RNA containing modified nucleosides and methods of use thereof

Patent metadata

Field	Value
Publication number	US-9750824-B2
Application number	US-201414456302-A
Country	US
Kind code	B2
Filing date	Aug 11, 2014
Priority date	Aug 23, 2005
Publication date	Sep 5, 2017
Grant date	Sep 5, 2017

Abstract

This invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising same, methods of synthesizing same, and methods for gene replacement, gene therapy, gene transcription silencing, and the delivery of therapeutic proteins to tissue in vivo, comprising the molecules. The present invention also provides methods of reducing the immunogenicity of RNA, oligoribonucleotide, and polyribonucleotide molecules.

First claim

What is claimed is:

1. A method for reducing the immunogenicity of in vitro-synthesized RNA comprising an open reading frame that encodes a functional protein, the method comprising:
replacing a nucleotide of the in vitro-synthesized RNA with at least one modified nucleotide comprising a modified nucleoside selected from the group consisting of:
pseudouridine (Ψ), 1-methylpseudouridine (m 1 Ψ), 5-methyluridine (m 5 U), 2-thiouridine (s 2 U), and 5-methylcytidine (m 5 C),
wherein when pseudouridine (Ψ) is selected, it replaces uridine, and
wherein when 1-methylpseudouridine (m 1 Ψ) is selected, it replaces uridine, and
wherein when 5-methyluridine (m 5 U) is selected, it replaces uridine, and
wherein when 2-thiouridine (s 2 U) is selected, it replaces uridine, and
wherein when 5-methylcytidine (m 5 C) is selected, it replaces cytidine,
thereby generating in vitro-synthesized RNA comprising a modified nucleotide and reducing the immunogenicity of said in vitro-synthesized RNA.

2. The method of claim 1 , wherein the reduced immunogenicity is detected by at least one method selected from the group consisting of:
(i) detecting a decrease in immunogenicity such that the in vitro-synthesized RNA comprising a modified nucleotide can be repeatedly administered without eliciting an immune response sufficient to detectably reduce expression of said functional protein, whereas repeatedly administering the same quantity of in vitro-synthesized RNA that does not comprise said modified nucleotide does detectably reduce expression of said functional protein;
(ii) measuring expression of a dendritic cell (DC) activation marker in response to in vitro-synthesized RNA comprising a modified nucleotide compared to the same quantity of in vitro-synthesized RNA that does not comprise said modified nucleotide;
(iii) measuring secretion of a cytokine secreted in response to administration of in vitro-synthesized RNA comprising a modified nucleotide relative to the same quantity of in vitro-synthesized RNA comprising the unmodified uridine or cytidine nucleotide; or
(iv) detecting a higher level of activation of toll-like receptor (TLR) signaling molecules in response to in vitro-synthesized RNA comprising a modified nucleotide compared to the same quantity of in vitro-synthesized RNA that does not comprise said modified nucleotide.

3. The method of claim 2 , wherein said DC activation marker in (ii) is selected from the group consisting of CD83, HLA-DR; CD80 and CD86; said at least one cytokine in (iii) is selected from the group consisting of: IL-12, IFN-α, TNF-α, RANTES, MIP-1α, MIP-1β, IL-6, IFN-βand IL-8; or said TLR signaling molecule in (iv) is selected from the group consisting of TLR3, TLR7 and TLR8 signaling molecules.

4. The method of claim 1 , wherein said replacing comprises replacing 100% of said uridine nucleotides with a modified nucleotide comprising a nucleoside selected from the group consisting of Ψ, m 1 Ψ, m 5 U, and s 2 U and replacing 100% of said cytidine nucleotides with m 5 C modified nucleotide.

5. The method of claim 1 , wherein said replacing comprises replacing 10% to 100% of said uridine nucleotides with a modified nucleotide comprising a nucleoside selected from the group consisting of Ψ, m 1 Ψ, m 5 U, and s 2 U.

6. The method of claim 5 , wherein said replacing further comprises replacing 10% to 100% of said cytidine nucleotides with m 5 C modified nucleotide.

7. The method of claim 1 , wherein said replacing comprises replacing 10% to 100% of said cytidine nucleotides with m 5 C modified nucleotide.

8. The method of claim 1 , wherein said replacing further comprises replacing 10% to 100% of the adenosine nucleotides with N 6 -methyladenosine (m 6 A).

9. The method of claim 1 , wherein said replacing comprises synthesizing said in vitro-synthesized RNA comprising a modified nucleotide in an in vitro transcription reaction comprising a DNA template comprising an open reading frame encoding said functional protein, an RNA polymerase, and a mixture of nucleoside triphosphates (NTPs) in which a desired percentage of a triphosphate derivative of each desired modified nucleoside is substituted for the unmodified NTP.

10. The method of claim 1 , wherein said in vitro-synthesized RNA comprises at least one selected from the group consisting of:
A. a poly-A tail;
B. an m7GpppG cap; and
C. a cap-independent translational enhancer.

11. The method of claim 1 , wherein said protein encoded by said in vitro-synthesized RNA is selected from the group consisting of:
erythropoietin (EPO); a detectable enzyme selected from firefly luciferase, Renilla luciferase, bacterial beta-galactosidase (lacZ), and green fluorescent protein (GFP); a transcription factor selected from MYC and SRY or MCOP; a growth factor or cytokine selected from the group consisting of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-betal (TGF-betal), insulin-like growth factor (IGF), alpha-melanocyte -stimulating hormone (alpha-MSH); insulin-like growth factor-I (IGF-I); IL-4; IL-13; and IL-10; inducible nitric oxide synthase (iNOS); a heat shock protein; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR); an enzyme with antioxidant activity selected from among catalase, phospholipid hydroperoxide glutathione peroxidase, superoxide dismutase-1, and superoxide dismutase-2; Bruton's tyrosine kinase; adenosine deaminase; ecto-ucleoside triphosphate diphosphydrolase; ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4A1; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG; C8A; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A; CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5; FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; GBA; GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2; KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ; MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1; OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPOX; PPTO; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD; SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2; TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5; ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS; BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COLAA3; COL4A4; COL6A3; CPS1; CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; EDAR; EFEMP1; EIF2AK3; ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC; IHH; IRS1; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC; NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB; SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24; WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3; BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRV; CTNNB1; DEM; ETM1; FANCD2; FIH; FOXL2; GBE1; GLB1; GLCLC; GNAI2; GNAT1; GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCC1; MDS1; MHS4; MITF; MLH1; MYL3; MYMY; OPA1; P2RY12; PBXP1; PCCB; POU1F1; PPARG; PROS1; PTHR1; RCA1; RHO; SCA7; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2; THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC; ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK; DSPP; DTDP2; ELONG; ENAM; ETFDH; EVC; F11; FABP2; FGA; FGB; FGFR3; FGG; FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF; JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1; PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2; WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3B1; APC; A

Assignees

• Univ Pennsylvania

Inventors

• Kariko Katalin
• Weissman Drew

Classifications

• A61P7/06

  Antianaemics · CPC title

• C12N2310/335

  Modified T or U · CPC title

• C12N15/117

  Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs · CPC title

• A61K48/0041 Primary

  the non-active part being polymeric · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title