Granted Patent
B2
US 12,533,422 · App. 17/136,724 · Granted Jan 27, 2026
Modified RNA with decreased immunostimulatory properties
Assignee:
CureVac SE
View Patent ↗
Loading inventors, assignments & file history…
Abstract
The present invention provides a method for providing modified mRNAs of reduced immunogenicity and/or immunostimulatory capacity for use in protein replacement therapy. The invention further provides modified mRNAs and pharmaceutical compositions comprising the modified mRNAs according to the invention for use in protein replacement therapy.
Claims (25)
1 . A method for expressing a biologically active polypeptide in a mammalian subject comprising administering to the subject a pharmaceutical composition comprising a purified RNA encoding the biologically active polypeptide formulated in a pharmaceutically acceptable carrier, said purified RNA comprising:
(i) a m7G 5′ Cap;
(ii) a 5′ untranslated region (UTR);
(iii) a coding sequence encoding the biologically active polypeptide, wherein said coding sequence is a modified coding sequence that has been modified to replace at least 70% of the cytosine-content optimizable codons with replacement codons having a higher cytosine content, wherein said replacement codons do not have a higher G-content than said cytosine-content optimizable codons;
(iv) a 3′ UTR; and
(v) a poly-A sequence of at least 60 nucleotides.
2 . The method of claim 1 , wherein at least 80% of the cytosine-content optimizable codons are replaced with codons having a higher cytosine content in the modified coding sequence.
3 . The method of claim 2 , wherein at least 90% of the cytosine-content optimizable codons are replaced with codons having a higher cytosine content in the modified coding sequence.
4 . The method of claim 3 , wherein 100% of the cytosine-content optimizable codons are replaced with codons having a higher cytosine content in the modified coding sequence.
5 . The method of claim 1 , wherein the RNA comprises at least one nucleotide substituted with a nucleotide analogue having a base modification.
6 . The method of claim 5 , wherein the nucleotide analogue is pseudouridine or 1-methyl-pseudouridine.
7 . The method of claim 5 , wherein the poly-A sequence is no more than 200 adenosine nucleotides and is located at the 3′ end of the RNA.
8 . The method of claim 1 , wherein the poly-A sequence comprises at least 70 nucleotides.
9 . The method of claim 8 , wherein the RNA is a mRNA.
10 . The method of claim 1 , wherein the subject has a hereditary disease.
11 . The method of claim 1 , wherein the subject has a disease caused by an amino acid disorder, carbohydrate metabolism disorder, cholesterol biosynthesis disorder, fatty acid oxidation defect, fat metabolism disorder, lactic acidosis, glycogen storage disease, mitochondrial disorder, organic acid disorder, urea cycle disorder or lysosomal storage disease disorder.
12 . The method of claim 1 , wherein the modified coding sequence encodes a wild type human polypeptide.
13 . The method of claim 12 , wherein the modified coding sequence has a cytoside content that is increased by at least 10% relative to a wild type coding sequence encoding the polypeptide.
14 . The method of claim 13 , wherein the modified coding sequence has a cytoside content that is increased by at least 15% relative to the wild type coding sequence encoding the polypeptide.
15 . The method of claim 12 , wherein the modified coding sequence has a codon adaptation index (CAI) that is increased by at least 0.05 relative to a wild type coding sequence encoding the polypeptide.
16 . The method of claim 15 , wherein the modified coding sequence has a CAI that is increased by at least 0.15 relative to the wild type coding sequence encoding the polypeptide.
17 . The method of claim 1 , wherein the biologically active polypeptide is an enzyme.
18 . The method of claim 1 , wherein the subject has methylmalonic acidemia.
19 . The method of claim 1 , wherein the subject has propionic acidemia.
20 . The method of claim 1 , wherein the biologically active polypeptide is at least 50 amino acids in length.
Assignments (1)
Priority Claims (1)
WO PCT/EP2013/003293
· Nov 1, 2013
· international
Continuity (3)
Continuation
PCTEP2014002931
· Oct 31, 2014
References Cited (400)
US 3906092A
· Hilleman et al.
· 1975
[cited by applicant]
US 4373071A
· Itakura
· 1983
[cited by applicant]
US 4401796A
· Itakura
· 1983
[cited by applicant]
US 4415732A
· Caruthers et al.
· 1983
[cited by applicant]
US 4458066A
· Caruthers et al.
· 1984
[cited by applicant]
US 4500707A
· Caruthers et al.
· 1985
[cited by applicant]
US 4588585A
· Mark et al.
· 1986
[cited by applicant]
US 4668777A
· Caruthers et al.
· 1987
[cited by applicant]
US 4737462A
· Mark et al.
· 1988
[cited by applicant]
US 4816567A
· Cabilly et al.
· 1989
[cited by applicant]
US 4879111A
· Chong
· 1989
[cited by applicant]
US 4959314A
· Mark et al.
· 1990
[cited by applicant]
US 4973679A
· Caruthers et al.
· 1990
[cited by applicant]
US 5017691A
· Lee et al.
· 1991
[cited by applicant]
US 5047524A
· Andrus et al.
· 1991
[cited by applicant]
US 5116943A
· Koths et al.
· 1992
[cited by applicant]
US 5132418A
· Caruthers et al.
· 1992
[cited by applicant]
US 5153319A
· Caruthers et al.
· 1992
[cited by applicant]
US 5262530A
· Andrus et al.
· 1993
[cited by applicant]
US 5580859A
· Felgner et al.
· 1996
[cited by applicant]
US 5663153A
· Hutcherson et al.
· 1997
[cited by applicant]
US 5700642A
· Monforte et al.
· 1997
[cited by applicant]
US 5928649A
· Daley et al.
· 1999
[cited by applicant]
US 5965720A
· Gryaznov et al.
· 1999
[cited by applicant]
US 5965726A
· Pavlakis et al.
· 1999
[cited by applicant]
US 6214804B1
· Felgner et al.
· 2001
[cited by applicant]
US 6239116B1
· Krieg et al.
· 2001
[cited by applicant]
US 6265387B1
· Wolff et al.
· 2001
[cited by applicant]
US 6322967B1
· Parkin
· 2001
[cited by applicant]
US 6376248B1
· Hawley-Nelson et al.
· 2002
[cited by applicant]
US 6406705B1
· Davis et al.
· 2002
[cited by applicant]
US 6500919B1
· Adema et al.
· 2002
[cited by applicant]
US 6514948B1
· Raz et al.
· 2003
[cited by applicant]
US 6552006B2
· Raz et al.
· 2003
[cited by applicant]
US 6589940B1
· Raz et al.
· 2003
[cited by applicant]
US 6610661B1
· Carson et al.
· 2003
[cited by applicant]
US 6664066B2
· Parks
· 2003
[cited by applicant]
US 6924365B1
· Miller et al.
· 2005
[cited by applicant]
US 7001890B1
· Wagner et al.
· 2006
[cited by applicant]
US 7208478B2
· Carson et al.
· 2007
[cited by applicant]
US 7268120B1
· Horton et al.
· 2007
[cited by applicant]
US 7276489B2
· Agrawal et al.
· 2007
[cited by applicant]
US 7316925B2
· Draghia-Akli et al.
· 2008
[cited by applicant]
US 8217016B2
· Hoerr et al.
· 2012
[cited by applicant]
US 8383340B2
· Ketterer et al.
· 2013
[cited by applicant]
US 8703906B2
· Baumhof et al.
· 2014
[cited by applicant]
US 8968746B2
· Baumhof et al.
· 2015
[cited by applicant]
US 9155788B2
· Hoerr et al.
· 2015
[cited by applicant]
US 9226959B2
· Kramps et al.
· 2016
[cited by applicant]
US 9234013B2
· Thess et al.
· 2016
[cited by applicant]
US 9314535B2
· Baumhof et al.
· 2016
[cited by applicant]
US 9352028B2
· Barner et al.
· 2016
[cited by applicant]
US 9402887B2
· Probst et al.
· 2016
[cited by applicant]
US 9421255B2
· Baumhof et al.
· 2016
[cited by applicant]
US 9433669B2
· Hoerr et al.
· 2016
[cited by applicant]
US 9433670B2
· Hoerr et al.
· 2016
[cited by applicant]
US 9439956B2
· Hoerr et al.
· 2016
[cited by applicant]
US 9447431B2
· Thess et al.
· 2016
[cited by applicant]
US 9463228B2
· Hoerr et al.
· 2016
[cited by applicant]
US 9572874B2
· Fotin-Mleczek et al.
· 2017
[cited by applicant]
US 9616084B2
· Mutzke
· 2017
[cited by applicant]
US 9623095B2
· Kallen et al.
· 2017
[cited by applicant]
US 9655955B2
· Hoerr et al.
· 2017
[cited by applicant]
US 9669089B2
· Thess et al.
· 2017
[cited by applicant]
US 9683233B2
· Thess
· 2017
[cited by applicant]
US 9688729B2
· Kramps et al.
· 2017
[cited by applicant]
US 9737595B2
· Lorenz et al.
· 2017
[cited by applicant]
US 9839697B2
· Thess et al.
· 2017
[cited by applicant]
US 9890391B2
· Thess et al.
· 2018
[cited by applicant]
US 9907862B2
· Baumhof et al.
· 2018
[cited by applicant]
US 9974845B2
· Fotin-Mleczek et al.
· 2018
[cited by applicant]
US 20020123099A1
· Weiner et al.
· 2002
[cited by applicant]
US 20020132788A1
· Lewis et al.
· 2002
[cited by applicant]
US 20030008342A1
· Scholler et al.
· 2003
[cited by applicant]
US 20030077604A1
· Sun et al.
· 2003
[cited by applicant]
US 20030143204A1
· Lewis et al.
· 2003
[cited by applicant]
US 20040005667A1
· Ratti et al.
· 2004
[cited by applicant]
US 20040106567A1
· Hagstrom et al.
· 2004
[cited by applicant]
US 20050064596A1
· Riemen et al.
· 2005
[cited by applicant]
US 20050112141A1
· Terman
· 2005
[cited by applicant]
US 20050250723A1
· Hoerr et al.
· 2005
[cited by applicant]
US 20060188490A1
· Hoerr et al.
· 2006
[cited by applicant]
US 20060241076A1
· Uhlmann et al.
· 2006
[cited by applicant]
US 20070280929A1
· Hoerr et al.
· 2007
[cited by applicant]
US 20080025944A1
· Hoerr et al.
· 2008
[cited by applicant]
US 20080171711A1
· Hoerr et al.
· 2008
[cited by applicant]
US 20080267873A1
· Hoerr et al.
· 2008
[cited by applicant]
US 20090324584A1
· Hoerr et al.
· 2009
[cited by applicant]
US 20100047261A1
· Hoerr et al.
· 2010
[cited by applicant]
US 20100203076A1
· Fotin-Mleczek et al.
· 2010
[cited by applicant]
US 20100291156A1
· Barner et al.
· 2010
[cited by applicant]
US 20100305196A1
· Probst et al.
· 2010
[cited by applicant]
US 20110053829A1
· Baumhof et al.
· 2011
[cited by applicant]
US 20120258046A1
· Mutzke
· 2012
[cited by applicant]
US 20130195867A1
· Hoerr et al.
· 2013
[cited by applicant]
US 20130295043A1
· Kallen et al.
· 2013
[cited by applicant]
US 20150037326A1
· Butler-Ransohoff et al.
· 2015
[cited by applicant]
US 20150118183A1
· Baumhof
· 2015
[cited by applicant]
US 20150118264A1
· Baumhof et al.
· 2015
[cited by applicant]
US 20150258214A1
· Baumhof et al.
· 2015
[cited by applicant]
US 20150306249A1
· Baumhof et al.
· 2015
[cited by applicant]
US 20160166668A1
· Kallen et al.
· 2016
[cited by applicant]
US 20160168227A1
· Kallen et al.
· 2016
[cited by applicant]
US 20170239372A1
· Baumhof et al.
· 2017
[cited by applicant]
US 20170326225A1
· Rauch et al.
· 2017
[cited by applicant]
US 20180126003A1
· Hoerr
· 2018
[cited by applicant]
US 20180126005A1
· Fotin-Mleczek et al.
· 2018
[cited by applicant]
US 20180161451A1
· Fotin-Mleczek et al.
· 2018
[cited by applicant]
US 20180161454A1
· Fotin-Mleczek et al.
· 2018
[cited by applicant]
US 20180169267A1
· Fotin-Mleczek et al.
· 2018
[cited by applicant]
US 20180207295A1
· Fotin-Mleczek et al.
· 2018
[cited by applicant]
US 20180208957A1
· Roos et al.
· 2018
[cited by applicant]
US 20180256694A1
· Barner et al.
· 2018
[cited by applicant]
US 20180312545A1
· Baumhof et al.
· 2018
[cited by applicant]
US 20190008954A1
· Baumhof
· 2019
[cited by applicant]
US 20190017100A1
· Wochner et al.
· 2019
[cited by applicant]
US 20190024096A1
· Schmid et al.
· 2019
[cited by applicant]
US 20190049414A1
· Wochner et al.
· 2019
[cited by applicant]
US 20190134222A1
· von der Mülbe et al.
· 2019
[cited by applicant]
US 20190151438A1
· Kallen et al.
· 2019
[cited by applicant]
US 20190175756A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190175757A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190175758A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190175759A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190175760A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190177714A1
· Kunze et al.
· 2019
[cited by applicant]
US 20190185859A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190194760A1
· Koch et al.
· 2019
[cited by applicant]
US 20190225971A1
· Williams
· 2019
[cited by applicant]
US 20190255161A1
· Thess et al.
· 2019
[cited by applicant]
US 20190336608A1
· Baumhof et al.
· 2019
[cited by applicant]
US 20190336611A1
· Baumhof et al.
· 2019
[cited by applicant]
US 20190343942A1
· Fotin-Mleczek et al.
· 2019
[cited by applicant]
US 20190345504A1
· Grund et al.
· 2019
[cited by applicant]
US 20190381180A1
· Baumhof et al.
· 2019
[cited by applicant]
US 20200016264A1
· Hoerr et al.
· 2020
[cited by applicant]
US 20200040370A1
· Eber et al.
· 2020
[cited by applicant]
US 20200085852A1
· Fotin-Mleczek
· 2020
[cited by applicant]
US 20200085942A1
· Kramps et al.
· 2020
[cited by applicant]
US 20200085944A1
· Heidenreich et al.
· 2020
[cited by applicant]
US 20200163878A1
· Baumhof et al.
· 2020
[cited by applicant]
US 20200216878A1
· Wochner
· 2020
[cited by applicant]
US 20200338215A1
· Baumhof et al.
· 2020
[cited by applicant]
US 20200399322A1
· Baumhof et al.
· 2020
[cited by applicant]
US 20210023199A1
· Kallen et al.
· 2021
[cited by applicant]
US 20210030866A1
· Kallen et al.
· 2021
[cited by applicant]
US 20210040526A1
· Wochner et al.
· 2021
[cited by applicant]
US 20210046179A1
· Fotin-Mleczek et al.
· 2021
[cited by applicant]
US 20210060175A1
· Fotin-Mleczek et al.
· 2021
[cited by applicant]
US 20210060181A1
· Thess et al.
· 2021
[cited by applicant]
US 20210170017A1
· Lutz et al.
· 2021
[cited by applicant]
US 20210251898A1
· English et al.
· 2021
[cited by applicant]
US 20210260178A1
· Jasny et al.
· 2021
[cited by applicant]
US 20210308238A1
· Hoerr et al.
· 2021
[cited by applicant]
US 20210361761A1
· Lutz et al.
· 2021
[cited by applicant]
US 20210361764A1
· Fotin-Mleczek et al.
· 2021
[cited by applicant]
US 20210393755A1
· Thess et al.
· 2021
[cited by applicant]
US 20220040281A1
· Schwendt et al.
· 2022
[cited by applicant]
CA 2854685
· 2002
[cited by applicant]
CA 2473135
· 2003
[cited by applicant]
CA 2376634
· 2005
[cited by applicant]
DE 10119005
· 2001
[cited by applicant]
DE 10229872
· 2004
[cited by applicant]
EP 0175960
· 1986
[cited by applicant]
EP 0839912
· 1998
[cited by applicant]
EP 1083232
· 2001
[cited by applicant]
EP 1393745
· 2004
[cited by applicant]
EP 3062798
· 2020
[cited by applicant]
JP 2000509281
· 2000
[cited by applicant]
JP 7503372
· 2007
[cited by applicant]
WO WO1990011092
· 1990
[cited by applicant]
WO WO1993014778
· 1993
[cited by applicant]
WO WO1995024485
· 1995
[cited by applicant]
WO WO1995026204
· 1995
[cited by applicant]
WO WO1997041210
· 1997
[cited by applicant]
WO WO1997048370
· 1997
[cited by applicant]
WO WO1998012207
· 1998
[cited by applicant]
WO WO1998034640
· 1998
[cited by applicant]
WO WO1998055495
· 1998
[cited by applicant]
WO WO1999014346
· 1999
[cited by applicant]
WO WO1999020774
· 1999
[cited by applicant]
WO WO1999052503
· 1999
[cited by applicant]
WO WO2000006723
· 2000
[cited by applicant]
WO WO2000029561
· 2000
[cited by applicant]
WO WO2000075304
· 2000
[cited by applicant]
WO WO2001004313
· 2001
[cited by applicant]
WO WO2001014416
· 2001
[cited by applicant]
WO WO2001021810
· 2001
[cited by applicant]
WO WO2001093902
· 2001
[cited by applicant]
WO WO2002008435
· 2002
[cited by applicant]
WO WO2002064799
· 2002
[cited by applicant]
WO WO2002085434
· 2002
[cited by applicant]
WO WO2002098443
· 2002
[cited by applicant]
WO WO2003028656
· 2003
[cited by applicant]
WO WO2003051401
· 2003
[cited by applicant]
WO WO2003059381
· 2003
[cited by applicant]
WO WO2003066649
· 2003
[cited by applicant]
WO WO2003086280
· 2003
[cited by applicant]
WO WO2004058159
· 2004
[cited by applicant]
WO WO2004092329
· 2004
[cited by applicant]
WO WO2006008154
· 2006
[cited by applicant]
WO WO2006024518
· 2006
[cited by applicant]
WO WO2006049777
· 2006
[cited by applicant]
WO WO2007024708
· 2007
[cited by applicant]
WO WO2009127230
· 2009
[cited by applicant]
WO WO2011069528
· 2011
[cited by applicant]
WO WO2011069587
· 2011
[cited by applicant]
WO WO2011144358
· 2011
[cited by applicant]
WO WO2012013326
· 2012
[cited by applicant]
WO WO2012019780
· 2012
[cited by applicant]
WO WO2012089338
· 2012
[cited by applicant]
WO WO2012113513
· 2012
[cited by applicant]
WO WO2012116810
· 2012
[cited by applicant]
WO WO2012116811
· 2012
[cited by applicant]
WO WO2013113501
· 2013
[cited by applicant]
WO WO2013113502
· 2013
[cited by applicant]
WO WO2013113736
· 2013
[cited by applicant]
WO WO2013120626
· 2013
[cited by applicant]
WO WO2013120627
· 2013
[cited by applicant]
WO WO2013120628
· 2013
[cited by applicant]
WO WO2013120629
· 2013
[cited by applicant]
WO WO2013143698
· 2013
[cited by applicant]
WO WO2013143699
· 2013
[cited by applicant]
WO WO2013143700
· 2013
[cited by applicant]
WO WO2013174409
· 2013
[cited by applicant]
WO WO2014127917
· 2014
[cited by applicant]
WO WO2015062738
· 2015
[cited by applicant]
WO WO2015149944
· 2015
[cited by applicant]
WO WO2016091391
· 2016
[cited by applicant]
WO WO2016097065
· 2016
[cited by applicant]
WO WO2016107877
· 2016
[cited by applicant]
Aissani et al., “CpG islands, genes and isochores in the genomes of vertebrates,”
[cited by applicant]
Akashi, “Gene expression and molecular evolution,”
[cited by applicant]
Alberts et al.,
[cited by applicant]
Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,”
[cited by applicant]
Anichini et al., “Cytotoxic T cells directed to tumor antigen not expressed on normal melanocytes dominate HLA-A2 1-restricted immune repertoire to melanoma,”
[cited by applicant]
Annex to Ngumbela et al., “Quantitative Effect of Suboptimal Codon Usage on Translational Efficiency of mRNA Encoding HIV-1 gag in Intact T Cells”,
[cited by applicant]
Aota et al., “Diversity in G+C content at the third position of codons in vertebrate genes and its cause,”
[cited by applicant]
Apostolopoulos et al., “Cellular mucins: targets for immunotherapy,”
[cited by applicant]
Ashley et al., “Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors,”
[cited by applicant]
Aurup et al., “Translation of 2′-modified mRNA in vitro and in vivo,”
[cited by applicant]
Austyn, “New insights into the mobilization and phagocytic activity of dendritic cells,”
[cited by applicant]
Bernardi, “Isochores and the evolutionary genomics of vertebrates,”
[cited by applicant]
Bernardi, “The vertebrate genome: isochores and evolution,”
[cited by applicant]
Berneman et al., “T-Cell Stimulatory Capacity of Different Types of In Vitro Cultured Monocyte-Derived Dendritic Cells Following Electroporation with RNA Encoding Defined Antigens,” Laboratory of Experimental Hematology…
[cited by applicant]
Bernhard et al., “Generation of immunostimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow and peripheral blood,”
[cited by applicant]
Bettinger et al., “Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells,”
[cited by applicant]
Bevan, “Antigen presentation to cytotoxic T lymphocytes in vivo,”
[cited by applicant]
Bevilacqua et al., “Post-transcriptional regulation of gene expression by degradation of messenger RNAs,”
[cited by applicant]
Bieler und Wagner (in: Schleef), Plasmids for Therapy and Vaccination, Kapitel 9, Seiten 147-168, Wiley-VCH, Weinbeim, 2001.
[cited by applicant]
Binder et al., “Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3′ UTR and does not involve poly(A) tail shortening,”
[cited by applicant]
Bird, “CpG-rich islands and the function of DNA methylation,”
[cited by applicant]
Boczkowski et al., “Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo,”
[cited by applicant]
Boczkowski et al., “Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells,”
[cited by applicant]