INHIBITION OF INNATE IMMUNE RESPONSE
20210079346 ยท 2021-03-18
Inventors
Cpc classification
A61L2300/00
HUMAN NECESSITIES
C12N2710/24122
CHEMISTRY; METALLURGY
C12N5/0658
CHEMISTRY; METALLURGY
A61K38/1774
HUMAN NECESSITIES
C12N2760/16122
CHEMISTRY; METALLURGY
C12N2710/20022
CHEMISTRY; METALLURGY
C12N2710/22022
CHEMISTRY; METALLURGY
C12N5/0696
CHEMISTRY; METALLURGY
A61K31/7105
HUMAN NECESSITIES
A61K31/711
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
International classification
A61K31/7105
HUMAN NECESSITIES
A61K31/711
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
C07K14/705
CHEMISTRY; METALLURGY
C07K14/715
CHEMISTRY; METALLURGY
Abstract
The present invention provides methods, kits, and compositions for reducing an innate immune system response in a human or animal cell, tissue or organism. One embodiment comprises: introducing an Agent mRNA comprising in vitro-synthesized mRNA encoding one or more proteins that affect the induction, activity or response of an innate immune response pathway; whereby, the innate immune response in the cell, tissue or organism is reduced compared to the innate immune response in the absence of the Agent mRNA. Other embodiments are methods, compositions and kits for using an Agent mRNA for treating a disease or medical condition in a human or animal that exhibits symptoms of an elevated innate immune system, or for reducing an innate immune response that is induced in a human or animal cell, tissue or organism by a Foreign Substance that is administered to the cell, tissue or organism.
Claims
1. A method for reducing an innate immune response in a human or animal cell, tissue or organism, comprising: introducing into the cell, tissue or organism an Agent mRNA comprising in vitro-synthesized mRNA encoding one or more proteins that affect the induction, activity and/or response of an innate immune response pathway; whereby the innate immune response in the cell, tissue or organism is reduced compared to the innate immune response that results or would have resulted in the absence of introducing said Agent mRNA.
2. The method of claim 1, wherein said Agent mRNA encodes one or more proteins that inhibits the activity of an innate immune effector protein in a signaling pathway mediated by a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10, or a biologically active fragment, analog or variant of any of said effector proteins.
3. The method of claim 1, wherein said Agent mRNA encodes one or more proteins that is a regulator or inhibitor of type I-interferon signaling, induction, or response selected from the group consisting of: a) a biologically inactive fragment, mutant, analog or variant or a dominant negative functional inhibitor of TP53, TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1, STAT2, EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2, OAS1, OAS2, OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2, IFIT3, IFIT5, PKR, RIG-1, MDA5, NF-B, TRIF, Tyk2 and IRF7; and b) Vaccinia virus B18R protein, Vaccinia virus E3L protein, Vaccinia virus K3L protein, Influenza A virus NS1 protein, human papilloma virus 18 protein E6, human interferon alpha/beta binding proteins, a soluble form of a human interferon alpha receptors, or a biologically active fragment, analog or variant of any of said proteins.
4. The method of claim 1, wherein the cell is a human or animal cell t selected from the group consisting of: a fibroblast cell, a fetal fibroblast, a neonatal fibroblasts, adult fibroblasts, an hematopoietic cell, a B cell, a T cell, a dendritic cell, a macrophage cell, a Langerhans cell, a Kuppfer cell, an artificial APC, a monocyte, mononuclear cells, a keratinocyte cell, a primary keratinocyte, a keratinocyte derived from hair, an adipose cell, an epithelial cell, an epidermal cell, a chondrocyte, a cumulus cell, a neural cell, a glial cell, an astrocyte, a cardiac cell, an esophageal cell, a muscle cell, a melanocyte, and an osteocyte.
5. The method of claim 1, wherein said introducing is into said organism, wherein said organism is a human, has a disease or medical condition comprising an elevated type I IFN-mediated innate immune response.
6. The method of claim 5, wherein said human has psoriasis or systemic lupus erythematosus (SLE).
7. The method of claim 1, wherein said innate immune response that is reduced is caused by introduction of a Foreign Substance that is capable of causing an innate immune response in said cell, tissue or organism by affecting the induction, activity and/or response of an innate immune response pathway in said cell, tissue or organism; wherein said Foreign Substance is selected from the group consisting of: a) Exogenous RNA; b) Exogenous siRNA or Exogenous miRNA; c) dsRNA that is transfected into said cell, tissue or organism; and d) a lipopolysaccharide (LPS) that is introduced to said cell, tissue or organism.
8. The method of claim 7, wherein said Exogenous RNA comprises mRNA, and wherein the expression of said Agent mRNA: a) increases the translation of said Exogenous RNA in said cells; and/or b) decreases the cell toxicity to said cells; and/or c) increases the survival of the cells.
9. The method of any claim 7, wherein said cell, tissue or organism comprises an antigen-presenting cell selected from the group consisting of: a dendritic cell, a macrophage, a Langerhans cell, a Kuppfer cell, and an artificial APC, from a human or animal patient, and wherein said Foreign Substance that is transfected into said cell is Exogenous mRNA comprising or consisting of one or multiple mRNAs derived from a cancer cell from a human or animal patient by in vitro transcription (IVT) of cDNA generated from substantially all of the mRNA isolated from one or more cancer cells.
10. The method of claim 7, wherein said Foreign Substance comprises Exogenous mRNA encoding a non-defective form of a protein that is defective or lacking in a cell of a human or animal patient, and said method further comprises transfecting said Exogenous mRNA into a cell of said cell, tissue or organism.
11. The method of claim 7, wherein said Foreign Substance is Exogenous mRNA encoding one or more proteins selected from the group consisting of: OCT3/4, SOX2, KLF4, c-MYC, c-MYC(T58A), L-MYC, NANOG, LIN28, SV40 Large-T antigen, hTERT, E-Cadherin, MYOD1, SHH, GLI1, RAR, LRH1, GLIS1, NURR1, MASH1, LMX1A, BRN2, MYT1L, GATA4, MEF2C, TBX5, HAND2, FOXA1, FOXA2, FOXA3, HNF1, HNF4, PAX3 and PAX7.
12. The method of claim 1, wherein, prior to introducing to the cell, tissue or organism said Agent mRNA, the method comprises the step of contacting the cell, tissue, or organism with an effective amount of a protein that is capable of reducing an innate immune response due to said Agent mRNA.
13. A system comprising: a) an Agent mRNA that reduces the innate immune response in a cell that is induced a Foreign Substance, and b) a Foreign Substance that induces said innate immune response, particularly.
14. The system of claim 13, wherein said Agent mRNA encodes one or more proteins that inhibits the activity of an innate immune effector protein in a signaling pathway mediated by a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10, or a biologically active fragment, analog or variant of any of said proteins.
15. The system of claim 13, wherein said Agent mRNA encodes one or more proteins is selected from the group consisting of: a) a biologically inactive fragment, mutant, analog or variant or a dominant negative functional inhibitor of TP53, TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1, STAT2, EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2, OAS1, OAS2, OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2, IFIT3, IFIT5, PKR, RIG-1, MDA5, NF-B, TRIF, Tyk2, IRF7, or a biologically active fragment, analog or variant of any of said proteins; and b) Vaccinia virus B18R protein, Vaccinia virus E3L protein, Vaccinia virus K3L protein, Influenza A virus NS1 protein, human papilloma virus 18 protein E6, human interferon alpha/beta binding proteins, a soluble form of a human interferon alpha receptors (e.g., INFAR1, INFAR2), or a biologically active fragment, analog or variant of any of said proteins.
16. The system of claim 13, further comprising a human or animal cell selected from the group consisting of: a fibroblast cell, a fetal fibroblast, a neonatal fibroblast, adult fibroblast, an hematopoietic cell, a B cell, a T cell, a dendritic cell, a macrophage cell, a Langerhans cell, a Kuppfer cell, an artificial APC, a monocyte, a mononuclear cell, a keratinocyte cell, a primary keratinocyte, a keratinocyte derived from hair, an adipose cell, an epithelial cell, an epidermal cell, a chondrocyte, a cumulus cell, a neural cell, a glial cell, an astrocyte, a cardiac cell, an esophageal cell, a muscle cell, a melanocyte, and an osteocyte.
17. The system of claim 1, wherein said Foreign Substance is Exogenous mRNA encoding one or more proteins selected from the group consisting of: OCT3/4, SOX2, KLF4, c-MYC, c-MYC(T58A), L-MYC, NANOG, LIN28, SV40 Large-T antigen, hTERT, E-Cadherin, MYOD1, SHH, GLI1, RAR, LRH1, GLIS1, NURR1, MASH1, LMX1A, BRN2, MYT1L, GATA4, MEF2C, TBX5, HAND2, FOXA1, FOXA2, FOXA3, HNF1, HNF4, PAX3 and PAX7.
18. The system of claim 13 further comprising an effective amount of a protein that is capable of reducing an innate immune response due to said Agent mRNA.
19. A composition comprising: a) an Agent mRNA that reduces the innate immune response in a cell that is induced by a Foreign Substance, and b) a Foreign Substance RNA that induces said innate immune response when transfected into said cell.
20. The composition of claim 19, wherein said Agent mRNA encodes one or more proteins that inhibits the activity of an innate immune effector protein in a signaling pathway mediated by a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10, or a biologically active fragment, analog or variant of any of said proteins.
21. The composition of claim 19, wherein said Agent mRNA encodes one or more proteins that is a regulator or inhibitor of type I-interferon signaling, induction, or response selected from the group consisting of: a) a biologically inactive fragment, mutant, analog or variant or a dominant negative functional inhibitor of TP53, TLR3, TLR4, TLR7, TLR8, RARRES3, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNK, IFNB1, IL6, TICAM1, TICAM2, MAVS, STAT1, STAT2, EIF2AK2, IRF3, TBK1, CDKN1A, CDKN2A, RNASEL, IFNAR1, IFNAR2, OAS1, OAS2, OAS3, OASL, RB1, ISG15, MX1, IRF9, ISG20, IFIT1, IFIT2, IFIT3, IFIT5, PKR, RIG-1, MDA5, NF-B, TRIF, Tyk2, IRF7; and b) Vaccinia virus B18R protein, Vaccinia virus E3L protein, Vaccinia virus K3L protein, Influenza A virus NS1 protein, human papilloma virus 18 protein E6, human interferon alpha/beta binding proteins, soluble forms of the human interferon alpha receptors, INFAR1, INFAR2, or a biologically active fragment, analog or variant of any of said proteins.
22. The composition of claim 19, wherein said Foreign Substance is Exogenous mRNA encoding one or more proteins selected from the group consisting of: OCT3/4, SOX2, KLF4, c-MYC, c-MYC(T58A), L-MYC, NANOG, LIN28, SV40 Large-T antigen, hTERT, E-Cadherin, MYOD1, SHH, GLI1, RAR, LRH1, GLIS1, NURR1, MASH1, LMX1A, BRN2, MYT1L, GATA4, MEF2C, TBX5, HAND2, FOXA1, FOXA2, FOXA3, HNF1, HNF4, PAX3 and PAX7.
23. The composition of claim 19, further comprising a human or animal cell selected from the group consisting of: a fibroblast cell, a fetal fibroblast, a neonatal fibroblasts, adult fibroblast, a hematopoietic cell, a B cell, a T cell, a dendritic cell, a macrophage cell, a Langerhans cell, a Kuppfer cell, an artificial APC, a monocyte, mononuclear cells, a keratinocyte cell, a primary keratinocyte, a keratinocyte derived from hair, an adipose cell, an epithelial cell, an epidermal cell, a chondrocyte, a cumulus cell, a neural cell, a glial cell, an astrocyte, a cardiac cell, an esophageal cell, a muscle cell, a melanocyte, and an osteocyte.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0046] The following figures form part of the present specification and are included as examples to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein, but are not intended to limit the invention.
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DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention provides methods, kits, and compositions for increasing translation of Exogenous mRNA in cells using an Agent mRNA that reduces or suppresses an innate immune response induced, for example, by the introduction of Exogenous mRNA. In certain embodiments, the Agent mRNA encodes a protein that inhibits or reduces type I interferon-induced cellular toxicity and/or inhibition of translation resulting from the introduction of Exogenous mRNA into a human or animal cell.
Definitions and General Aspects of the Invention
[0059] If the same terms or similar terms have been used with different meaning by others, including those cited in the section entitled Background herein, the terms when used to describe the present invention, shall nevertheless be interpreted to have the meanings presented below and in the sections related to the specification and claims, unless otherwise expressly stated to the contrary.
[0060] When used in describing an aspect of the present invention, the terms such as, including, for example, e.g., and the like shall be interpreted to mean without limitation.
[0061] As used herein, an Agent mRNA means in vitro-synthesized mRNA encoding one or more proteins that affect the induction, activity or response of an innate immune response pathway, whereby the innate immune response in a cell (e.g., a cell in a tissue or organism) is reduced, suppressed or prevented compared to the innate immune response in the absence of introducing said in vitro-synthesized mRNA.
[0062] As used herein, Exogenous RNA means RNA that is synthesized in an in vitro transcription reaction by an RNA polymerase using a DNA template that exhibits an RNA polymerase promoter sequence recognized by said RNA polymerase upstream of a DNA sequence encoding the sequence of an RNA which is desired to cause a biological or medical effect, which effect does not include functioning as an Agent mRNA, and wherein said RNA induces an innate immune response upon introduction into a cell, tissue or organism; Exogenous RNA includes, for example, RNA that exhibits a sequence encoding at least one protein and which is capable of being translated into protein upon introduction into a living cell that has a functional translation system, and also includes RNA that exhibits an mRNA cap structure and a poly(A) tail. Exogenous RNA may also include-undesired RNA molecules that are synthesized in the in vitro transcription reaction, including truncated RNA due to abortive transcription or incomplete synthesis, uncapped in vitro transcription products, and dsRNA. In some cases herein, we refer to Exogenous RNA which encodes at least one protein (e.g., one or more proteins), including wherein the Exogenous RNA exhibits a cap structure and a poly(A) tail, as Exogenous mRNA. One important benefit of the methods, kits and compositions of the present invention is that the Agent mRNA reduces or suppresses an innate immune response which would be induced by the Exogenous RNA in the absence of such Agent mRNA.
[0063] As used herein, Exogenous siRNA and Exogenous miRNA mean a siRNA or miRNA, respectively, that is synthesized in vitro using any method known in the art, and that is for the purpose of causing a biological or medical effect in a cell, tissue or organism into which it is introduced, which effect does not include functioning as an Agent mRNA. In some embodiments, the Exogenous miRNA or Exogenous siRNA is synthesized by in vitro transcription of a DNA template, including in either one or two in vitro transcription reactions using either one or two DNA templates or one RNA polymerase that recognizes one RNA polymerase promoter sequence or two different RNA polymerases, each or which recognizes a different RNA polymerase promoter sequence, or by chemical synthesis on an oligonucleotide synthesizer using methods known in the art.
[0064] Agent mRNA and Exogenous RNA (e.g., mRNA) can be made using similar methods. For example, in some embodiments of the methods, kits, systems or compositions of the invention, the Agent mRNA or Exogenous RNA, or other RNA, is synthesized by in vitro transcription (IVT) of a DNA template using an RNA polymerase (e.g., SP6, T3 or T7 RNA polymerase) and nucleoside-5-triphosphates (NTPs). In some embodiments, the NTPs used for IVT comprise or consist of only GTP ATP, UTP, and CTP (canonical NTPs), and the Agent mRNA or Exogenous RNA product is described as GAUC. In other embodiments, a modified NTP is used in place of some or all of one or more of the respective canonical NTPs. In some preferred embodiments, the modified NTP, pseudouridine-5-triphosphate (TP) is used for IVT in place of some or all of the UTP; if TP is used for IVT in place of all of the UTP, the Agent mRNA or Exogenous RNA product is described as GAC. In some preferred embodiments, the modified NTP, 5-methylcytidine-5-triphosphate (m.sup.5CTP or 5mCTP) is used for IVT in place of some or all of the CTP. In some preferred embodiments wherein TP is used for IVT in place of some or all of the UTP, m.sup.5CTP is also used in place of some or all of the CTP. In some preferred embodiments, both TP and m.sup.5CTP are used for IVT in place of all of the corresponding UTP and CTP, and the Exogenous RNA product is described as GAm.sup.5C or GA5mC. In most embodiments, the Agent mRNA or Exogenous RNA is mRNA, meaning that it exhibits a cap on its 5-terminus and a poly(A) tail on its 3-terminus, as will be generally understood by those with knowledge in the art. In some preferred embodiments, Agent mRNA or Exogenous RNA that is mRNA is synthesized by IVT, followed by addition of the cap using a capping enzyme system comprising RNA guanyltransferase activity and addition of a poly(A) tail using a poly(A) polymerase (e.g., using an T7 mScript Standard mRNA Production System, as described elsewhere herein). In some other embodiments, the cap is added by incorporation of a dinucleotide cap analog (e.g., m7GpppG or the 3-O-methyl-m7GpppG ARCA) during IVT. In some embodiments, the poly(A) tail is added to the 3-terminus during IVT of a DNA template that encodes the poly(A) tail.
[0065] In some preferred embodiments of the methods, kits, systems and compositions, the Agent mRNA, Exogenous RNA (e.g., Exogenous mRNA), Exogenous miRNA or Exogenous siRNA comprises or consists of GAC RNA. In other preferred embodiments of the methods, kits, systems and compositions, the Agent mRNA, Exogenous RNA (e.g., Exogenous mRNA), Exogenous miRNA or Exogenous siRNA comprises or consists of GAm.sup.5C RNA.
[0066] In some preferred embodiments, Agent mRNA is further purified. In some embodiments, Exogenous RNA (e.g., Exogenous mRNA) is also further purified, in which embodiments, the same purification methods, purity quality standards, and assays for purity, as described herein may be used. In certain embodiments, the Agent mRNA is purified so that the mRNA is substantially free, virtually free, essentially free, or free of contaminants (or of a particular RNA contaminant, such as dsRNA). By substantially free, virtually free, essentially free, or free of contaminants (or of a particular RNA contaminant, such as dsRNA), it is meant that less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01%, respectively, of the total mass or weight of the RNA in the Agent mRNA is composed of contaminants (or of a particular RNA contaminant, such as dsRNA). The amounts and relative amounts of non-contaminant mRNA molecules and RNA contaminant molecules (or of a particular RNA contaminant, such as dsRNA) may be determined by HPLC or other methods used in the art to separate and quantify RNA molecules. In some preferred embodiments wherein the Agent mRNA (including GAUC, GAC or GAm.sup.5C Agent mRNA) is substantially free, virtually free, essentially free, or free of contaminant dsRNA, the relative amounts of non-contaminant mRNA and of contaminant dsRNA are assayed using the J2 dsRNA-specific antibody (English & Scientific Consulting, Szirk, Hungary); by substantially free, virtually free, essentially free, or free of dsRNA it is meant that less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01%, respectively, of the total mass or weight of the RNA in the Agent mRNA consists of dsRNA of a size greater than about 40-basepairs in length when assayed by dot blot immunoassay as described below using the J2 dsRNA-specific antibody or using another assay that gives equivalent results to the assay described herein. It shall be understood herein that the results of the dot blot immunoassays using the J2 dsRNA-specific antibody will be based on comparing the assay results obtained using the Agent mRNA with the assay results of J2 dsRNA-specific antibody dot blot immunoassays performed at the same time with dsRNA standards comprising known quantities of dsRNA of the same or equivalent size and J2 antibody binding.
[0067] As defined herein, Agent mRNA (or Exogenous mRNA) may be analyzed for the amount or relative amount of contaminant dsRNA by performing the following dot blot immunoassay using a dsRNA-specific antibody, such as the J2 dsRNA-specific antibody, or another antibody that gives equivalent results: RNA samples are spotted (5 l/dot) on Nytran SPC positively charged nylon membranes and then allowed to dry on the nylon membrane for 30 minutes. The membrane is then blocked in blocking buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 5% W/V dry milk) at room temperature for 1 hour on a rotating platform. The primary antibody (e.g., J2 antibody; English & Scientific Consulting, Hungary) is added at 1 g/ml in blocking buffer at room temperature for 1 hour on a rotating platform. The membranes are then washed 6 times for 5 minutes in 20 mls of wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20). The secondary antibody (anti-mouse HRP (Cell Signaling Technologies, Danvers, Mass.) is added at 1:1000 in blocking buffer at room temperature for 1 hour on a rotating platform. The membranes are then washed 6 times for 5 minutes in 20 mls of wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Then, equal volumes of Supersignal West Pico Chemiluminescent Substrates (Cat #34080, Thermo Scientific) are added and the color is allowed to develop for 5 minutes on a rotating platform. The dots are imaged by exposing film in the dark room and developing the film in Kodak Developer for 1 minute and Kodak Fixer for 1 minute.
[0068] The present invention is not limited with respect to the purification methods used to purify the Agent mRNA or Exogenous mRNA, and the invention includes use of any method that is known in the art or developed in the future in order to purify the mRNA and remove contaminants, including RNA contaminants, that interfere with the intended use of the mRNA. For example, in preferred embodiments, the purification of the mRNA removes contaminants that are toxic to the cells (e.g., by inducing an innate immune response in the cells, or, in the case of RNA contaminants comprising dsRNA, by inducing an interferon response or by inducing RNA interference (RNAi), e.g., (via siRNA, miRNA or long RNAi molecules) and contaminants that directly or indirectly decrease translation of the mRNA in the cells. In some embodiments, the mRNA is purified by HPLC. In certain embodiments, the mRNA is purified using on a polymeric resin substrate comprising a C18 derivatized styrene-divinylbenzene copolymer and a triethylamine acetate (TEAA) ion pairing agent is used in the column buffer along with the use of an acetonitrile gradient to elute the mRNA and separate it from the RNA contaminants in a size-dependent manner; in some embodiments, the mRNA purification is performed using HPLC, but in some other embodiments a gravity flow column is used for the purification. In some embodiments, the mRNA is purified using a method described in the book entitled RNA Purification and Analysis (Gjerde et al., 2009). In some embodiments, the mRNA purification is carried out in a non-denaturing mode (e.g., at a temperature less than about 50 C., e.g., at ambient temperature). In some embodiments, the mRNA purification is carried out in a partially denaturing mode (e.g., at a temperature less than about 50 C. and 72 C.). In some embodiments, the mRNA purification is carried out in a denaturing mode (e.g., at a temperature greater than about 72 C.). Those with knowledge in the art will know that the denaturing temperature depends on the melting temperature (Tm) of the mRNA that is being purified as well as on the melting temperatures of RNA, DNA, or RNA/DNA hybrids which contaminate the mRNA. In some other embodiments, the mRNA is purified as described (Mellits et al., 1990). These authors used a three step purification to remove the contaminants which may be used in embodiments of the present invention. Step 1 was 8% polyacrylamide gel electrophoresis in 7M urea (denaturing conditions). The major RNA band was excised from the gel slice and subjected to 8% polyacrylamide gel electrophoresis under nondenaturing condition (no urea) and the major band recovered from the gel slice. Further purification was done on a cellulose CF-11 column using an ethanol-salt buffer mobile phase which separates double stranded RNA from single stranded RNA (Barber, 1966; Franklin, 1966; Zelcer et al., 1981) and the final purification step was cellulose chromatography. In some other embodiments, the mRNA is purified using an hydroxylapatite (HAP) column under either non-denaturing conditions or at higher temperatures as described in (Andrews-Pfannkoch et al., 2010; Clawson and Smuckler, 1982; Lewandowski et al., 1971; Pays, 1977). In some other embodiments, the mRNA is purified by weak anion exchange liquid chromatography under non-denaturing conditions as described by (Easton et al., 2010). In some embodiments, the mRNA is purified using a combination of any of the above methods or another method known in the art or developed in the future. In still another embodiment, the mRNA used in the compositions and methods of the present invention is purified using a process which comprises treating the mRNA with an enzyme that specifically acts on (e.g., digests) one or more contaminant RNA or contaminant nucleic acids (e.g., including DNA), but which does not act on (e.g., does not digest) the desired mRNA. For example, in some embodiments, the mRNA used in the compositions and methods of the present invention is purified using a process which comprises treating the mRNA with a ribonuclease III (RNase III) enzyme (e.g., E. coli RNase III) and the mRNA is then purified away from the RNase III digestion products. A ribonuclease III (RNase III) enzyme herein means an enzyme that digests dsRNA greater than about twelve basepairs to short dsRNA fragments. In some embodiments, the mRNA used in the compositions, kits and methods of the present invention is purified using a process which comprises treating the mRNA with one or more other enzymes that specifically digest one or more contaminant RNAs (e.g., dsRNA) or contaminant nucleic acids (e.g., including DNA).
[0069] Differentiation or cellular differentiation means the naturally occurring biological process by which a cell that exhibits a less specialized state of differentiation or cell type (e.g., a fertilized egg cell, a cell in an embryo, or a cell in a eukaryotic organism) becomes a cell that exhibits a more specialized state of differentiation or cell type. Scientists, including biologists, cell biologists, immunologists, and embryologists, use a variety of methods and criteria to define, describe, or categorize different cells according to their cell type, differentiated state, or state of differentiation. In general, a cell is defined, described, or categorized with respect to its cell type, differentiated state, or state of differentiation based on one or more phenotypes exhibited by that cell, which phenotypes can include shape, a biochemical or metabolic activity or function, the presence of certain biomolecules in the cell (e.g., based on stains that react with specific biomolecules), or on or in the cell (e.g., based on binding of one or more antibodies that react with specific biomolecules inside the cell or on the cell surface). For example, in some embodiments, different cell types are identified and sorted using a cell sorter or fluorescent-activated cell sorter (FACS) instrument. Differentiation or cellular differentiation can also occur to cells in culture. In some embodiments, the term reprogramming is used herein to refer to differentiation or cellular differentiation, including de-differentiation or transdifferentiation, that occurs in response to delivery of one or more reprogramming factors into the cell, directly (e.g., by delivery of protein or polypeptide reprogramming factors into the cell) or indirectly (e.g., by delivery of an exogenous RNA preparation of the present invention which consists of one or more mRNA molecules, each of which encodes a reprogramming factor) and maintaining the cells under conditions (e.g., medium, temperature, oxygen and CO.sub.2 levels, matrix, and other environmental conditions) that are conducive for differentiation. The term reprogramming when used herein is not intended to mean or refer to a specific direction or path of differentiation (e.g., from a less specialized cell type to a more specialized cell type) and does not exclude processes that proceed in a direction or path of differentiation than what is normally observed in nature. Thus, in different embodiments of the present invention, reprogramming means and includes any and all of the following: [0070] (1) Dedifferentiation, meaning a process of a cell that exhibits a more specialized state of differentiation or cell type (e.g., a mammalian fibroblast, a keratinocyte, a muscle cell, or a neural cell) going to a cell that exhibits a less specialized state of differentiation or cell type (e.g., an iPS cell); [0071] (2) Transdifferentiation, meaning a process of a cell that exhibits one specialized state of differentiation or cell type (e.g., a mammalian fibroblast, a keratinocyte, or a neural cell) going to a different specialized state of differentiation or cell type (e.g., from a fibroblast or keratinocyte to a muscle cell); and [0072] (3) Expected or Natural Differentiation, meaning a process of a cell that exhibits any particular state of differentiation or cell type going to another state of differentiation or cell type as would be expected in nature if the cell was present in its natural place (e.g., in an embryo or an organism).
[0073] In some embodiments, the Agent mRNAs in the methods, compositions, systems, and kits of the present invention comprise or consist of the B18R, E3L, and K3L mRNAs that exhibit the nucleic acid sequences in
[0074] Briefly, in carrying out site directed mutagenesis of a DNA template, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA.
[0075] PCR mutagenesis is also suitable for making nucleic acid or amino acid sequence variants in the DNA template that is used for IVT (Vallette et al., 1989). Briefly, a small amount of the starting DNA template that one wishes to mutate is amplified by PCR using at least one PCR primer that exhibits a desired variant nucleic acid sequence compared to the corresponding region in the starting DNA template to generate a relatively large quantity of a specific DNA fragment that differs from the starting DNA template sequence only at the positions where the at least one PCR primers differed from the starting DNA template. This PCR mutagenesis process can be repeated using the product of a prior PCR mutagenesis reaction to introduce additional desired mutations in the DNA template.
[0076] Another method for preparing sequence variants, known as cassette mutagenesis, is based on the technique described by (Wells et al., 1985). The starting material is the plasmid (or other vector) comprising the starting DNA template to be mutated. The codon(s) in the starting DNA template to be mutated are first identified. There should be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they are generated in the starting DNA template using the above-described oligonucleotide-mediated mutagenesis method. The plasmid DNA is then cut with the restriction enzyme(s) to linearize it at these sites. Two oligonucleotides that exhibit the sequences of each strand of the DNA between the restriction sites but containing the one or more desired mutations are synthesized using standard procedures, and then hybridized together using standard techniques to generate a double-stranded DNA referred to as the cassette. This cassette is designed to have 5 and 3 ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated into the plasmid DNA from which the corresponding unmutated DNA was removed. This plasmid DNA now contains the mutated DNA sequence and can be used to prepare the DNA template for in vitro transcription of mRNA that exhibits the desired variant sequence.
[0077] Alternatively, or additionally, the desired amino acid sequence encoding one or more polypeptide variants can be determined, and a nucleic acid sequence encoding such amino acid sequence variant(s) can be generated synthetically. Conservative modifications in the amino acid sequences of the proteins may also be made. Naturally occurring residues are divided into classes based on common side-chain properties: [0078] (1) hydrophobic: norleucine, met, ala, val, leu, ile; [0079] (2) neutral hydrophilic: cys, ser, thr; [0080] (3) acidic: asp, glu; [0081] (4) basic: asn, gln, his, lys, arg; [0082] (5) residues that influence chain orientation: gly, pro; and [0083] (6) aromatic: trp, tyr, phe.
Conservative substitutions will entail exchanging a member of one of these classes for another member of the same class. The expected activity of the variant protein is confirmed following introduction of the Agent mRNA variant (e.g., from in vitro transcription of the variant DNA template) into the cell using methods disclosed herein.
[0084] Variant Agent mRNAs that encode variant B18R, E3L, or K3L proteins are generated (e.g., by truncation, deletion, or insertion into the DNA template for IVT) and screened as described in the Examples herein to determine if they function to reduce or suppress the innate immune response induced by a Foreign Substance (e.g., by the introduction of an Exogenous RNA into a cell). In this regard, any variant of an Agent mRNA that is constructed can be screened to identify variants suitable for use as a composition of the present invention or for use in a kit or method of the present invention.
[0085] Some embodiments of the invention comprise a kit or composition comprising or consisting of the mRNA encoding the antibody or artificial antibody. In some embodiments of any of the methods, kits and compositions of the invention, the mRNA is Agent mRNA encoding an antibody or artificial antibody. In some embodiments of any of the methods, kits and compositions of the invention, the mRNA is Exogenous mRNA encoding an antibody or artificial antibody.
[0086] Still another embodiment of the invention is a composition comprising or consisting of Exogenous mRNA encoding an antibody or artificial antibody for any desired function for which an antibody comprising protein is used in a cell, tissue or organism. For example, in some embodiments, the Exogenous mRNA encodes one or more antibodies or artificial antibodies that binds to a cell-specific or disease-specific or pathogen-specific protein that is expressed in a human or animal cell, tissue or organism. For example, in some embodiments, the Exogenous mRNA encodes one or more antibodies or artificial antibodies that binds to a cancer-specific or tumor-specific protein. In some embodiments of the method, Exogenous mRNA encoding one or more antibodies or artificial antibodies that is or are specific for a condition, disease or pathogen infecting a human or animal patient is administered to the patient to treat the condition, disease or pathogen-induced state (e.g., by administering the Exogenous mRNA to a cell, tissue or organism in the patient, e.g., by transfection, electroporation, or by intravenous, interperitoneal, intradermal, subdermal, or internodal injection). In some embodiments, the sequence of an mRNA encoding an antibody that reduces an innate immune response is first made in a non-human species and then, using any of the methods known in the art, the Agent mRNA in made by modifying the sequence so that the protein encoded by said Agent mRNA is similar to an antibody which would be produced naturally in humans; the antibody encoded by said Agent mRNA is then said to be humanized because it is has been adapted to be suitable for use in humans with minimal chance of inducing an active immune response. Agent mRNA encoding antibodies intended for use in other species can be similarly adapted for use in those species.
Examples
[0087] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
[0088] These examples demonstrate that Agent mRNA comprising or consisting of B18R mRNA, E3L mRNA, and K3L mRNA, alone or in combination, decrease cell toxicity (or increase cell survival) and increase translation of Exogenous mRNAs that are transfected into human or other mammalian cells. For example, introducing these Agent mRNAs into cells at the same time as or prior to (e.g., in some embodiments, 8-24 hours prior to) transfecting the cells with Exogenous mRNAs encoding one or more other proteins of interest enhances the translation or activity of the proteins encoded by those other Exogenous mRNAs. These Examples also demonstrate that introducing these Agent mRNAs into cells inhibits the biological activity of type I interferons (IFN, IFN) but not type II interferons (INF). The Examples also demonstrate that introducing Agent mRNAs comprising E3L mRNA, K3L mRNA, or both E3L mRNA and K3L mRNA together with an Exogenous mRNA comprising in vitro-transcribed MYOD mRNA resulted in reprogramming of mouse C3H10T1/2 mesenchymal stem cells to myoblast cells, whereas no reprogramming occurred when the C3H10T1/2 mesenchymal stem cells were transfected only with Exogenous mRNA comprising MYOD mRNA in the absence of these Agent mRNAs.
Materials and Methods
[0089] Templates for In Vitro Transcription
[0090] A B18R DNA template for preparing Agent mRNA comprising or consisting of B18R mRNA (GAC) was prepared as follows: a B18R coding sequence (cds) was cloned into a pUC-based plasmid DNA that contained a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR), a cloning site (into which the B18R cds was inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with NotI.
[0091] An E3L DNA template for preparing Agent mRNA comprising or consisting of E3L mRNA (GA.sub.Wm.sup.5C) was prepared as follows: an E3L cds was cloned into a pUC19-based plasmid DNA that contained a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR), a cloning site (into which the E3L cds was inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with SalI.
[0092] A K3L DNA template for preparing Agent mRNA comprising or consisting of K3L mRNA (GAm.sup.5C) was prepared as follows: a K3L cds was cloned into a pUC19-based plasmid DNA that contained a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR), a cloning site (into which the K3L cds was inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with SalI.
[0093] EGFP and c-MYC DNA templates for preparing EGFP or c-MYC mRNA (GAm.sup.5C), respectively, for use as negative control mRNAs in place of Agent B18R, E3L or K3L mRNAs, were prepared as follows: the respective EGFP or c-MYC cds was cloned into a pUC19-based plasmid DNA that contained a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR), a cloning site (into which the respective cds was inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with SalI.
[0094] A mouse Alkaline Phosphatase (ALKP) DNA template for preparing Exogenous mRNA comprising or consisting of mouse ALKP mRNA (GAUC) was prepared as follows: the mouse ALKP cds was cloned into a pUC19-based plasmid DNA that contained a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR),
a cloning site (into which the mouse ALKP cds was inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with SalI.
[0095] A mouse MYOD DNA template for preparing mouse Exogenous mRNA comprising or consisting of mouse MYOD mRNA (GAUC) for use in reprogramming mouse mesenchymal stem cells or somatic cells (e.g., fibroblasts) to myoblast cells was prepared as follows: the MYOD cds was cloned into a pUC19-based plasmid DNA that contained a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR), a cloning site (into which the MYOD cds was inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with SalI.
[0096] A luciferase (luc2) DNA template for preparing Exogenous mRNA comprising or consisting of firefly luciferase luc2 (Photinus pyralis luc2) mRNA (GAUC) was obtained by linearizing a commercially available plasmid (Promega, Madison, Wis.).
[0097] DNA templates for preparing other Exogenous mRNAs are similarly prepared as follows: the cds is cloned into a pUC19-based plasmid DNA that contains a T7 RNA polymerase promoter followed by 5 Xenopus Beta Globin (UTR), a cloning site (into which the cds is inserted), and a 3 Xenopus Beta Globin 3 UTR, and then linearized with SalI (or another restriction enzyme if the cds contains a SalI restriction site). For example, DNA templates were thus prepared for use in making mRNAs encoding the human and mouse transcription factors OCT4, SOX2, KLF4, LIN28, NANOG, c-MYC, L-MYC, and c-MYC(T58A), and Exogenous mRNAs are prepared using these templates as described herein, and used for reprogramming somatic cells to pluripotent stem cells. For example, in some embodiments, a total of about 1 to 1.2 microgram per transfection of mRNAs encoding human or mouse OCT4, SOX 2, KLF4 and a MYC protein selected from c-MYC, L-MYC and c-MYC(T58A) at a ratio of 3:1:1-3:1, respectively, (or 1 to 1.2 microgram per transfection of mRNAs encoding human or mouse OCT4, SOX 2, KLF4 and a MYC protein selected from c-MYC, L-MYC and c-MYC(T58A), and LIN28 and/or NANOG at a ratio of 3:1:1-3:1:1:(1) are transfected into human or animal somatic cells once daily for about 18 days, whereby the somatic cells are reprogrammed to pluripotent stem cells. In some embodiments, the use of an Agent mRNA encoding B18R, E3L and/or K3L proteins, or encoding other proteins inhibitors of innate immune response that are disclosed herein, facilitates or enhances the reprogramming of somatic cells to pluripotent stem cells by these Exogenous mRNAs. In still other embodiments, said Agent mRNA facilitates or enhances the reprogramming (e.g., differentiation, transdifferentiation) of one type of cell to another type of cell).
[0098] In Vitro Transcription, Capping and Polyadenylation to Make mRNAs
[0099] Each of the mRNAs used for transfection in the various experiments was prepared by in vitro transcription of the respective linear DNA template using the in vitro transcription components, the capping enzyme components, and the polyadenylation components provided in a T7 mScript Standard mRNA Production System (or the IVT components of an INCOGNITO T7 -RNA Transcription Kit or an INCOGNITO T7 5mC- & -RNA Transcription Kit) as described by the manufacturer (CELLSCRIPT, Inc., Madison, Wis.) unless otherwise stated herein. In some experiments in which a T7 mScript Standard mRNA Production System was used, pseudouridine triphosphate (TP) and/or m.sup.5CTP was used for in vitro transcription instead the corresponding UTP or CTP, respectively. The DNA templates for in vitro transcription were prepared as generally described in the T7 mScript or INCOGNITO product literature (e.g., by linearization of plasmid containing the mRNA coding sequence or by PCR of said gene).
[0100] NotI-linearized B18R DNA template was used as a template in in vitro transcription reactions using either the INCOGNITO T7 -RNA Transcription Kit, which contains pseudouridine triphosphate (TP) instead of UTP (CELLSCRIPT, Inc.), or the in vitro transcription components in the T7 mScript Standard mRNA Production System, except that TP was used in place of the UTP, to generate GAC RNA, which was subsequently capped and tailed to make GAFC mRNA.
[0101] E3L or K3L DNA templates were used as templates in in vitro transcription reactions containing TP and m.sup.5CTP to generate GAm.sup.5C RNAs, which were subsequently capped and tailed to make GAm.sup.5C mRNAs. Similarly, in some experiments, EGFP RNA or c-MYC mRNA was made for use as a negative control in place of Agent B18R, E3L or K3L mRNA by in vitro transcription of the respective linearized DNA template using either an INCOGNITO T7 5mC- & -RNA Transcription Kit (CELLSCRIPT, Inc.), which contains m.sup.5CTP and TP, or the T7 mScript Standard mRNA Production System, but with m.sup.5CTP (Trilink, San Diego, Calif.) and TP in place of standard CTP and UTP, respectively; these were subsequently capped and tailed to make GAm.sup.5C mRNAs.
[0102] Prior to capping and poly(A) tailing, Agent mRNAs E3L mRNA and K3L mRNA, as well as EGFP and c-MYC mRNAs, which were used as negative controls in place of Agent mRNA in the Examples herein, were treated with RNase III to remove interferon-inducing dsRNA, as described in the literature provided with the MINiMMUNE Kit (CELLSCRIPT, Inc).
[0103] All mRNAs used herein as Exogenous mRNAs that were not Agent mRNAs for inhibiting an innate immune response or control mRNAs for replacing an agent comprising mRNA for inhibiting an innate immune response were made by in vitro transcription of the respective DNA templates using the IVT components of the T7 mScript Standard mRNA Production System and only the canonical nucleotides GTP, ATP, UTP and CTP (GAUC). For example, firefly luciferase luc2 mRNA and mouse ALKP mRNA, which were used as Exogenous mRNAs for expressing proteins in cells whose activities could be easily detected and quantified, and MYOD mRNA, which was used as Exogenous mRNAs for expression in cells in order to induce reprogramming of the cells to myoblasts, were made by in vitro transcription of the respective DNA templates using the IVT components of the T7 mScript Standard mRNA Production System and only the canonical nucleotides GTP, ATP, UTP and CTP to generate GAUC RNA. For example, in some experiments, firefly luciferase luc2 (Photinus pyralis luc2) RNA was made for use as Exogenous mRNA by in vitro transcription of a linearized plasmid (Promega, Madison, Wis.) with only GTP, ATP, UTP and CTP (i.e., without substitution by TP or m.sup.5CTP). The Exogenous mRNAs containing GAUC disclosed herein are solely for the purpose of examples and are not intended to limit the application of the methods, compositions or kits disclosed herein. For example, in other embodiments, these Exogenous mRNAs are made by in vitro transcription using TP in place of UTP, and in still other embodiments, the Exogenous mRNAs are made by in vitro transcription using m.sup.5CTP in place of CTP, including wherein TP is used in place of UTP.
[0104] In order to make mRNA, in vitro-transcribed RNAs are capped using the ScriptCap m.sup.7G Capping Enzyme System (CELLSCRIPT, Inc.) to make cap0 RNA or using both the ScriptCap m.sup.7G Capping Enzyme System and the ScriptCap 2-O-Methyltransferase (CELLSCRIPT, Inc.) to make capl RNA, or with the same capping enzyme components in the T7 mScript Standard mRNA Production System; unless otherwise stated herein, all of the capped mRNAs used in the Examples herein exhibited a capl structure. For example, B18R RNA was capped with the ScriptCap m.sup.7G Capping Enzyme System and ScriptCap 2-O-Methyltransferase (CELLSCRIPT, Inc.) or with the corresponding capping enzyme components in the T7 mScript Standard mRNA Production System, as described in the respective product literature.
[0105] The resulting capped RNAs were polyadenylated using either the A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT, Inc.) or the poly(A) tailing components of theT7 mScript Standard mRNA Production System, as described in the respective product literature. For example, the resulting Cap 1-capped B18R RNA was polyadenylated using either the A-Plus Poly(A) Polymerase Tailing Kit or the poly(A) tailing components of theT7 mScript Standard mRNA Production System to generate B18R mRNA. For example, a 30-minute reaction using the A-Plus Poly(A) Polymerase Tailing Kit generated mRNAs with a poly(A) tail comprising approximately 150 A residues.
[0106] The in vitro-transcribed and capped and poly(A)-tailed Agent mRNAs or Exogenous mRNAs were made and purified as described in the literature provided with the T7 mScript Standard mRNA Production System. Briefly, after completion of the IVT reaction, the DNA template for IVT was digested by adding RNase-free DNase I to the in vitro transcription reaction and incubating for 15 minutes at 37 C. Then, the RNA was phenol-chloroform extracted, then precipitated by adding an equal volume of 5M ammonium acetate, incubated on ice for 10 minutes and spinning at 13,000 rpms for 10 minutes. The RNA pellet was washed with 70% ethanol and dissolved in water. Following capping and poly(A) tailing, the mRNA was again phenol-chloroform extracted, precipitated with ammonium acetate, washed with 70% ethanol and dissolved in water.
[0107] mRNA Transfections
[0108] Transfections were performed using commercially available transfection reagents, including the TransIT mRNA transfection reagent (Mirus Biosciences) and RNAiMax (Invitrogen), as described in the manufacturers' literature. For example,
for TransIT, the RNA was diluted in 250 ls Opti-MEMI (Invitrogen) and mixed with 5 ls TransIT BOOST reagent and 5 ls TransIT transfection reagent and the mixture was immediately applied to the cells. The present invention is not limited to use of these transfection reagents for delivering the Agent mRNA into cells. Any reagent or method (e.g., electroporation) that results in efficient delivery of the Agent mRNA into the cells and that does not result in high toxicity can be used in or with the compositions, kits or methods of the present invention.
[0109] Luciferase Assays
[0110] In all experiments with firefly luciferase luc2, except the pre-treatment time course, cells were washed in 2 mls 1PBS and incubated with 500 ls 1 Reporter Lysis Buffer (Promega). Plates were frozen for at least 1 hour, as the buffer requires a freeze-thaw cycle, and thawed at room temperature. Lysates were then transferred into microcentrifuge tubes and activity was determined using the Luciferase Assay System (Promega), where 20 ls of lysate was incubated with 100 ls of the kit luciferase reagent. Light emission was measured for 10 seconds with no lag time on a Lumiskan Ascent luminometer (Thermo Scientific). Protein concentration was determined for lysate samples using the Pierce BCA Protein Assay Kit (Fisher) and used to determine the total amount of protein present in 20 ls of lysate. Luminescence readings were normalized to the amount of protein used for the luciferase assays. In the pre-treatment time course experiment, cells were lysed using 1 Passive Lysis Buffer (Promega), in which a freeze thaw cycle was not required. Cells in these experiments were washed with 2 mls 1PBS, incubated with 500 pis 1 Passive Lysis Buffer for 2 minutes at room temperature, and transferred to microcentrifuge tubes. The lysate was then used in activity assays as described above.
[0111] Assays of the Effects of Purified B18R Protein on Expression of Exogenous
[0112] Luciferase mRNA
[0113] BJ fibroblasts (ATCC) were plated onto 6-well dishes coated with 0.1% gelatin (Millipore) at 110.sup.5 cells per well. Cells were fed fibroblast media consisting of Advanced MEM (Invitrogen), 10% Hyclone Heat Inactivated FBS (Fisher), 2 mM GLUTAMAX (Invitrogen), and 0.1 mM beta-mercaptoethanol (Sigma). Purified B18R protein (eBiosciences) was added to make final concentrations of 0, 50, 100, 200 or 400 g/ml. Cells were transfected with luciferase mRNA at a final concentration of 1.4 g/ml as described above. After 20 hours, cells were lysed and assayed for luciferase activity as described above.
[0114] Assays for Effects of B18R-Conditioned Medium on Luciferase Expression
[0115] Either BJ or 1079 fibroblast cells (both from ATCC) were plated 110.sup.5 cells per well in a 6-well dish coated with 0.1% gelatin. Both cell types were transfected with a plasmid that expresses the B18R protein under control of the constitutive CMV promoter at a final concentration of 2.7 g/ml using Lipofectamine 2000 (Invitrogen). A control plasmid that expressed EGFP under control of the CMV promoter was co-transfected at 0.5 g per reaction to check how well the transfection procedure worked. In the DNA transfections, 0.5 l of Lipofectamine 2000 per g of DNA was mixed with 12.5 ls per g DNA of Opti-MEMI and incubated at room temperature for 5 minutes. The mixture was then added to a solution of the DNA plus 12.5 ls per gs of DNA of Opti-MEMI. The transfection mix was incubated at room temperature for 20 minutes before application to cells fed with 1.5 mls of fibroblast media. Transfection medium was removed 4 to 5 hours after transfection, and cells were fed with 2.5 mls fresh fibroblast media per well. In the case of 1079 cells, the medium was conditioned for 48 hours, while for the BJ fibroblasts, the medium was conditioned for 20 hours. Conditioned media were collected from the cells and fed to a new plate of the same cell type. Control conditioned medium was made by transfecting cells with the same amount of the plasmid that expressed EGFP as was used to tranfect the cells with the plasmid that expressed the B18R protein. Luciferase mRNA was transfected into the cells at a final concentration of 1.4 g/ml in the presence of the conditioned medium, and cells were assayed for luciferase activity 24 hours later according to the procedures described above. Mock transfections with only the transfection reagent without any luciferase mRNA present were done as controls.
[0116] Assays for Effects of B18R mRNA on Luciferase Expression
[0117] Either BJ or 1079 fibroblast cells were plated at 110.sup.5 cells per well in a 6-well dish coated with 0.1% gelatin. Both cell types were transfected with various amounts of Agent mRNA consisting of B18R mRNA as indicated in
[0118] Time Course of B18R Pre-Treatment on Expression of Exogenous mRNA
[0119] 1079 fibroblast cells were plated at 110.sup.5 cells per well in a 6-well dish coated with 0.1% gelatin. B18R mRNA was transfected at a final concentration of 0.4 g/ml in fibroblast medium using the procedure described above. At various time points, as indicated in
[0120] Use of a Hela Cell Line Containing ISREs to Assay B18R mRNA Inhibition of Specific Interferon Responses
[0121] In some embodiments, Interferon Stimulated Response Elements (ISRE) (e.g., DNA containing four ISRE sites that exhibit the following sequence (SEQ ID NO:1):
TABLE-US-00001 5-CAGTTTCACTTTCCCCAGTTTCACTTTCCCCAGTTTCACTTTCCC CAGTTTCACTT-3
are inserted into the XhoI and BglII sites of pGL4.26 plasmid (Promega, Madison, Wis.) upsteam of a minimal promoter and the luc2 luciferase gene.
[0122] In some experiments, a human or animal cell line (e.g., a Hela human cell line) containing the ISREs is generated by using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) to transfect the cells (e.g., standard Hela cells from ATCC, Manassas, Va.) with the pGL4.264x ISREs-luc2-containing plasmid and clones in which the ISREs-luc2 are integrated are isolated by serial dilution of cells in 96-well dishes and selection with Hygromycin B (InivoGen, San Diego, Calif.; e.g., at 200 g/ml), followed by confirmation that the cell lines are responsive to recombinant Interferons (e.g., INF cat #11100-1, INFO cat #11415-1, and INF cat #285-IF from R&D Systems, Minneapolis, Minn.).
[0123] The ISREs-luc2 cell line is then used to assay the interferon response that results from the various interferons following transfection of the cell line with Agent B18R mRNA, compared to the interferon response that results following transfection of the cell line with EGFP mRNA as a negative control for the Agent mRNA comprising B18R mRNA. These results indicate if and to what extent the Agent B18R mRNA inhibits or reduces the interferon response by each of the respective interferons (e.g., INF, INFO or INF), thereby showing the specificity of interferon responses and their levels of response at various times (e.g., 8 hours, 16 hours, 24 hours) after transfection with Agent B18R mRNA compared to the EGFP mRNA as a negative control for the Agent B18R mRNA (e.g., by performing luciferase assays using the Bright-Glo Luciferase Assay Reagent from Promega, Madison, Wis., and a SpectraMax M3 luminometer from Molecular Devices, Sunnyvale, Calif.). For example, in some experiments, a Hela line containing the ISREs upstream of the luciferase gene are transfected with B18R mRNA or EGFP mRNA (e.g., each at 0.5 g-1/ml), followed by treatment 8 hours later with recombinant INF (2777 U/ml), INF (333 U/ml) or INF (300 g/ml) proteins and assay for luciferase activity at various times (e.g., 8 hours, 16 hours, 24 hours, or 48 hours) after the addition of the recombinant interferons to the cell culture media.
[0124] Use of a Hela Cell Line Containing ISREs to Assay E3L mRNA Inhibition of Specific Interferon Responses
[0125] The ISRE-luc2 Hela cell line was also used to test the effect of an Agent mRNA comprising E3L mRNA on the induction of an innate immune response by co-transfected LIN28 dsRNA. The LIN28 coding sequence was cloned in a pUC29-based plasmid downstream of T7 and T3 RNA polymerase promoters, and then different aliquots of the plasmid, which were linearized with BamHI or EcoRl, respectively, were used as templates for in vitro transcription with T7 and T3 RNA polymerases, respectively, in standard in vitro transcription reactions using GAUC canonical NTPs. These complementary RNAs were hybridized to generate dsRNA using the following hybridization parameters; 10 minutes at 70 C., 10 minutes at 60 C., 10 minutes at 50 C., 10 minutes at 40 C., then allowing the RNA to anneal for another 30 minutes at room temperature (22 C.). RNAiMax (Invitrogen) was used to transfect ISRE-luc2 cells with 0.2 g/ml of LIN28 dsRNA along with 0.5 g/ml of either Agent mRNA comprising E3L mRNA or human c-MYC mRNA as a negative control for the Agent E3L mRNA. Bright-Glo luciferase assays were performed 18 hours post transfection.
[0126] Effects of Agent mRNAs Comprising E3L or K3L mRNA on Expression of Exogenous mRNA Comprising Mouse Alkaline Phosphatase mRNA
[0127] In order to assay for the effects of Agent mRNA on expression of Exogenous mRNA consisting of mouse ALKP mRNA, 0.2 g/ml of ALKP GAUC mRNA was transfected using RNAiMax into mouse C3H10T1/2 mesenchymal stem cells or 1079 human fibroblasts, either alone or together with 0.5 g/ml of Agent mRNA comprising EGFP mRNA, E3L mRNA, K3L mRNA, or both E3L mRNA and K3L mRNA (each at 0.5 g/ml). Cells were lysed and ALKP reporter assays were performed 18 hours post transfection. Absorbance was read on a spectrophotometer at 405 nm as a readout of ALKP activity.
[0128] Use of Agents Comprising E3L or K3L mRNA to Facilitate MYOD mRNA-Induced Reprogramming of Mesenchymal Stem Cells to Myoblast Cells
[0129] Mouse C3H10T1/2 cells (Passage 16) were plated at 210.sup.5 cells per well of a gelatin-coated 6-well dish and grown overnight in DMEM, 10% FBS, GLUTAMAX, and pen/strep. The next day, the cells were switched to differentiation media (DMEM+2% horse serum, GLUTAMAX, and pen/strep). MYOD mRNAs were in vitro transcribed using the T7 mScript Standard mRNA Production System with GAUC nucleotides while E3L, K3L, EGFP mRNAs were all made using the T7 mScript RNA Transcription Kit with GAm.sup.5C nucleotides. Cells were transfected with 0.6 g/ml of the MYOD GAUC mRNA, and an Agent mRNA comprising E3L GAm.sup.5C mRNA (5 g/ml), K3L GAm.sup.5C mRNA (5 g/ml), or both E3L GAm.sup.5C mRNA and K3L GAm.sup.5C mRNA (5 g/ml of each) using RNAiMax in differentiation media. mRNA was added to a tube containing OptiMEM media with the total volume equaling 60 l in tube A. 5 l of RNAiMax was added to tube B for every g mRNA totaling 60 l in tube B. Tube A and Tube B were mixed and incubated at room temperature for 15 minutes. After 15 minutes the mRNA/RNAiMax mix was added to 2 ml of differentiation media already on the cells. The media were changed 4 hours post transfection with new differentiation media. Twenty-four hours after the first transfection another MYOD mRNA transfection was administered. The media were again changed 4 hours post transfection. Forty-eight hours after the first transfection, the cells were fixed and immunofluorescence assays were performed to detect Myosin Heavy Chain (MHC) expression, which is a marker for myoblast muscle differentiation.
[0130] Immunofluorescence.
[0131] C3H10T1/2 cell plates were washed with PBS and fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature. The cells were then washed 3 times for 5 minutes each wash with PBS followed by three washes in PBS+0.1% Triton X-100. The cells were then blocked in blocking buffer (PBS+0.1% Triton, 2% FBS, and 1% BSA) for 1 hour at room temperature. The cells were then incubated for 2 hours at room temperature with the primary antibody (mouse anti-human MHC Cat #05-716, Millipore, Temecula, Calif.), at a 1:1000 dilution in blocking buffer. After washing 5 times in PBS+0.1% Triton X-100, the C3H10T1/2 cells were incubated for 2 hours with the anti-mouse Alexa Fluor 555 (Cat #4409, Cell Signaling Technology, Danvers, Mass.) at 1:1000 dilutions in blocking buffer. Images were taken on a Nikon TS100F inverted microscope (Nikon, Tokyo, Japan) with a 2-megapixel monochrome digital camera (Nikon) using NIS-elements software (Nikon).
[0132] Results
[0133] B18R Protein Increased Activity of Firefly Luciferase mRNA in Cells.
[0134] BJ fibroblast cells transfected with luciferase mRNA in the presence of varying concentrations of purified recombinant B18R protein showed an increase in luciferase activity compared to cells transfected in the absence of B18R protein in the same medium (
[0135] Luciferase Activity by Luciferase mRNA is Increased in Cells Expressing B18R Protein.
[0136] Two cells lines, BJ fibroblasts and 1079 fibroblasts, were tested for the effects of transfecting luciferase mRNA using media conditioned by cells expressing B18R protein. In both BJ fibroblast (
[0137] Introducing B18R mRNA Prior to Transfection of Luciferase mRNA Increased Luciferase Activity.
[0138] Transfection of B18R mRNA before transfection of luciferase mRNA increased luciferase activity in both BJ fibroblast (
[0139] Introduction of B18R mRNA Prior to Transfection of Luciferase mRNA Increases Luciferase Activity in Cells.
[0140] Transfection of B18R mRNA 10, 24, 36, and 48 hours prior to luciferase mRNA transfection increased luciferase activity (
[0141] Introduction of B18R mRNA into Cells Inhibits Type I but not Type II Interferon Activity.
[0142] IFN, IFN and IFN have all been shown previously to activate Jak/Stat signaling cascades, ultimately resulting in Interferon Response Factor (IRF) binding to Interferon Stimulated Response Elements (ISREs) eliciting interferon responsive transcriptional activation (Nelson et al., 1993). B18R protein has previously been shown to bind to and inhibit type I interferons (IFN and IFN), but not type II interferons (IFN) (Symons et al., 1995). Similarly, based on assays using a Hela cell line that contains ISREs linked to the luc2 gene, B18R mRNA made with TP substituted for UTP (and/or with m.sup.5CTP substituted for CTP) results in inhibition IFN and IFN activity, but has no effect on IFN activity, whereas other Exogenous mRNAs (e.g., EGFP mRNA as a negative control for Agent mRNA comprising B18R mRNA) does not detectably inhibit the activity of any of the interferons (
[0143] Introduction of E3L or K3L mRNA Prior to Transfection of Alkaline Phosphatase mRNA Increased Alkaline Phosphatase Activity in Cells.
[0144] The vaccinia virus E3L and K3L intracellular proteins have been shown to inhibit innate immune system activation elicited by the introduction of dsRNA into the cytoplasm (Carroll et al., 1993; Chang et al., 1992; Davies et al., 1992; Rice et al., 2011; Xiang et al., 2002). Inhibition of the innate immune system by expression of E3L or K3L proteins increases transcription activation by blocking interferon induction through IRF3, the 2-5A/RNaseL pathway, and the PKR pathway (Carroll et al., 1993; Rice et al., 2011; Xiang et al., 2002). Transfection of ALKP mRNA alone or along with EGFP mRNA as a negative control for Agent mRNA comprising E3L or K3L mRNA results in similar ALKP reporter activity in C3H10T1/2 mouse mesenchymal stem cells (
[0145] Introduction of E3L mRNA Inhibits dsRNA-Induced Interferon Stimulation.
[0146] Transfection of dsRNA or in vitro transcribed RNA containing unwanted dsRNA contamination is known to bind to Toll-like receptor 3 (TLR3) and activate the immune system resulting in interferon production (Alexopoulou et al., 2001; Kariko et al., 2004). Transfection of dsRNA (e.g., LIN28 dsRNA) into the stable Hela cell line expressing Interferon Stimulated Response Elements (ISRE) driving luciferase 2 expression (ISRE-luc2) resulted in enhanced luciferase reporter activity as a readout of interferon pathway activation (
[0147] Introduction of E3L or K3L mRNA into Mesenchymal Stem Cells Facilitates
[0148] Their Reprogramming to Myoblasts by MYOD mRNA.
[0149] Mouse mesenchymal stem cells can be induced to form muscle myoblasts by overexpression of the master regulatory transcription factor, MYOD (Davis et al., 1987). Myoblast induction results in the formation of multinucleated myoblasts expressing myosin heavy chain (MHC) as a marker of muscle differentiation (Davis et al., 1987). We found that MYOD mRNA containing canonical nucleosides (GAUC) could not reprogram C3H10T1/2 mesenchymal stem cells into myoblasts after two successive transfections (
[0150] Introduction of E3L or K3L mRNA into Human or Mouse Fibroblasts or Keratinocytes Facilitates their Reprogramming to iPS Cells
[0151] Human or mouse fibroblasts or keratinocytes that are transfected daily for 18 days (at a total daily combined dose of 0.6-1.2 g (preferably 1-1.2 g) for all Exogenous mRNA (comprising a 3:1:1:1:1:1 molar ratio of GAC mRNAs encoding OCT3/4, SOX2, KLF4, NANOG, LIN28 and one protein selected from c-MYC(T58A), c-MYC and L-MYC) per approximately 10.sup.5 cells) in the presence of E3L GAm.sup.5C mRNA (5 g/ml), K3L GAm.sup.5C mRNA (5 g/ml), or both E3L GAm.sup.5C mRNA and K3L GAm.sup.5C mRNA (5 g/ml of each) complexed with RNAiMax transfection reagent would result in induction of a higher number of iPS cells compared to the same cells that are similarly transfected with the same Exogenous mRNA in the absent of the Agent mRNA. Thus, Agent mRNA comprising E3L GAm.sup.5C mRNA and/or K3L GAm.sup.5C mRNA can increase iPSC induction. Without being bound by theory, it is believed that this increased level of iPSC induction is due to a reduction in the innate immune response and/or an increase in the translation of the Exogenous mRNAs during the reprogramming period.
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[0192] All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.