource International) were measured by
`ELISA.
`
`Gene array analysis
`Human DCs from three donors were generated in 5%
`FCS. Cells (1 106 DCs/well of a 6-well plate) were
`treated with TransIT-complexed TEVRenA51 RNA with
`or without modification and with or without purification.
`Six hours later, RNA was isolated using RNeasy (Qiagen).
`RNA was amplified with the TargetAmp Nano-g
`Biotin-aRNA labeling kit (Epicentre) and analyzed on
`an Illumina Human HT12v4 chip in an Illumina
`BeadStation 500GX. Raw data was processed by the
`Bead Studio v.3.0 software. Levels in untreated DC were
`used as the baseline for the calculation of fold increase.
`
`Northern blot
`
`Samples were processed and analyzed as previously
`described (6). Probes were derived from plasmids and
`were specific for the coding regions of human IFN-a13,
`IFN-b (Open Biosystems), TNF-a, or GAPDH (ATCC).
`
`RESULTS
`
`A dot blot assay with J2 and K1 monoclonal antibodies
`(mAbs) that recognize dsRNA (19) was used to determine
`whether in vitro-transcribed RNA contains dsRNA. These
`mAbs recognize continuous double stranded structure
`of at least 40 bp in length (20), which is not found in
`any of the coding sequences or UTRs in the mRNAs
`analyzed in this study. Testing mammalian and re-
`porter protein-encoding in vitro transcripts containing
`either no nucleoside modifications, pseudouridine- ( ),
`or 5-methylcytidine- (m5C) and - (m5C/ ) nucleoside
`modifications, we found that all
`samples contained
`dsRNA contamination (Figure 1A and data not shown).
`Recognition of dsRNA by J2 mAb was not affected by the
`presence of modified nucleosides, while K1 had reduced
`binding to dsRNA containing or m5C/ nucleoside
`modifications.
`Others and we have previously demonstrated that in-
`corporation of modified nucleotides into RNA reduced
`its ability to activate RNA sensors including Toll-like
`receptor (TLR)3, TLR7 and TLR8 (21), retinoic acid
`inducible gene I
`(RIG-I)
`(22) and RNA-dependent
`protein kinase (PKR)
`(6,23). Monocyte-derived DCs
`that express these and all other known RNA sensors
`(24) were used to measure residual
`immune activation
`present in modified nucleoside-containing RNA. A series
`
`

`

`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`HPLC-purified - or m5C/ -modified RNAs that were
`complexed with Lipofectin or TransIT,
`respectively
`(Figure 3C and D and data not shown). Similarly, no
`cytokine induction could be detected when HPLC-
`purified modified nucleoside-containing RNAs were trans-
`fected into murine DCs. HPLC purification similarly
`ablated IFN-a secretion from DCs transfected with the
`clinically relevant -nucleoside modified mRNAs com-
`plexed to TransIT used in Figure 3B. However,
`HPLC-purified RNA without nucleoside modification
`inducers of TNF-a and IFN-a
`remained potent
`(Figure 3C and D).
`The impact of HPLC purification of unmodified, - and
`m5C/ -modified RNA on gene expression in human DCs
`was analyzed using gene arrays. Total cellular RNA
`isolated from DCs from three different donors 6 h after
`cells were transfected with TransIT-complexed RNA, were
`analyzed on an Illumina Human HT12v4 chip. RNA
`modified with or m5C/ nucleosides induced less ex-
`pression of type I interferons, interleukins, tumor necrosis
`factor (TNF) family members, chemokines and markers
`associated with DC activation, while HPLC purification
`of - and m5C/ -modified RNA further reduced induc-
`tion of these genes to the levels observed in cells treated
`only with TransIT (Figure 4A). The same sets of total
`RNA from DCs that were tested on the gene arrays
`were also analyzed for levels of IFN-a, IFN-b and
`TNF-a mRNA by northern blot. Lower levels of IFN-a,
`IFN-b and TNF-a mRNAs were detectable in DCs
`treated with nucleoside modified as compared to unmodi-
`fied RNA. More importantly, none of these cytokine
`mRNAs were detectable when DCs were transfected
`with HPLC-purified RNAs containing or m5C/ modi-
`fication. However, HPLC purified RNA without nucleo-
`side modification remained a potent inducer of IFN-a,
`IFN-b and TNF-a mRNAs (Figure 4B).
`To determine whether HPLC purification affected
`translatability of in vitro transcripts, a series of mRNAs
`were tested following cell delivery. HPLC-purified Renilla
`and mouse
`erythropoietin (mEPO) mRNAs were
`translated at 2- to 20-fold higher levels compared to un-
`purified RNA when delivered to 293 T cells by TransIT
`(Figure 5A). In primary human DCs, the translational
`enhancement was more robust, resulting in up to a
`1000-fold increase when the same sets of unpurified and
`HPLC-purified mRNAs were transfected with Lipofectin
`(Figure 5B) or TransIT (Figure 5C). Similar increases in
`translation were observed for other mRNAs after HPLC
`purification, including mRNAs encoding firefly luciferase,
`human EPO, macaque EPO and Metridia luciferase, and
`other cell types, including mouse embryonic fibroblasts
`and human primary keratinocytes. Translation levels
`were much higher with - and m5C/ -modified eGFP
`mRNA when the mRNA was HPLC-purified prior to
`transfection of human DCs (Figure 5D and data not
`shown).
`To characterize the contaminants being removed by
`HPLC purification, three fractions corresponding to RNAs
`eluting from the column prior to the major transcription
`product (fraction I), the full-length transcription product
`(fraction
`II),
`and RNAs
`eluting
`after
`the
`
`CUREVAC EX2031
`Page 4
`
`4 Nucleic Acids Research, 2011
`
`TransIT induced low levels of TNF-a and high levels of
`IFN-a (data not shown) with some donor-dependent vari-
`ation. Typically, Lipofectin-complexed RNA with or
`m5C/ modifications induced less TNF-a. (Figure 1B),
`while TransIT-complexed RNA with or without nucleo-
`side modification induced variable, sequence-dependent
`effects on IFN-a secretion (Figure 1C). These data
`suggest
`that
`the presence of dsRNA and potentially
`other contaminants in in vitro-transcribed RNA could be
`responsible for innate immune activation.
`Multiple HPLC bead matrix compositions and buffer
`systems were
`screened and alkylated non-porous
`polystyrene-divinylbenzene
`copolymer matrix
`and
`triethylammonium acetate buffer with an acetonitrile
`gradient was identified as a system capable of removing
`dsRNA and other contaminants from in vitro-transcribed
`RNA. The HPLC chromatogram of -modified mRNA
`encoding enhanced green fluorescent protein (eGFP)
`demonstrated a major peak (Figure 2), which was col-
`lected and identified as the expected RNA product using
`agarose gel electrophoresis. Additional UV-absorbing
`products with shorter and longer retention times relative
`to the main RNA product could also be observed.
`Reanalysis of the purified RNA by HPLC demonstrated
`a single peak with the same retention time. RNAs with or
`without nucleoside modification encoding different se-
`quences yielded similar patterns with varying relative
`heights for the preceding and succeeding peaks.
`HPLC purification of both unmodified and nucleoside-
`modified RNA reduced staining by dsRNA-specific mAb
`to baseline levels (Figure 3A). Analysis of -modified
`RNA encoding clinically relevant proteins demonstrated
`that the amounts of dsRNA contamination in the in vitro
`transcripts were dependent on the sequence, but HPLC
`could successfully remove the contaminants from all of
`them (Figure 3B). Next, the HPLC-purified RNAs were
`tested on human DCs. No TNF-a or type-I interferons
`(IFN-a and b) were induced following transfection of
`
`Figure 2. HPLC purification of RNA identifies contaminants eluting
`before and after the expected product. Chromatogram of -modified
`TEVeGFPAn mRNA. RNA was applied to the HPLC column and
`eluted using a linear gradient of Buffer B (0.1 M TEAA, pH 7.0,
`25% acetonitrile) in Buffer A (0.1 M TEAA, pH 7.0). The gradient
`spanned 38–55% Buffer B over 22 min (red line). Absorbance at
`260 nm was analyzed (black line), which demonstrated the expected
`sized RNA as well as smaller and larger RNA species. Data shown
`are from one experiment that is representative of over 200.
`
`

`

`Nucleic Acids Research, 2011
`
`5
`
`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`Figure 3. HPLC purification of in vitro-transcribed nucleoside modified mRNA removes dsRNA contaminants and eliminates immunogenicity.
`(A) 200 ng of RNA encoding the indicated protein and containing the indicated modified nucleosides with or without HPLC purification were
`blotted and analyzed with the J2 dsRNA-specific mAb. (B) 200 ng of RNA encoding the indicated protein and containing -modifications with or
`without HPLC purification were blotted and analyzed with the J2 dsRNA-specific mAb. Blots were reprobed with a 32P-labeled probe for the
`0
`-UTR of the RNAs to control for amount of RNA analyzed. (C) DCs were treated with TEVRenA51 RNA containing the indicated nucleoside
`3
`modifications with or without HPLC purification and complexed to Lipofectin. TNF-a levels were measured in the supernatants at 24 h. Differences
`in the effect of nucleoside modification on immunogenicity of Renilla-encoding mRNA compared to Figure 1B is likely due to donor variation and
`differences in UTRs of the RNAs. (D) DCs were treated with TEVLucA51 RNA containing the indicated nucleoside modifications with or without
`HPLC purification and complexed to TransIT. IFN-a levels were measured in the supernatants at 24 h. Error bars are standard error of the mean.
`Data shown is from one experiment that is representative of 3 or more.
`
`expected transcription product (fraction III) were col-
`lected (Figure 6A). Nucleoside-modified RNA occasional-
`ly demonstrated a second smaller peak overlying the large
`peak and isolation and purification of both peaks
`demonstrated similar RNA lengths on denaturing and
`non-denaturing agarose gel electrophoresis and RNAs
`with similar levels of translation and immunogenicity.
`RNA purified from the three fractions was analyzed for
`immunogenicity. RNA in fractions I and III induced
`IFN-a secretion from transfected DCs, while the purified
`full-length RNA (fraction II) was not immunogenic when
`it contained - or m5C/ -nucleoside modifications
`(Figure 6B). Fraction I RNA had low levels of staining
`with the J2 mAb, while fraction III RNA had high levels
`of staining similar to the unpurified RNA (Figure 6C).
`Primary human keratinocytes and murine fibroblasts
`treated once with unpurified, unmodified RNA delivered
`by TransIT complexing demonstrated detachment from
`the collagen-coated plastic base as evidence of cell death.
`A second delivery of unmodified RNA 24 h later resulted
`in the termination of the culture. Substantially less tox-
`icity was observed when the RNA contained - or m5C/
` -nucleoside modifications, but repeated daily delivery
`of m5C/ -nucleoside modified mRNA to keratino-
`cytes reduced the final cell number by 75% on day 11
`
`the RNA greatly
`(Figure 7). HPLC purification of
`reduced toxicity in treated keratinocytes where unmodi-
`fied mRNA caused minimal cell rounding and death.
`Daily treatment of keratinocytes
`for 10 days with
`HPLC-purified m5C/ -modified mRNA complexed to
`TransIT showed no signs of cell toxicity and the rate of
`proliferation was similar to that obtained with TransIT
`alone treated cells (Figure 7).
`
`DISCUSSION
`
`Modified nucleoside-containing mRNA has previously
`been used for the induction of iPS cells from fibroblasts
`with very high efficiency (3). The authors determined that
`m5C/ -nucleoside modified mRNA yielded the least
`amount of RNA sensor activation and the highest level
`of translation, but needed to add the B18R protein, a
`vaccinia virus decoy receptor for type I interferon (25),
`for optimal iPS cell generation. We previously reported
`that modified nucleoside-containing mRNA was efficient-
`ly delivered to primary dividing and non-dividing cells and
`produced high levels of encoded protein in an easily
`controlled manner
`(5).
`In addition,
`the RNA had
`reduced innate immune sensor activation. The residual
`amount of activation of modified nucleoside-containing
`
`CUREVAC EX2031
`Page 5
`
`

`

`6 Nucleic Acids Research, 2011
`
`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`Figure 4. HPLC purification of in vitro-transcribed nucleoside-modified mRNA eliminates activation of genes associated with RNA sensor activa-
`tion. (A) Heatmap representing changes in expression of genes activated by RNA sensors were derived from microarray analyses of DCs treated for 6
`hr with TransIT alone or transit-complexed TEVRenA51 RNA with the indicated modifications with or without HPLC purification. RNA from
`medium-treated cells was used as the baseline for comparison. (B) Northern blot of RNA from DCs treated with medium or TransIT alone or
`TransIT-complexed TEVRenA51 RNA with the indicated modifications with or without HPLC purification and probed for IFN-a, IFN-b, TNF-a
`and GAPDH mRNAs.
`
`mRNA depended on the sequence. In this report, we
`identify that m5C/ -nucleoside-modified RNA often has
`the least ability to induce RNA sensor activation, but even
`with these modifications, certain RNA sequences induce
`high levels of cytokine production (Figure 1B and C).
`HPLC purification removes dsRNA and other contamin-
`ants from in vitro-transcribed RNAs containing or
`m5C/ nucleosides, yielding RNA with the highest
`levels of
`translation, up to 1000-fold more
`than
`non-HPLC purified RNA with no release of type I IFNs
`
`or TNF-a and no significant induction of genes associated
`with RNA sensor activation. The purification procedure
`can be easily scaled to produce large amounts of RNA
`necessary for
`therapeutic applications and can be
`completed quickly and efficiently.
`The data suggests that different types of immunogenic
`contaminants are present in in vitro-transcribed mRNA. A
`series of RNAs that eluted before the major transcription
`product, suggesting a smaller size, induced high levels of
`IFN-a, but had minimal staining with dsRNA-specific
`
`CUREVAC EX2031
`Page 6
`
`

`

`Nucleic Acids Research, 2011
`
`7
`
`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`Figure 5. HPLC purification of in vitro transcribed mRNA enhances translation. 293T (A) and human DCs (B and C) were transfected with
`TransIT- (A and C) or Lipofectin- (B) complexed TEVRenA51 or TEVmEPOA51 mRNA with the indicated modifications with or without
`HPLC purification and analyzed for Renilla luciferase activity or levels of supernatant-associated mEPO protein at 24 h. (D) Human DCs were
`transfected with -modified TEVeGFPAn mRNA with or without HPLC purification (0.1 mg/well) complexed with Lipofectin or TransIT and
`analyzed 24 h later. Error bars are standard error of the mean. Data shown is from one experiment that is representative of three or more.
`
`mAbs. A second series of RNAs that eluted after the main
`transcription product were immunogenic when the RNA
`was unmodified or contained -modifications and had
`levels of dsRNA staining similar to the unpurified RNA
`(Figure 6). Long RNA (2 kb) with an added shorter length
`(276 nt) of complimentary RNA eluted at high concentra-
`tions of acetonitrile, i.e. had a longer retention time, sug-
`gesting that ds structures delay elution from the column
`matrix (K.K., H.M. and D.W., preliminary data). Three
`of the known mechanisms that produce contaminants
`0
`during in vitro transcription, self-complementary 3
`exten-
`sion (9), RNA-primed transcription from RNA templates
`(10) and RNA-dependent RNA polymerase activity (11)
`can result in dsRNA of various lengths. If these contam-
`inants contain the main transcription product, they would
`likely elute after
`the main transcription product.
`Interestingly, fraction III RNA from the purification of
`m5C/ -modified RNA stained with dsRNA mAb, but
`induced little IFN-a (Figure 6). dsRNA containing nu-
`cleoside modifications, including , has reduced ability
`
`to activate PKR (23), but whether m5C/ -nucleoside
`modifications in dsRNA block its ability to induce type
`I IFNs through RNA sensors is unknown. The nature of
`the contaminants that elute prior to the main product is
`unknown. They have minimal staining with dsRNA
`specific mAb, but these mAbs require extended lengths
`of dsRNA for binding (20). We cannot rule out that
`shorter segments of dsRNA are present in the RNA that
`elutes prior to the main transcription product.
`HPLC purification of mRNAs enhanced their transla-
`tion up to a 1000-fold in primary cells. The level of en-
`hancement was greatest when the mRNA was unmodified,
`decreased when was incorporated and decreased fur-
`ther when m5C and were present (Figure 5A–C).
`These differences in translational enhancement of the
`mRNAs are likely due to the RNA sensors PKR and
`oligoadenylate synthetase (OAS) that directly decrease
`translation with activation. We previously demonstrated
`that incorporation of m5C and into long mRNA
`reduced its ability to activate PKR (6). Similarly, we
`
`CUREVAC EX2031
`Page 7
`
`

`

`8 Nucleic Acids Research, 2011
`
`Downloaded from
`
`nar.oxfordjournals.org
`
` at Aerztegemeinschaft fuer Anaesthesie on September 12, 2011
`
`Figure 7. Daily transfection with HPLC-purified m5C/ -modified
`mRNA does not reduce cell proliferation. Primary keratinocytes were
`or m5C/ -modified
`transfected
`daily with
`TransIT
`alone
`RNA-encoding Renilla luciferase with or without HPLC purification
`complexed with TransIT. Every 2–3 days, cultures were split and equal
`numbers of cells for each condition were plated. Total cell numbers for
`each condition were divided by the total cell number in untreated cells
`to calculate the percent of control proliferation.
`
`dsRNA contaminants could involve RNA interference.
`dsRNA greater than 27 bp in length is a substrate for
`Dicer (27), whose action can lead to a specific suppression
`of translation through the RNAi pathway (28). dsRNA
`contaminants homologous to the desired mRNA would
`lead to a specific suppression of translation in addition
`to the non-specific suppression through RNA sensors of
`the innate immune system.
`The observation that the complexing agent used with
`identical RNAs alters the type of cytokines released
`from DCs could be caused by an effect of the complexing
`agent on the interaction between the RNA and endosomal
`RNA sensors, the location of RNA after cytoplasmic
`entrance, or the amount of time the RNA remains in the
`endosome after endocytosis. RNA sensing TLRs require
`acidification of endosomes to signal (29). A complexing
`agent
`that allows
`endocytosed RNA to exit
`the
`endosome and enter the cytoplasm before acidification
`and TLR signaling would result in reduced TNF-a secre-
`tion, as was observed with TransIT-complexed mRNA. In
`a
`comparison
`of
`cationic
`liposomes
`to
`linear
`polyethyleneimine (PEI) (a cationic polymer) delivery of
`CpG containing DNA,
`it was found that PEI-DNA
`induced less TNF-a, which was associated with a faster
`exit from endosomes, while cationic lipid delivery resulted
`in DNA remaining in vesicular structures for extended
`periods of time and high levels of TNF-a (30). We simi-
`larly observed that cationic liposome complexed RNA
`induced higher levels of TNF-a compared to the cationic
`polymer with lipid TransIT. Another possibility for the
`discrepant cytokine response could be due to the sizes of
`the complexed mRNA particles. A recent report by
`Retting et al. (31) demonstrated that nanometric particles
`
`CUREVAC EX2031
`Page 8
`
`Figure 6. Analysis of RNA contaminants removed by HPLC purifica-
`tion. (A) One hundred microgram of -modified T7TSLucA30 RNA
`was applied to the HPLC column and 3 fractions were collected, all
`RNAs eluting before the main transcription product (I), the expected
`RNA (II), and all RNAs eluting after the main transcription product
`(III). The gradient began at 38% Buffer B and increased to 43% Buffer
`B over 2.5 min and then spanned 43–65% Buffer B over 22 min.
`Unmodified and m5C/ -modified T7TSLucA30 RNA had similar frac-
`tions obtained. (B) The RNAs from each fraction were complexed to
`TransIT and added to DCs and IFN-a in the supernatant was
`measured 24 h later. Error bars are standard error of
`the mean.
`(C) 200 ng of RNA from the 3 fractions and the starting unpurified
`RNA were blotted and analyzed with the J2 dsRNA-specific mAb.
`
`recently demonstrated that RNA with -modifications in
`the absence of dsRNA contaminants induced less activa-
`tion of OAS compared to unmodified mRNA (26). It is
`also possible that RNA samples with nucleoside modifica-
`tions contain a smaller amount of short dsRNA contam-
`inants as the dsRNA-specific mAbs do not recognize
`dsRNA shorter than 40 bp. An additional mechanism
`for the inhibition of translation by mRNA containing
`
`

`

`Downloaded f