RU2811940C2 - 5'-cap-trinucleotide compounds or 5'-cap compounds with large number of nucleotides and their use for rna stabilization, protein expression and in therapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention discloses new 5'-cap compounds and can be used to stabilize RNA with such 5'-cap compounds, as well as to increase their expression in cells.

EFFECT: obtaining 5'-cap-trinucleotide compounds or 5'-cap compounds with a large number of nucleotides.

33 cl, 5 dwg, 1 tbl, 5 ex

Description

Field of technology to which the invention relates

The present invention relates to 5'-cap compounds, in particular to trinucleotides or higher homologs (i.e. homologs with more than 3 nucleotides), where the 5'-cap compounds contain at least one phosphorothioate, phosphoselenoate and/or boranophosphate moiety in the phosphate bridge between the first and second nucleotide, and where the second nucleotide is locked in its 2' position. In particular, the present invention relates to the stabilization of RNA by such 5' cap compounds, particularly in the context of using RNA to express a peptide or protein of interest, for example in the context of vaccination, and provides compositions, such as pharmaceutical compositions, and cells containing RNA , modified with such a 5' cap compound, as well as methods for producing the peptide or protein of interest using the compositions or cells of the present invention. In addition, the present invention provides RNA, compositions or cells for use in therapy, in particular for use in a method of treating a disease or disorder using protein replacement therapy, genome engineering, genetic reprogramming or immunotherapy; a method for increasing RNA stability in cells; a method for increasing RNA expression in cells; and a method for producing RNA with a 5' cap structure.

Prerequisites for creating the invention

The concept of nucleic acid-encoded therapeutics originated in 1990 when Wolff et al. (Science, 247: 1465-1468) showed that direct intramuscular injection of in vitro transcribed (IVT) mRNA or plasmid DNA (pDNA) into the skeletal muscle of mice resulted in expression of the encoded proteins in the injected muscle. This discovery served as a major stimulus for further study of the applicability of nucleic acids in therapy, in particular in immunotherapy. DNA-based vaccines against infectious pathogens were first studied (Cox et al., 1993, J. Virol. 67: 5664-5667; Davis et al., 1993, Hum. Mol. Genet. 2: 1847-1851; Ulmer et al. , 1993, Science 259: 1745-1749; Wang et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 4156-4160). In addition, the utility of nucleic acids in gene therapy against tumors and for the induction of specific antitumor immunity has been studied (Conry et al., 1994, Cancer Res. 54: 1164-1168; Conry et al., 1995, Gene Ther. 2: 59-65 ; Spooner et al., 1995, Gene Ther. 2: 173-180; Wang et al., 1995, Hum. Gene Ther. 6: 407-418).

Nucleic acid-based therapy has several advantages. For example, the production of nucleic acid-based therapeutics is simple, relatively inexpensive, and DNA-based therapeutics are shelf stable. However, DNA-based therapeutics in particular are associated with many potential safety risks, such as the induction of anti-DNA antibodies (Gilkeson et al., 1995, J. Clin. Invest. 95: 1398-1402) and potential transgene integration into the host genome. This may lead to cellular gene inactivation, uncontrolled long-term transgene expression, or tumorigenesis, and thus is not usually done for tumor-associated antigens with oncogenic potential such as erb-B2 (Bargmann et al., 1986, Nature 319: 226-230) and p53 (Greenblatt et al., 1994, Cancer Res. 54: 4855-4878).

The use of RNA represents an attractive alternative to circumvent the potential risks of DNA-based therapeutics. Some of the advantages of RNA-based therapy are transient expression and non-transforming properties. In addition, for transgene expression, RNA does not need to be transported into the nucleus, and, moreover, it cannot be integrated into the host genome.

Two different strategies have been used for IVT RNA therapy, both of which have been successfully tested in different animal models. Either the RNA is administered directly to the patient by various routes (Hoerr et al., 2000, Eur. J. Immunol. 30: 1-7), or dendritic cells are transfected with IVT RNA using conventional in vitro transfection methods, and then the transfected dendritic cells are administered to the patient ( Heiser et al., 2000, J. Immunol. 164: 5508-5514). Immunization with RNA-transfected dendritic cells has been shown to induce antigen-specific cytotoxic T lymphocytes (CTL) in vitro and in vivo (Su et al., 2003, Cancer Res. 63: 2127-2133; Heiser et al., 2002 ), J. Clin. Invest. 109: 409-417). In addition, it has been shown that direct injection of deproteinized RNA into the lymph nodes of laboratory animals (intranodal injection) results in the uptake of said RNA mainly by immature dendritic cells, probably through a process called macropinocytosis (see DE 10 2008 061 522.6). It is assumed that the RNA is translated and the expressed protein is presented on MHC molecules on the surface of antigen-presenting cells, causing an immune response.

The main disadvantage of RNA-based therapies is the instability of RNA in vivo. Degradation of long-chain RNA from the 5' end is induced in the cell by the so-called decapping enzyme Dcp2, which cleaves m ⁷ GDP from the RNA chain. Thus, cleavage is predicted to occur between the alpha and beta phosphate groups of the RNA cap.

Eukaryotic messenger RNAs (mRNAs) carry a special structure at the 5' end, the so-called cap structure. It consists of an N ⁷ -methylated guanosine moiety that is added to the first transcribed RNA nucleotide, usually guanosine, through a 5'-5'-triphosphate bridge. Accordingly, this structure is often called m ⁷ GpppG. The m ⁷ GpppG structure is required, among other things, for the translation of mRNA into the encoded protein.

Cellular mRNAs in higher eukaryotes are further modified at the 5' end by methylation at the 2'-O position of the first nucleotide after the m ⁷ Gppp fragment. This structure is called cap-1 (compared to cap-0 for the unmethylated form). Although this modification was described more than 40 years ago, its function remained unknown until recently. It was only in 2010 that 2'-O cap methylation was first reported to evade recognition by cap-0 structure recognition proteins such as the IFIT proteins, especially IFIT1. Binding of IFIT1 to cap-0 mRNA disrupts the binding of eIF4E to initiate translation upon cap binding, resulting in decreased translation efficiency.

Synthetic mRNAs are typically produced by in vitro transcription from a suitable DNA template (eg, linearized plasmid DNA) using phage RNA polymerase (mainly T7 or SP6 RNA polymerase). Capped mRNAs can be produced by in vitro transcription by adding an excess cap dinucleotide, such as m ⁷ GpppG, to the reaction. However, it has been reported that the cap dinucleotide m ⁷ GpppG can be incorporated during in vitro transcription in two orientations, only one of which is functional. Therefore, anti-inverted cap analogs (ARCAs) have been developed that cannot be inserted in the reverse orientation due to modifications at the 2' or 3' position of m ⁷ -guanosine. Accordingly, it has been demonstrated in rabbit reticulocyte lysate and dendritic cells that ARCA-capped RNAs exhibit higher translation efficiency compared to m ⁷ GpppG-capped RNAs.

In the last decade, ARCAs have been further modified in an attempt to stabilize mRNAs against decapping enzymes and increase translational efficiency by increasing affinity for eIF4E. Modifications include various substitutions at the bridging and non-bridging oxygens in the phosphate bridge, extended phosphate groups, and guanosine modifications. The task is complicated by the fact that analogues of the cap structure, inert towards the decapping enzyme Dcp1-Dcp2, are not always good substrates for the initiation factor and, as a result, are poorly translated. However, the use of phosphorothioate-modified β-phosphate cap analogues (β-S-ARCA or β-S-ARCA) has resulted in mRNAs with both increased translation efficiency and an extended half-life, for example in dendritic cells, compared to ARCA or m ⁷ GpppG. β-S-ARCA is synthesized as a mixture of two diastereomers, called D1 and D2, depending on their HPLC elution pattern due to the introduction of a stereogenic P-site due to sulfur modification. Interestingly, these diastereomers have been shown to have different biological properties, particularly with respect to resistance to enzymatic degradation (e.g., Dcp2 degradation) and/or binding to eIF4E. While m ⁷ GpppG was commonly used in the past, ARCA-capped mRNAs are increasingly moving into preclinical and now clinical studies.

Because modification of the 2'-O position in a cap dinucleotide inhibits incorporation by phage RNA polymerase (which is predominantly used in ARCA), only cap-0 structures can be cotranscriptionally added in vitro when using a cap dinucleotide. In vitro capping of transcribed RNA can also be achieved post-transcriptionally using appropriate enzymes, for example from vaccinia virus. Here the cap-1 structure can be obtained. However, this synthesis process consists of two stages: transcription and capping, which makes it more labor-intensive. Moreover, the 5' RNA sequence itself has a strong influence on the efficiency of capping enzymes. In addition, this method is limited to the use of unmodified caps due to the specificity of the enzymes. Thus, no useful modifications as described above (eg phosphorothioate substitutions) can be incorporated in this manner.

As a quick summary, RNAs are particularly well suited for clinical use. However, the use of RNA in therapy is primarily limited by the short half-life of RNA, particularly in the cytoplasm, and/or recognition of RNA by proteins that recognize the cap-0 structure, such as the IFIT proteins, in particular IFIT1 (which ultimately leads to impaired RNA binding to eIF4E), ultimately both lead to low and/or insufficient protein expression. Thus, increasing RNA stability and/or RNA expression in cells is of particular importance for RNA therapy. It is therefore an object of the present invention to provide RNA that is particularly well suited for RNA therapy, that is, to provide a means for particularly potently stabilizing RNA and/or increasing RNA expression in cells. This technical problem has been solved by the present invention using the claimed subject matter.

The essence of the invention

In a first aspect, the present invention provides a 5' cap compound having a 5' cap structure according to formula (I):

formula (I)

where R ¹ is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl;

R ² and R ³ are independently selected from the group consisting of H, halogen, OH and optionally substituted alkoxy, or R ² and R ³ together form OXO, where X is selected from the group consisting of optionally substituted CH ₂ , optionally substituted CH ₂ CH ₂ , optionally substituted CH ₂ CH ₂ CH ₂ , optionally substituted CH ₂ CH(CH ₃ ) and optionally substituted C(CH ₃ ) ₂ , or R ² combined with a hydrogen atom at the 4' position of the ring to which R ² is attached, with the formation of –O-CH ₂ or –CH ₂ -O-;

R ⁴ and R ⁶ are independently selected from the group consisting of O, S, Se and BH ₃ ;

R ⁵ is selected from the group consisting of S, Se and BH ₃ ;

R ⁷ is a mononucleotide or oligonucleotide containing from 2 to 9 bases;

R ⁸ represents H, halogen or optionally substituted alkoxy;

n is 1, 2 or 3; And

B is a purine or pyrimidine base moiety.

In a second aspect, the present invention provides a composition or kit comprising the 5' cap compound of the first aspect. Such a kit or composition can be used to produce RNA with the 5' cap structure of the present invention.

In a third aspect, the present invention provides RNA that is modified with a 5' cap compound from the first aspect.

In a fourth aspect, the present invention provides a composition or cell containing RNA from the third aspect.

In a particularly preferred embodiment of the third and fourth aspects of the present invention, the RNA further comprises a nucleotide sequence encoding a peptide or protein of interest.

In a fifth aspect, the present invention provides a method for producing a peptide or protein of interest, comprising the step of using RNA from a particularly preferred embodiment of the third aspect, or a composition or cell from a particularly preferred embodiment of the fourth aspect.

In a sixth aspect, the present invention provides a method of expressing a peptide or protein of interest in an individual, comprising the step of administering to said individual an RNA from a particularly preferred embodiment of the third aspect, or a composition or cell from a particularly preferred embodiment of the fourth aspect.

In a seventh aspect, the present invention provides an RNA from a particularly preferred embodiment of the third aspect, or a composition or cell from a particularly preferred embodiment of the fourth aspect, for use in therapy.

In an eighth aspect, the present invention provides a method of treating a disease or disorder in a subject, comprising the step of administering to said subject RNA from a particularly preferred embodiment of the third aspect or a composition or cell from a particularly preferred embodiment of the fourth aspect. Treatment of the disease or disorder is preferably selected from the group consisting of protein replacement therapy, genomic engineering, genetic reprogramming and immunotherapy.

In a ninth aspect, the present invention provides an RNA from a particularly preferred embodiment of the third aspect, or a composition or cell from a particularly preferred embodiment of the fourth aspect, for use in a method of treating a disease or disorder in a subject. Treatment of the disease or disorder is preferably selected from the group consisting of protein replacement therapy, genomic engineering, genetic reprogramming and immunotherapy.

In a tenth aspect, the present invention provides a method of increasing the stability of RNA in cells (such as immature antigen presenting cells) and/or increasing the expression of RNA in cells (such as immature antigen presenting cells), wherein said method comprises providing said RNA with a structure according to formula (I), as defined in the first aspect; and transferring said RNA modified with the structure according to formula (I) into cells.

In an eleventh aspect, the present invention provides a method for producing RNA with a 5' cap structure, the method comprising performing a transcription reaction using a template nucleic acid in the presence of a 5' cap compound from the first aspect.

In additional aspects, the present invention provides the following:

- a method of inducing an immune response in an individual, comprising the step of administering to said individual an RNA from a preferred embodiment of the third aspect or a composition (preferably in the form of a vaccine composition) or a cell (preferably an immature antigen presenting cell) from a preferred embodiment of the fourth aspect; in one embodiment, the method is designed to induce an immune response against a virus, such as influenza virus (A, B or C), cytomegalovirus (CMV) or respiratory syncytial virus (RSV);

- a method of increasing the proportion of MHC molecules that present an antigen of interest on the surface of an antigen-presenting cell, wherein said method includes providing an RNA containing a nucleotide sequence encoding a peptide or protein containing said antigen of interest or an antigenic peptide thereof, wherein said RNA is modified with a structure according to the formula (I) as defined in the first aspect; and transferring said RNA modified with a structure corresponding to formula (I) into an immature antigen-presenting cell; in one embodiment, the antigen of interest is an antigen of a virus (such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof;

- a method of stimulating and/or activating immune effector cells, comprising providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest or an antigenic peptide thereof, wherein said RNA is modified with a structure according to formula (I) as defined in the first aspect; transferring said RNA modified by the structure of formula (I) into immature antigen-presenting cells; and ensuring contact of antigen-presenting cells with immune effector cells; in one embodiment, the antigen of interest is an antigen of a virus (such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof;

- a method of inducing an immune response in an individual, comprising providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest or an antigenic peptide thereof, said RNA modified with a structure according to formula (I) as defined in the first aspect; and administering said RNA modified with the structure according to formula (I) to said individual; in one embodiment, the antigen of interest is an antigen of a virus (such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof; And

- a method for inducing an immune response in an individual, comprising providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest or an antigenic peptide thereof, said RNA modified with a structure according to formula (I) as defined in the first aspect; transfer of said RNA, modified with the structure according to formula (I), into immature antigen-presenting cells; and administering antigen presenting cells to said individual; in one embodiment, the antigen of interest is an antigen of a virus (such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof.

Additional aspects as well as advantages and novel features of the present invention will become apparent from the following detailed description, when necessary in combination with the accompanying drawings.

Brief description of drawings

Fig. 1: Synthesis of a P-imidazolide precursor (Im-pm ^2'-O GpG) for the synthesis of an exemplary 5'-cap compound of the present invention, m2 ^7,2'-O GppSpm ^2'-O GpG (hereinafter "Compounds 1"; OR = OCH ₃ ).

Fig. 2: Synthesis (A) and HRMS spectrum (B) of Compound 1 (m2 ^7,2'-O GppSpm ^2'-O GpG; OR = OCH ₃ ).

Fig. 3: Comparison of the translational ability of RNAs capped cotranscriptionally with different analogues of the 5' cap structure. D1 beta-S-ARCA: D1 diastereomer of beta-S-ARCA; D2 beta-S-ARCA: D2 diastereomer of beta-S-ARCA; D1 Compound 1: D1 diastereomer of Compound 1; D2 Compound 1: Diastereomer of D2 Compound 1. Luciferase RNAs containing the appropriate cap structures were electroporated into hiDC. Luciferase activity was recorded for 72 hours.

Fig. 4: in vivo translation of RNAs modified with various analogues of the 5' cap structure. Beta-S-ARCA D1/D2 RNA: RNA was prepared by IVT using the D1 or D2 beta-S-ARCA diastereomer; D1/D2 RNA of Compound 1: RNA was prepared by IVT using the D1 or D2 diastereomer of Compound 1; Cap-0/Cap-1 enzymatic RNA: RNA was prepared by IVT in the absence of any cap analogue and then, in a second step, enzymatically capped using either the vaccinia capping enzyme alone (enzymatic Cap-0 RNA) or using a capping enzyme vaccinia together with methyltransferase (enzymatic Cap-1 RNA). In FIG. Figure 4 shows the luciferase signal at 6 hours (A), 24 hours (B), or 48 hours (C) after administration.

Fig. 5: In vivo translation of murine erythropoietin (mEPO) RNAs modified with various 5' cap analogues having a cap-0 structure (A) or a cap-1 structure (B). ARCA G: RNA co-transcriptionally capped with ARCA G; D1: RNA cotranscriptionally capped with the D1 beta-S-ARCA diastereomer; Esar-0: enzymatically capped RNA providing the structure of cap-0; ARCA G + Ecar-1: RNA cotranscriptionally capped with ARCA G, then enzymatically capped using vaccinia virus capping enzyme and vaccinia methyltransferase, which provide cap-1 structure; D1+Esar-1: RNA co-transcriptionally capped with the D1 beta-S-ARCA diastereomer, then enzymatically capped using vaccinia virus capping enzyme and vaccinia virus methyltransferase, which provide the structure of cap-1; Esar-1: Enzymatically capped RNA that provides the structure of cap-1. mEPO mRNA (3 μg) containing 1-methylpseudouridine (m1Ψ) was prepared in TransIT® and administered intraperitoneally to mice. In FIG. Figure 5 shows the plasma levels of erythropoietin in mice at 6 hours, 24 hours, 48 hours or 72 hours after injection.

Detailed Description of the Present Invention

Although the present invention is described in more detail below, it should be understood that this invention is not limited to the specific methodologies, protocols and reagents described herein, as they may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used in this application have the same meanings as commonly understood by one skilled in the art.

In the following, elements of the present invention will be described in more detail. These elements are listed with specific embodiments, but it should be understood that they may be combined in any manner and in any quantities to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to only those embodiments expressly described. It should be understood that the present description supports and covers embodiments that combine the explicitly described embodiments with any number of disclosed and/or preferred elements. In addition, any permutations and combinations of all described elements in this application are to be deemed disclosed in the specification of this application unless the context indicates otherwise. For example, if in a preferred embodiment R ¹ is methyl and in another preferred embodiment R ⁵ is S, then in one preferred embodiment R ¹ is methyl and R ⁵ is S. Likewise, if in a preferred embodiment In one preferred embodiment, R ⁷ is *pm ^2'-O GpN, and in another preferred embodiment, R ⁸ is OCH ₃ , then in one preferred embodiment, R ⁷ is *pm ^2'-O GpN, and R ⁸ represents OCH ₃ .

Preferably, terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will use, unless otherwise indicated, conventional chemistry, biochemistry and recombinant DNA techniques as explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J Sambrook et al Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout the present specification and the claims that follow, unless the context otherwise requires, the word “comprise” and its variants such as “comprises” and “comprising” will be understood to include the specified term, integer or step, or a group of members, integers or stages, but not to the exclusion of any other member, integer or stage, or a group of members, integers or stages. The term "consisting essentially of" means the exclusion of other elements, integers, or steps of any significant significance. The term “comprising” covers the term “consisting essentially of,” which in turn covers the term “consisting of.” Thus, in each case in this application, the term “comprising” may be replaced by the term “consisting essentially of” or “consisting of”. Likewise, in each instance in this application, the term “consisting essentially of” may be replaced by the term “consisting of.”

Terms in the singular (the articles "a", "an" and "the" in the original) and similar references used in the context of the description of the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural if unless otherwise stated in this application or unless clearly inconsistent with the context. The listing of ranges of values in this application is intended solely as a shorthand method for individually referring to each individual value falling within that range. Unless otherwise specified herein, each such individual value is included herein as if it were individually stated herein. All methods described in this application can be performed in any suitable order, unless otherwise indicated in this application or unless clearly inconsistent with the context. The use of any and all examples or illustrative language (eg, “such as”) herein is intended merely to better illustrate the invention and is not intended to limit the scope of the claimed invention. Nothing in the specification should be construed as indicating any element not claimed as being essential to the practice of the invention.

Several documents are cited throughout this description. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), both above and below, is incorporated herein by reference in its entirety. Nothing in this application should be construed as an admission that the invention is not entitled to precede such disclosure as a prior invention.

According to the invention, the term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the invention, the nucleic acid can be single-stranded or double-stranded, linear or covalently closed in a circle. According to the invention, the nucleic acid can be isolated. The term "isolated nucleic acid" in accordance with the invention means that the nucleic acid (i) has been amplified in vitro, for example by polymerase chain reaction (PCR) for DNA or in vitro transcription (using, for example, RNA polymerase) for RNA ; (ii) was obtained recombinantly by cloning; (iii) has been purified, for example, by digestion and separation by gel electrophoresis; or (iv) was synthesized, for example, by chemical synthesis.

In the context of the present invention, the term "DNA" refers to a molecule that contains deoxyribonucleotide residues and preferably consists entirely or substantially of deoxyribonucleotide residues. "Deoxyribonucleotide" refers to a nucleotide that lacks a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. The term "DNA" includes isolated DNA, such as partially or completely purified DNA, substantially pure DNA, synthetic DNA and recombinantly produced DNA, and includes modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such changes may include the addition of non-nucleotide material, for example, to the end(s) of DNA or within DNA, for example, to one or more DNA nucleotides. Nucleotides in DNA molecules may also contain non-standard nucleotides, such as unnatural nucleotides or chemically synthesized nucleotides. These altered DNAs can be called analogs, or analogues, of natural DNA.

In the context of the present invention, the term "RNA" refers to a molecule that contains ribonucleotide residues and preferably consists entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. The term "RNA" includes isolated RNA, such as partially or fully purified RNA, substantially pure RNA, synthetic RNA and recombinantly generated RNA, and includes modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such changes may include the addition of non-nucleotide material, for example, to the end(s) of the RNA or within, for example, one or more nucleotides of the RNA. Nucleotides in RNA molecules may also contain non-standard nucleotides, such as unnatural nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered/modified nucleotides may be referred to as natural nucleotide analogs, and the corresponding RNAs containing such altered/modified nucleotides (ie, altered/modified RNAs) may be referred to as natural RNA analogs. A molecule is “substantially composed of ribonucleotide residues” if the molecule contains at least 40% ribonucleotide residues (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (ie, naturally occurring) nucleotide residues or their analogues). In the context of the present invention, the RNA, preferably mRNA, is modified with a 5' cap compound of the present invention and preferably contains one or more additional modifications to further stabilize the RNA, as described below.

According to the invention, the RNA has a length of at least 20, preferably at least 50, in particular at least 100 nucleotides, for example from 100 to 15000, more preferably from 50 to 10000, more preferably from 100 to 5000, in particular from 200 to 1000 nucleotides. The RNA (in particular mRNA) that encodes a peptide or protein preferably has a length of at least 50, more preferably at least 150, in particular at least 200 nucleotides, for example from 100 to 15,000, more preferably from 50 to 10,000, more preferably from 100 to 5000, in particular from 200 to 1000 nucleotides.

According to the invention, "RNA" includes mRNA, tRNA, rRNA, snRNA, ssRNA, dsRNA and inhibitory RNA, and is preferably mRNA.

According to the invention, "dsRNA" means double-stranded RNA and is RNA with two partially or fully complementary strands.

According to the present invention, the term "mRNA" means "messenger RNA" and refers to a "transcript" that can be produced using a DNA template and can encode a peptide or protein. Typically, mRNA includes a 5'UTR, a peptide/protein coding region, and a 3'UTR. In the context of the present invention, the mRNA is preferably generated by in vitro transcription (IVT) from a DNA template. As stated above, in vitro transcription methodology is known to those skilled in the art, and many in vitro transcription kits are commercially available.

mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and join together to form double helices.

mRNA has a limited half-life in cells and in vitro. Thus, according to the invention, the stability and/or translation efficiency of RNA can be modified as necessary. For example, the mRNA may be stabilized and/or its translation may be enhanced by one or more modifications that have a stabilizing effect and/or increase the efficiency of translation of the mRNA. Such modifications are described, for example, in WO 2007/036366, the disclosure of which is incorporated herein by reference in its entirety. To increase the expression of the mRNA according to the present invention, it can be modified in the coding region, that is, in the sequence encoding the expressed peptide or protein, preferably without changing the sequence of the expressed peptide or protein, for example, to increase the GC content in order to increase the stability of the mRNA and to optimize codons and thus to increase translation in cells.

RNA can be isolated from cells, can be obtained from a DNA template, or can be chemically synthesized using methods known in the art. In preferred embodiments, the RNA is synthesized in vitro from a DNA template. In one particularly preferred embodiment, the RNA, in particular mRNA, is produced by in vitro transcription from a DNA template. The methodology for in vitro transcription is known to one skilled in the art; see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. In addition, various in vitro transcription kits are commercially available, such as those from Thermo Fisher Scientific (such as TranscriptAid ^TM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (e.g. HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), from Promega (e.g. RiboMAX™, HeLaScribe®, Riboprobe® systems), from Jena Bioscience (e.g. SP6 or T7 transcription kits) and from Epicenter (e.g. like AmpliScribe™). In one particularly preferred embodiment, the RNA is in vitro transcribed RNA (IVT RNA). To provide modified RNA, appropriate modified nucleotides, such as modified natural nucleotides, non-natural nucleotides, and/or modified non-natural nucleotides, can be included during synthesis (preferably in vitro transcription), or can be added to the RNA and/or to the RNA after transcription. certain modifications may be added.

The RNA of the present invention is at least modified with a 5' cap compound of the present invention.

In a preferred embodiment, the RNA of the present invention includes a nucleic acid sequence encoding a peptide or protein, preferably a pharmaceutically active peptide or protein, and is capable of expressing said peptide or protein, particularly when transferred into a cell or subject. Thus, the RNA of the present invention preferably contains a coding region (open reading frame (ORF)) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with a start codon and ending with a stop codon.

In accordance with the invention, the term “pharmaceutically active peptide or protein” means a peptide or protein that can be used to treat an individual where expression of the peptide or protein may be beneficial, for example to alleviate the symptoms of a disease or disorder. Preferably, the pharmaceutically active peptide or protein has therapeutic or palliative properties and may be administered to ameliorate, alleviate, mitigate, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. Preferably, the pharmaceutically active peptide or protein has a beneficial or beneficial effect on a disease or condition of an individual when administered to the individual in a therapeutically effective amount. The pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or disorder or reduce the severity of such a disease or disorder. The term "pharmaceutically active peptide or protein" includes entire proteins or polypeptides and may also refer to pharmaceutically active fragments thereof. It may also include pharmaceutically active peptide or protein analogues.

Specific examples of pharmaceutically active peptides and proteins include cytokines, adhesion molecules (eg, integrins), immunoglobulins (eg, antibodies), immunologically active compounds (eg, antigens), hormones, growth factors, protease inhibitors (eg, alpha-1 antitrypsin ), enzymes (eg, herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes and lactase), receptors (eg, growth factor receptors), apoptosis regulators, transcription factors, tumor suppressor proteins , structural proteins, reprogramming factors, genetically engineered proteins and blood proteins, but are not limited to.

In accordance with the invention, the term “cytokines” refers to proteins that have a molecular weight of about 5 to 20 kDa and that are involved in cell signaling (eg, paracrine, endocrine and/or autocrine signaling). In particular, when released, cytokines influence the behavior of cells around the site of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNF). According to the present application, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they usually act in much more variable concentrations than hormones, and (ii) they are usually produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons usually have antiviral, antiproliferative and immunomodulatory effects. Interferons are proteins that modify and regulate the transcription of genes within a cell by binding to interferon receptors on the surface of the regulated cell, thereby preventing the virus from replicating in the cells. Interferons can be divided into two types. IFN-γ is the only type II interferon; all others are type I interferons. Type I and type II interferons differ in gene structure (type II interferon genes have three exons; type I - one exon), location on the chromosome (in humans, type II genes are located on chromosome 12; type II interferon genes I are linked and are located on chromosome 9), as well as by the types of tissues in which they are produced (type I interferons are synthesized everywhere, type II - by lymphocytes). Type I interferons competitively inhibit each other's binding to cellular receptors, whereas type II interferons have a separate receptor. According to the invention, the term "interferon" or "IFN" preferably refers to type I interferons, in particular interferon alpha and interferon beta. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic protein (BMP), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 11 (IL-11).

According to the invention, the term “hormones” refers to a class of signaling molecules produced by glands, the signaling typically involving the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) hormone receptor binding; (v) signal retransmission and amplification; and (vi) hormone breakdown. Hormones differ from cytokines in that (1) hormones typically act in less variable concentrations and (2) are typically produced by specific cell types. Specific examples of hormones include insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormones (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin , leptins, catecholamines, gonadotropins, trophic hormones and dopamine. In one embodiment, the "hormone" is a peptide or protein hormone such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormones (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin and leptins.

According to the invention, the term “adhesion molecules” refers to proteins that are located on the surface of a cell and are involved in the binding of the cell to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (eg, integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (eg, P-selectin), and addressins.

Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins modulate cellular signaling pathways, such as those of transmembrane protein kinases such as receptor tyrosine kinases (RTKs). Such regulation can lead to cell growth, division, survival, differentiation or apoptosis. Specific _{examples of integrins} include: _α1β1 , _{α2β1 , α3β1} , _{α4β1 , α5β1 , α6β1 , α7β1 , αLβ2} , _{αMβ2 ,} α _IIb β ₃ , α _V β ₁ , α _V β ₃ , α _V β ₅ , α _V β ₆ , α _V β ₈ , and α ₆ β ₄ .

According to the invention, the term “immunoglobulins” or “immunoglobulin superfamily” refers to molecules that are involved in the processes of cell recognition, binding and/or adhesion. Molecules belonging to this superfamily share the common feature that they contain a region known as an immunoglobulin domain or fold. Members of the immunoglobulin superfamily include antibodies (eg, IgA, IgD, IgE, IgG, and IgM), T cell receptors (TCR), major histocompatibility complex (MHC) molecules, coreceptors (eg, CD4, CD8, CD19), accessory antigen receptor molecules (eg CD-3γ, CD3-δ, CD-3ε, CD79a, CD79b), costimulatory or inhibitory molecules (eg CD28, CD80, CD86) and others (eg CD147, CD90, CD7).

In accordance with the invention, the term "immunologically active compound" refers to any compound that modifies the immune response, preferably by inducing and/or suppressing the maturation of immune cells, inducing and/or suppressing the biosynthesis of cytokines, and/or altering humoral immunity by stimulating the production of B-antibodies. cells. The immunologically active compounds have potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can also suppress other aspects of the immune response, such as shifting the immune response away from the TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants. Specific examples of immunologically active compounds include interleukins, colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins , homing receptors and antigens, in particular tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic or viral antigens (e.g. one or more (e.g. 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10) influenza virus antigens (A, B or C), CMV or RSV)), allergens and autoantigens.

In accordance with the invention, the term "autoantigen" or "self-antigen" refers to an antigen that originates from the body of a subject (ie, an autoantigen may also be referred to as "autologous antigen") and which causes an abnormally strong immune response against that normal part of the body. Such strong immune reactions against self-antigens may be the cause of “autoimmune diseases.”

According to the invention, the term "allergen" refers to a type of antigen that originates from outside the body of a subject (i.e., an allergen can also be called a "heterologous antigen") and that causes an abnormally strong immune response in which the subject's immune system fights the perceived threat, which would otherwise be harmless to the subject. "Allergy" is a disease caused by such a vigorous immune response to allergens. An allergen is usually an antigen that is capable of stimulating a type I hypersensitivity reaction in atopic individuals through immunoglobulin E (IgE). Specific examples of allergens include allergens derived from peanut proteins (eg, Ara h 2.02), egg albumin, grass pollen proteins (eg, Phl p 5), and dust mite proteins (eg, Der p 2).

According to the invention, the term "growth factors" refers to molecules that are capable of stimulating cellular growth, proliferation, healing and/or cellular differentiation. Typically, growth factors act as signaling molecules between cells. The term "growth factors" includes certain cytokines and hormones that bind to specific receptors on the surface of their target cells. Examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs) such as VEGFA, epidermal growth factor (EGF), insulin-like growth factor, ephrins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor , granulocyte-macrophage colony-stimulating factor, neuregulins, neurotrophins (eg, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti-apoptotic factor survival), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β)) and tumor necrosis factor alpha (TNF-α ). In one embodiment, the "growth factor" is a peptide or protein growth factor.

According to the invention, the term "enzymes" refers to macromolecular biological catalysts that accelerate chemical reactions. Like any catalyst, enzymes are not consumed in the reaction they catalyze and do not change the equilibrium of said reaction. Unlike many other catalysts, enzymes are much more specific. In one embodiment, the enzyme is essential to the homeostasis of the subject, for example, any impairment of function (specifically, decreased activity, which may be caused by any mutation, deletion, or decreased production) of the enzyme results in disease. Examples of enzymes include cholesterol biosynthesis or degradation enzymes, steroidogenic enzymes, kinases, nucleases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, and neuraminidases such as tissue plasminogen activator, streptokinase, herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes (eg amylase, lipase and protease or mixtures thereof (eg pancrelipase)), and lactase.

According to the invention, the term "receptors" refers to protein molecules that receive signals (in particular, chemical signals called ligands) from outside the cell. Binding of a signal (eg, a ligand) to a receptor causes some cellular response, such as intracellular kinase activation. Receptors include transmembrane receptors (such as ion channel-coupled (ionotropic) receptors, G-protein coupled (metabotropic) receptors, and enzyme-coupled receptors) and intracellular receptors (such as cytoplasmic receptors and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (ie, receptors whose ligands are peptides) such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptors" refers to receptors that bind to growth factors. Growth factor receptors are the first step in the signaling cascade for cell differentiation and proliferation. Growth factor receptors can use the JAK/STAT, MAP kinase, and PI3 kinase pathways.

According to the invention, the term "protease inhibitors" refers to molecules, in particular peptides or proteins, which inhibit the function of proteases. Protease inhibitors can be classified by the protease that is inhibited (eg, aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors, threonine protease inhibitors, trypsin inhibitors) or by their mechanism of action (eg, suicide inhibitors such as serpins). Specific examples of protease inhibitors include serpins such as alpha-1 antitrypsin, aprotinin and bestatin.

According to the present invention, the term "apoptosis regulators" refers to molecules, in particular peptides or proteins, that modulate apoptosis, i.e. which either activate or inhibit apoptosis. Regulators of apoptosis can be divided into two broad classes: those that modulate mitochondrial function and those that regulate caspases. The first class includes proteins (eg, BCL-2, BCL-xL) that act to maintain mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of proapoptotic proteins such as cytochrome C into the cytosol. Also included in this first class are proapoptotic proteins (eg, BAX, BAK, BIM), which promote the release of cytochrome C. The second class includes proteins such as inhibitors of apoptosis proteins (eg, XIAP) or FLIP, which block caspase activation. Specific examples of apoptosis regulators are BAX, BCL-2, BCL-xL, BAK, BIM, XIAP and FLIP, in particular BAX.

According to the invention, the term "transcription factors" refers to proteins that regulate the rate of transcription of genetic information from DNA to messenger RNA, in particular by binding to a specific DNA sequence. Transcription factors can regulate cell division, growth, and cell death throughout life; cell migration and organization during embryonic development; and/or in response to signals from outside the cell, such as a hormone. Transcription factors contain at least one DNA-binding domain that binds to a specific DNA sequence, typically adjacent to genes that are regulated by the transcription factors. Specific examples of transcription factors include hepatocyte nuclear factors, MECP2, insulin promoter factor 1, FOXP2, FOXP3, STAT family of proteins, p53, HOX family of proteins, and SOX proteins such as SOX2.

According to the invention, the term "tumor suppressor proteins" refers to molecules, in particular peptides or proteins, that protect a cell from one of the steps on the path to cancer. Tumor suppressor proteins (usually encoded by corresponding tumor suppressor genes) exhibit a weakening or repressive effect on cell cycle regulation and/or promote apoptosis. Their functions may be one or more of the following: repression of genes necessary for cell cycle continuation; linking the cell cycle to DNA damage (as long as damaged DNA is present in the cell, cell division should not occur); initiation of apoptosis if damaged DNA cannot be repaired; inhibition of metastasis (eg, preventing tumor cell spread, blocking loss of contact inhibition, and inhibiting metastasis); and DNA repair. Specific examples of tumor suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI/SNF (SWItch), von Hippel-Lindau tumor suppressor (pVHL) of adenomatous coli-polyposis (APC), CD95, tumor suppressor 5 ( ST5), suppressed carcinogenicity 5 (ST5), suppressed carcinogenicity 14 (ST14) and Yippee-like 3 (YPEL3).

According to the invention, the term "structural proteins" refers to proteins that impart rigidity and strength to biological components that would otherwise be fluid. Structural proteins are primarily fibrous (eg, collagen and elastin) but can also be globular (eg, actin and tubulin). Typically, globular proteins are soluble as monomers, but polymerize to form long fibers that, for example, may constitute the cytoskeleton. Other structural proteins are motor proteins (such as myosin, kinesin and dynein), which are capable of generating mechanical forces, and surfactant proteins. Specific examples of structural proteins include collagen, fibroin, fibrinogen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin and myosin.

In accordance with the invention, the term "reprogramming factors" or "transcription reprogramming factors" refers to molecules, in particular peptides or proteins, which when expressed in somatic cells, optionally together with other agents, such as additional reprogramming factors, lead to the reprogramming or dedifferentiation of said somatic cells into cells with stem cell characteristics, in particular pluripotency. Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

According to the invention, the term "genomically engineered proteins" refers to proteins that are capable of inserting, deleting or replacing DNA in a subject's genome. Specific examples of genome-engineered proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9).

According to the invention, the term "blood proteins" refers to peptides or proteins that are present in the blood plasma of a subject, in particular in the blood plasma of a healthy subject. Blood proteins have a variety of functions, such as transport (eg, albumin, transferrin), enzymatic activity (eg, thrombin or ceruloplasmin), blood clotting (eg, fibrinogen), defense against pathogens (eg, complement components and immunoglobulins), protease inhibitors ( for example, alpha-1-antitrypsin), etc. Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (G-CSF) , modified factor VIII and anticoagulants.

In accordance with the invention, the term "protein replacement therapy" refers to a medical treatment that supplements or replaces a peptide or protein that has reduced activity in a patient compared to a healthy subject. Reduced activity (including null activity, which may occur when the peptide or protein is not present in the patient) may result from (i) reduced expression of the peptide or protein (i.e. the peptide or protein is fully functional, but its quantity is reduced) or (ii) the presence of one or more mutations in the amino acid sequence of the expressed peptide or protein (i.e. the peptide or protein is not fully functional). For example, such a reduction in the activity of a peptide or protein may result from a gene encoding the peptide or protein but containing one or more mutations such that (i) the expression of said gene is reduced or suppressed, resulting in a reduced amount of the peptide or protein (which is still may be fully functional) and/or (ii) the amino acid sequence of the peptide or protein encoded by the gene contains one or more mutations, resulting in an incompletely functional (or non-functional) peptide or protein. Diseases or disorders caused by reduced activity of a peptide or protein in a patient can be treated by replacing or adding the peptide or protein (protein replacement therapy), for example by administering RNA (particularly the RNA of the present invention) to a patient suffering from such disease or disorder. containing a nucleotide sequence encoding a peptide or protein. The nucleotide sequence encoding the peptide or protein may be autologous or heterologous to the patient. However, if the reduced activity of a peptide or protein in a patient is due to one or more mutations (i.e., resulting in an incompletely functional (or non-functional) peptide or protein), preferably the nucleotide sequence encoding the peptide or protein is heterologous to the patient, in particular obtained from a healthy subject (of the same species) expressing the peptide or protein in its native (i.e., non-mutant) form. For example, such protein replacement therapy may include the step of administering to the patient (i) RNA (in particular, RNA of the present invention) containing a nucleotide sequence encoding said peptide or protein (wherein said nucleotide sequence is preferably heterologous and can be obtained from a healthy subject) or (ii) a composition, for example a pharmaceutical composition, containing such RNA, or, alternatively, step (a) of transferring an RNA (in particular, the RNA of the present invention) containing a nucleotide sequence encoding said peptide or protein (wherein said nucleotide sequence is preferably heterologous and may be obtained from a healthy subject) into a cell (wherein said cell may be autologous to the patient) and (b) administering said transfected cell to the patient. Alternatively, (i) the RNA is preferably taken up by cells (eg, antigen-presenting cells such as monocytes, macrophages or dendritic cells, or other cells), and a translation product of a nucleotide sequence encoding a peptide or protein is produced (and optionally post-translationally modified) with obtaining a peptide or protein. In an alternative embodiment (ii), upon administration of the transfected cells to a patient, the transfected cells preferably express the peptide or protein.

The term "genomic engineering" refers to the process in which DNA is inserted, deleted or replaced in a subject's genome, preferably using genetically engineered proteins. Specific examples of genome-engineered proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9).

The term "genetic reprogramming" refers to the resetting of a cell's genetic program. The reprogrammed cell preferentially exhibits pluripotency.

In one embodiment, the pharmaceutically active peptide or protein is a disease-associated peptide or protein, i.e. it is causally associated with a disease or disorder.

For example, a disease or disorder may be caused by decreased activity of a peptide or protein. Reduced activity may result from (i) reduced expression of the peptide or protein (i.e., the peptide or protein is fully functional, but its quantity is reduced) or (ii) the presence of one or more mutations in the amino acid sequence of the expressed peptide or protein (i.e. the peptide or protein is not fully functional). For example, such a decrease in the activity of a peptide or protein may result from a gene encoding the peptide or protein but containing one or more mutations such that (i) the expression of the gene is reduced or suppressed, resulting in a decrease in the amount of the peptide or protein (which is still may be fully functional) and/or (ii) the amino acid sequence of the peptide or protein encoded by the gene contains one or more mutations, resulting in an incompletely functional (or non-functional) peptide or protein. Such diseases or disorders caused by reduced activity of the peptide or protein in a patient can be treated by replacing or adding the peptide or protein (protein replacement therapy), for example, by administering RNA (in particular, the RNA of the present invention) to the patient suffering from such disease or disorder ), containing a nucleotide sequence encoding a peptide or protein. The nucleotide sequence encoding the peptide or protein may be autologous or heterologous to the patient. However, if the reduced activity of a peptide or protein in a patient is due to one or more mutations (i.e., resulting in an incompletely functional (or non-functional) peptide or protein), preferably the nucleotide sequence encoding the peptide or protein is heterologous, in particular, derived from a healthy subject (of the same species) expressing the peptide or protein in its native (i.e., non-mutant) form. For example, such protein replacement therapy may include the step of administering to the patient (i) RNA (in particular, RNA of the present invention) containing a nucleotide sequence encoding said peptide or protein (wherein said nucleotide sequence is preferably heterologous and can be obtained from a healthy subject) or (ii) a composition, for example a pharmaceutical composition, containing such RNA, or, alternatively, step (a) of transferring an RNA (in particular, the RNA of the present invention) containing a nucleotide sequence encoding said peptide or protein (wherein said nucleotide sequence is preferably heterologous and may be obtained from a healthy subject) into a cell (wherein said cell may be autologous to the patient) and (b) administering said transfected cell to the patient. Alternatively, (i) the RNA is preferably taken up by cells (eg, antigen-presenting cells such as monocytes, macrophages or dendritic cells, or other cells), and a translation product of a nucleotide sequence encoding a peptide or protein is produced (and optionally post-translationally modified) with obtaining a peptide or protein. In an alternative embodiment (ii), upon administration of the transfected cells to a patient, the transfected cells preferably express the peptide or protein.

Alternatively, such diseases or disorders caused by reduced activity of the peptide or protein in a patient can be treated using genomic engineering, for example, by replacing the DNA sequence encoding the peptide or protein (i.e., resulting in an incompletely functional (or non-functional) peptide or protein) in a patient suffering from such a disease or disorder, the DNA sequence encoding the peptide or protein in its native (ie, non-mutant) form. For example, such a genetically engineered therapy may include the step of administering to a patient (i) RNA (in particular, the RNA of the present invention) containing a nucleotide sequence encoding a genetically engineered protein, and (ii) DNA containing a nucleotide sequence encoding a peptide or protein in its native (i.e. non-mutant) form. Upon administration, it is preferred that the RNA containing the nucleotide sequence encoding the genetically engineered protein and the DNA containing the nucleotide sequence encoding the peptide or protein in its native (i.e., non-mutant) form are taken up by cells (particularly diseased cells) and the translation product of the nucleotide sequence encoding the genome-engineered protein was formed (and, if necessary, post-translationally modified) to obtain the genome-engineered protein, so that the genome-engineered protein, together with the DNA sequence encoding the peptide or protein in its native (i.e. non-mutant) form, acted to replace a mutated DNA sequence in a cell's genome with a DNA sequence encoding a peptide or protein in its native (i.e., non-mutant) form.

In another alternative, such diseases or disorders caused by reduced activity of the peptide or protein in a patient can be treated by genetic reprogramming, for example by reprogramming somatic cells (particularly autologous somatic cells) of a patient suffering from such disease or disorder and administering said reprogrammed cells to the patient. This therapeutic approach may be particularly beneficial for patients suffering from a disease or disorder that causes the depletion or disappearance of cells that produce an essential peptide or protein (eg, a hormone such as insulin). For example, such genetic reprogramming therapy may include the steps of (a) introducing RNA (in particular, RNA of the present invention) containing a nucleotide sequence encoding one or more reprogramming factors into somatic cells; (b) creating conditions for the development of cells having characteristics of stem cells; and (c) administering to the patient cells having characteristics of stem cells. In a preferred embodiment, the somatic cells are autologous to the patient. Upon administration, cells having stem cell characteristics preferentially differentiate into cells expressing the desired peptide or protein.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2) and interleukin 10 (IL-11) , more preferably EPO. A disease or disorder caused by decreased cytokine activity, or a disease or disorder in which increasing the amount of a cytokine (i) improves, alleviates, alleviates, or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset of the disease or disorder, and/ or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., cytokine replacement or supplementation), appropriate genomic engineering therapy as described herein, and/or therapy with genetic reprogramming as described in this application. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is an adhesion molecule, particularly an integrin. A disease or disorder caused by decreased activity of an adhesion molecule, or a disease or disorder wherein increasing the amount of adhesion molecule (i) improves, alleviates, alleviates, or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset of the disease or disorder, and /or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (eg, by replacing or supplementing an adhesion molecule), appropriate genome engineering therapy as described herein, and/or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a hormone, particularly vasopressin, insulin or growth hormone. A disease or disorder caused by decreased activity of a hormone, or a disease or disorder in which increasing the amount of a hormone (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (eg, hormone replacement or supplementation), appropriate genome engineering therapy as described herein, and/or therapy with using genetic reprogramming as described in this application. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a growth factor, particularly VEGFA. A disease or disorder caused by reduced growth factor activity, or a disease or disorder in which increasing the amount of growth factor (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder and/or (ii) delays the onset of the disease or disorder and /or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (eg, by replacing or supplementing a growth factor), appropriate genomic engineering therapy as described herein, and/ or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-related peptide or protein, is an enzyme, preferably selected from the group consisting of herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes glands and lactase. A disease or disorder caused by decreased enzyme activity, or a disease or disorder in which increasing the amount of enzyme (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset of the disease or disorder and/ or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., by enzyme replacement or supplementation), appropriate genomic engineering therapy as described herein, and/or therapy with genetic reprogramming as described in this application. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a receptor, in particular growth factor receptors. A disease or disorder caused by decreased receptor activity, or a disease or disorder, wherein increasing the amount of the receptor (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., receptor replacement or complementation), appropriate genomic engineering therapy as described herein, and/or therapy with using genetic reprogramming as described in this application. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a regulator of apoptosis, particularly BAX. A disease or disorder caused by decreased activity of an apoptosis regulator, or a disease or disorder in which increasing the amount of an apoptosis regulator (i) improves, alleviates, alleviates, or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated by appropriate protein replacement therapy as described herein (eg, by replacing or supplementing an apoptosis regulator), appropriate genomic engineering therapy as described herein, and/ or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a tumor suppressor protein, particularly p53. A disease or disorder caused by decreased activity of a tumor suppressor protein, or a disease or disorder wherein increasing the amount of a tumor suppressor protein (i) improves, alleviates, reduces, or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., by replacing or supplementing a tumor suppressor protein), appropriate genomic engineering therapy as described in this application, and/or genetic reprogramming therapy as described in this application. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a structural protein, particularly surfactant protein B. A disease or disorder caused by decreased activity of a structural protein, or a disease or disorder in which an increase in the amount of structural protein protein (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy, as described herein (eg, by replacement or addition of a structural protein), appropriate genome engineering therapy as described herein, and/or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a transcription factor, particularly FOXP3. A disease or disorder caused by decreased activity of a transcription factor, or a disease or disorder in which increasing the amount of a transcription factor (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder and/or (ii) delays the onset of the disease or disorder and /or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (eg, by replacing or supplementing a transcription factor), appropriate genomic engineering therapy as described herein, and/ or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a reprogramming factor, such as OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG. A disease or disorder caused by decreased activity of a reprogramming factor, or a disease or disorder in which increasing the amount of a reprogramming factor (i) improves, alleviates, alleviates, or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (eg, by replacing or supplementing a reprogramming factor), appropriate genomic engineering therapy as described herein, and /or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a genetically engineered protein, particularly clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9). A disease or disorder caused by reduced activity of a genetically engineered protein, or a disease or disorder in which increasing the amount of a genetically engineered protein (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder, and/or (ii) delays onset of a disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., by replacing or supplementing a genetically engineered protein), appropriate genetically engineered therapy as described herein, and/or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein, such as a disease-associated peptide or protein, is a blood protein, particularly fibrinogen or alpha-1 antitrypsin. A disease or disorder caused by decreased activity of a blood protein, or a disease or disorder in which increasing the amount of protein in the blood (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder, and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., by replacing or supplementing a blood protein), appropriate genomic engineering therapy as described herein, and/or genetic reprogramming therapy as described herein. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein is an immunoglobulin, in particular an antibody. A disease or disorder caused by decreased immunoglobulin activity, or a disease or disorder in which increasing the amount of immunoglobulin (i) improves, alleviates, reduces or reverses one or more symptoms of the disease or disorder and/or (ii) delays the onset of the disease or disorder and/or (iii) reduces the severity of the disease or disorder, can be treated with appropriate protein replacement therapy as described herein (e.g., by replacing or supplementing immunoglobulin), appropriate genomic engineering therapy as described herein, and/or genetic reprogramming therapy, as described in this application. A patient suffering from such a disease or disorder, or at risk of developing such a disease or disorder, can be treated accordingly.

In one embodiment, the pharmaceutically active peptide or protein is an immunologically active compound, in particular an antigen, such as a disease-associated antigen. Thus, another example of disease associated peptides or proteins is disease associated antigen, i.e. an antigen that is characteristic of a disorder or disease and which under normal conditions, that is, in a healthy person, is specifically expressed in a limited number of organs and/or tissues or at certain stages of development (for example, a disease-associated antigen may under normal conditions be specifically expressed in a nonviable tissue, in reproductive organs, such as the testes, in trophoblastic tissue, such as the placenta or germline cells), and is expressed or abnormally expressed in one or more affected tissues. In this context, "limited amount" preferably means no more than 3, more preferably no more than 2 or 1. Specific examples of a disease-associated antigen include tumor-associated antigens, pathogen-associated antigens (e.g., virus antigens (e.g., virus influenza (A, B or C), CMV or RSV) and allergens. A disease or disorder that is characterized by a disease-associated antigen can be treated by inducing an immune response against said disease-associated antigen in a patient who has said disease or disorder or is susceptible to risk of its development. For example, in case the disease-associated antigen is a tumor-associated antigen, immunotherapy can be considered as cancer immunotherapy; in case the disease-associated antigen is a pathogen-associated antigen (for example, an antigen of a virus (such as a virus influenza (A, B or C), CMV or RSV), immunotherapy may be considered pathogen immunotherapy; and in case the antigen associated with the disease is an allergen, immunotherapy may be considered an allergy tolerance-enhancing therapy, respectively. Thus, the RNA of the present invention can be used to produce a disease-associated antigen that vaccinates an individual against a malignant disease or infectious disease, or can be used to produce an allergen that leads to tolerance to an allergy.

The term "immunotherapy" refers to a treatment, preferably involving a specific immune response and/or immune effector function(s).

As used herein, the term “de-differentiation” refers to the loss of specialization in form or function. In cells, de-differentiation results in a less committed cell. The term “committed” refers to cells that are considered permanently committed to a specific function. Committed cells are also called "terminally differentiated cells".

As used herein, the term “differentiation” refers to the adaptation of cells to a particular form or function. In cells, differentiation results in a more committed cell.

A "differentiated cell" is a mature cell that has undergone progressive changes during development to a more specialized form or function. Cell differentiation is the process through which a cell matures into a distinctly specialized cell type. Differentiated cells have different characteristics, perform specific functions, and are less likely to divide than their less differentiated counterparts.

An "undifferentiated" cell, such as an immature, embryonic, or primitive cell, typically has a nonspecific appearance, may perform few nonspecific actions, and may perform poorly or not perform functions normally performed by differentiated cells.

"Somatic cell" refers to any and all differentiated cells and does not include stem cells, germ cells, or gametes. Preferably, the term “somatic cell” as used herein refers to a terminally differentiated cell.

A "stem cell" is a cell that has the ability to self-renew, remain undifferentiated, and differentiate. A stem cell can divide without restriction, at least throughout the life of the animal in which it lives naturally. The stem cell does not differentiate completely; it is not the last stage of the differentiation pathway. When a stem cell divides, each daughter cell can either remain a stem cell or begin a course leading to terminal differentiation.

The term “cells having stem cell characteristics” is used herein to refer to cells that, although derived from differentiated somatic non-stem cells, possess one or more characteristics typical of stem cells, in particular embryonic stem cells. Such features include embryonic stem cell morphology such as compact colonies, high nuclear to cytoplasmic ratios and prominent nucleoli, normal karyotypes, expression of telomerase activity, expression of cell surface markers characteristic of embryonic stem cells, and/or expression of genes that are characteristic of embryonic stem cells. stem cells. Cell surface markers that are characteristic of embryonic stem cells are selected, for example, from the group consisting of stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, tumor antigen-1-60 (TRA-1-60) , TRA-1-81 and TRA-2-49/6E. Genes specific for embryonic stem cells are selected, for example, from the group consisting of endogenous OCT4, endogenous NANOG, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic stem cell specific developmental pluripotency associated gene 1 (ESG1), developmental pluripotency associated gene 2 (DPPA2), DPPA4 and telomerase reverse transcriptase (TERT) cells. In one embodiment, one or more characteristics typical of stem cells include pluripotency. In one embodiment, cells having stem cell characteristics exhibit a pluripotent state. In one embodiment, cells having stem cell characteristics have the developmental potential to differentiate into developed derivatives of all three primary germ layers. In one embodiment, the primary germ layer is endoderm and the developed derivative is intestinal-like epithelial tissue. In a further embodiment, the primary germ layer is mesoderm and the developed derivative is striated muscle and/or cartilage. In yet another embodiment, the primary germ layer is ectoderm and the developed derivative is neural tissue and/or epidermal tissue. In one preferred embodiment, cells having stem cell characteristics have the potential to differentiate into cells expressing the peptide or protein of interest. In accordance with the invention, generally, the terms “cells having stem cell characteristics,” “cells having stem cell properties,” “reprogrammed cells,” and “dedifferentiated cells,” or similar terms, have similar meanings and are used interchangeably herein.

In one embodiment, the RNA, particularly the RNA that contains a nucleic acid sequence encoding a peptide or protein and that is to be expressed in a cell, is single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is positive-sense single-stranded RNA. In one embodiment, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In one embodiment, the self-replicating RNA is or is derived from alphaviral genomic RNA. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is a Semliki Forest virus. In one embodiment, the self-replicating RNA contains one or more transgenes, which in one embodiment, if the RNA is viral RNA, can partially or completely replace viral sequences, such as viral sequences encoding structural proteins.

The term "nucleoside" (abbreviated here as "N") refers to compounds that can be considered as nucleotides without a phosphate group. Although a nucleoside is a nitrogenous base linked to a sugar (such as ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine and guanosine.

The five standard nucleosides that commonly constitute naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are usually designated by their single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly referred to as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) rather than ribonucleic acid (RNA). Conversely, uridine is found in RNA, not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA they will be represented as A, C and G, whereas in DNA they will be represented as dA, dC and dG.

The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified with one or more alkyl groups, more preferably one or more C _1-4 alkyl groups, even more preferably one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N ⁷ -alkyl-guanine, N ⁶ -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N ⁷ -C _1-4 -alkyl-guanine, N ⁶ -C _1-4 -alkyl-adenine, 5-C _1-4 -alkyl-cytosine, 5-C _1-4 -alkyl-uracil and N(1)-C _{1- 4} -alkyl-uracil, preferably N ⁷ -methyl-guanine, N ⁶ -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

The term "in vitro transcription" or "IVT" as used herein means that transcription (i.e., RNA generation) occurs in a cell-free manner, i.e. IVT does not use live/cultured cells, but transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

The term “modification” in the context of modified RNA (preferably mRNA) according to the present invention includes any modification of RNA (preferably mRNA) that is not naturally present in said RNA. In particular, the term "modification" refers to providing RNA (preferably mRNA) with the 5' cap structure of the present invention. For example, providing RNA (preferably mRNA) with a 5' cap structure of the present invention can be achieved by in vitro transcription of a DNA template in the presence of a 5' cap compound of the present invention, wherein said 5' cap structure is co-transcriptionally incorporated into the generated RNA strand, or RNA (preferably mRNA) can be produced, for example, by in vitro transcription, and the 5' cap structure can be attached to the RNA post-transcriptionally using capping enzymes, for example vaccinia virus capping enzymes.

The RNA (preferably mRNA) may contain additional modifications, for example to increase its stability and/or reduce immunogenicity and/or reduce cytotoxicity. For example, additional modification of the RNA, preferably an mRNA modified with a 5' cap junction of the present invention, may be an extension or truncation of the naturally occurring poly(A) tail, alteration of the 5' or 3' untranslated regions (UTRs), such as introducing a UTR not associated with the coding region of said RNA, replacing one or more natural nucleotides with synthetic nucleotides, and/or codon optimization (eg, to change, preferably increase, the GC content of the RNA).

RNA (preferably mRNA) having an unmasked poly-A sequence is translated more efficiently than RNA (preferably mRNA) having a masked poly-A sequence. The term "poly(A) tail" or "poly-A sequence" refers to the sequence of adenosine (specifically adenylyl) (A) residues that is typically located at the 3' end of an RNA molecule (preferably mRNA), and the "unmasked poly sequence" -A" means that the poly-A sequence at the 3' end of the RNA molecule (preferably mRNA) ends with the A of the poly-A sequence, and is not followed by nucleotides other than the A located at the 3' end, that is, downstream poly-A sequences. In addition, the long poly-A sequence, which is approximately 120 nucleotides in length, results in optimal transcript stability and RNA (preferably mRNA) translation efficiency.

Therefore, in order to increase the stability and/or expression of the RNA, preferably mRNA, according to the present invention, it can be modified so that it is present together with a poly-A sequence, preferably having a length of from 10 to 500, more preferably from 30 to 300, even more preferably from 65 to 200 and especially from 100 to 150 adenosine (in particular adenylyl) residues. In a particularly preferred embodiment, the poly-A sequence is approximately 120 adenosine (particularly adenylyl) residues in length. To further improve the stability and/or expression of RNA, preferably mRNA, according to the invention, the poly-A sequence can be unmasked.

In addition, the inclusion of a 3'UTR in the 3'untranslated region of an RNA molecule (preferably an mRNA) can lead to increased translation efficiency. A synergistic effect can be achieved by including two or more of these 3'UTRs (which are preferentially located in a head-to-tail orientation; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'UTRs may be autologous or heterologous to the RNA (preferably mRNA) into which they are introduced. In one specific embodiment, the 3'-UTR is derived from a globin gene or mRNA, such as an alpha2-globin, alpha1-globin, or beta-globin gene or mRNA, preferably beta-globin, more preferably human beta-globin. For example, RNA (preferably mRNA) can be modified by replacing an existing 3'UTR or inserting one or more, preferably two copies of a 3'UTR derived from a globin gene, such as alpha2-globin, alpha1-globin, beta globin, preferably beta-globin, more preferably human beta-globin.

The RNA (preferably mRNA) according to the invention may contain modified ribonucleotides to increase its stability and/or reduce immunogenicity and/or reduce cytotoxicity. For example, in one embodiment, in the RNA (preferably mRNA) of the invention, 5-methylcytidine is partially or completely, preferably completely, replaced by cytidine. Alternatively or additionally, in one embodiment, in the RNA (preferably mRNA) according to the invention, pseudouridine or N(1)-methylpseudouridine or 5-methyluridine is partially or completely, preferably completely, replaced by uridine. RNA (preferably mRNA) that is modified with pseudouridine (replacing part or all, preferably all, of uridine) is referred to herein as “Ψ-modified,” while the term “m1Ψ-modified” means that the RNA (preferably mRNA) contains N( 1)-methylpseudouridine (substituting partially or completely, preferably completely, uridine). In addition, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (replacing part or all, preferably all, uridine). Such Ψ-, m1Ψ- or m5U-modified RNAs of the present invention generally exhibit reduced immunogenicity compared to their unmodified forms and are thus preferred in applications in which the induction of an immune response must be avoided or minimized (eg, protein replacement therapy , genomic engineering therapy and genetic reprogramming therapy, as described herein).

A combination of the modifications described above, i.e. inclusion of a poly-A sequence, unmasking of a poly-A sequence, inclusion of one or more 3'-UTRs, and replacement of one or more natural nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (Ψ) or N(1)- methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine) has a synergistic effect on RNA (preferably mRNA) stability and increases translation efficiency. Thus, in a preferred embodiment, the RNA (preferably mRNA) of the present invention is not only modified with a 5'-cap compound of the present invention, but also contains a combination of the above three modifications, that is, (i) inclusion of a poly-A sequence, unmasking of a poly-A sequence, A; (ii) inclusion of one or more 3'UTRs; and (iii) replacing one or natural nucleotides with synthetic nucleotides (eg, 5-methylcytidine for cytidine and/or pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine). If necessary, the codons of the RNA (preferably mRNA) of the present invention can be further optimized, for example, to increase the GC content of the RNA and/or to replace codons that are rare in the cell (or subject) where the peptide or protein of interest is to be expressed by the codons , which are synonymous with frequent codons in a specified cell (or subject).

The term "RNA polymerase" as used herein refers to a DNA-dependent RNA polymerase that produces the primary transcriptional RNA. Examples of RNA polymerases suitable for generating IVT RNA according to the present invention include T7, T3 and SP6 RNA polymerases. The preferred RNA polymerase is T7 RNA polymerase.

The term "traditional 5' cap" refers to the cap structure found at the 5' end of an mRNA molecule, usually consisting of guanosine 5'-triphosphate (Gppp), which, through its triphosphate moiety, is linked to the 5' end of the next nucleotide mRNA (i.e. guanosine is linked to the rest of the mRNA through a 5'-5' triphosphate bond). Guanosine can be methylated at position N7 (resulting in the cap structure of m7Gppp).

As used herein, the term “cap-0” means the structure “m ⁷ GpppN”, where N is any nucleoside bearing an OH moiety at the 2' position. As used herein, the term "cap-1" means the structure "m ⁷ GpppNm", where Nm is any nucleoside containing an OCH ₃ moiety at the 2' position. As used herein, the term “cap 2” means the structure “m ⁷ GpppNmNm”, where each Nm independently represents any nucleoside bearing an OCH ₃ moiety at the 2' position.

In the context of the present invention, the term "5' cap structure of the present invention" is a 5' cap analogue that resembles the structure of a conventional 5' cap, but is modified to have the ability to stabilize RNA (in a particular mRNA) and/or increase expression RNA (in particular, the expression of mRNA), when attached to it, preferably in vivo or in a cell. The cell can be any cell that can be transfected with RNA (preferably mRNA) of the present invention, and is preferably a cell obtained from a subject, for example, a stem cell (for example, a mesenchymal stem cell (MSC)) or an antigen presenting cell (for example, an immature antigen presenting cell). cell) such as a dendritic cell (eg, an immature dendritic cell). Preferably, the 5'-cap structure of the present invention at least includes the structure "1-(N ⁷ -(R1)-guanin-9-yl)-pentoso-5-yl-(phosphothioate bond)-N(R ⁸ )"-" any of the formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), ( IIe), (III) and (IIIa) (i.e. where the first guanine of the 5'-cap structure is replaced at position N ⁷ by R ¹ and connected at position N ⁹ with C ^1' of the substituents R ² and R ³ carrying pentose; the phosphorothioate bond has the structure -OP(O ^- )(R ⁴ )-OP(O ^- )(R ⁵ )-O[-P(O ^- )(R ⁶ )-O] _n ; and N is any nucleoside , bearing the base B and replaced at the 2' position by R ⁸ ). Additionally, if R ^{is 7} in any of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa) is a ribooligonucleotide in which the OH group at the 2' position of at least ribose at the 5' end of the ribooligonucleotide is replaced by an R ^8' substituent selected from the group consisting of H , halogen and optionally substituted alkoxy, and the ribose at the 3' end of the ribooligonucleotide has a free OH group at the 2' position, then the 5' cap structure of the present invention preferably includes in addition to the structure "1-(N ⁷ - (R ¹ )-guanin-9-yl)-pentoso-5-yl-(phosphothioate bond)-N(R ⁸ )-" any of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa), also any nucleoside substituted at the 2' position by an R ^8' group ( together with any internucleotide linkage between the N(R ⁸ ) fragment and the N(R ^8' ) fragment, as well as any internucleotide linkage between each pair of N(R ^8' ) fragments in the case of a ribooligonucleotide contains more than one N(R ^8' ) fragment) . For example, if, to provide a 5' cap structure of the present invention, a 5' cap compound of formula (I) wherein R ⁷ is a ribooligonucleotide of the formula [pN(R ^8' )] ₂ pN (i.e., only a nucleoside of 3 '-end of the ribooligonucleotide has a free OH group at position 2', while the other two nucleosides are replaced by R8' at position 2'), the 5'-cap structure of the present invention will include at least the structure "1-(N ⁷ -(R ¹ )-guanin-9-yl)-pentoso-5-yl-(phosphothioate bond)-N(R ⁸ )-[pN(R ^8' )] _2. ”

As used herein, the term “5'-capped RNA” means RNA that contains a cap structure at its 5' end.

As used herein, the term “RNA modified with a 5' cap compound of the present invention” means RNA that contains at its 5' end the 5' cap structure of the present invention. Likewise, the term “mRNA modified with a 5' cap compound of the present invention” means an mRNA that contains at its 5' end the 5' cap structure of the present invention. Thus, in a preferred embodiment, such RNA (e.g., mRNA) modified with a 5' cap compound of the present invention at least contains at its 5' end the structure "1-(N ⁷ -(R ¹ )-guanine -9-yl)-pentoso-5-yl-(phosphothioate bond)-N(R ⁸ )-" any of the formulas (I), (Ia), (Ib), (Ic), (Id), (Ie) , (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa) (i.e. where the first guanine of the 5'-cap structure is substituted at the N ⁷ position group R ¹ and is connected on N ⁹ with C ^1' pentose-containing substituents R ² and R ³ ; the phosphorothioate bond has the structure -OP(O ^- )(R ⁴ )-OP(O ^- )(R ⁵ )-O-[-P (O ^- )(R ⁶ )-O] _n ; and N is any nucleoside bearing the base B and substituted at the 2' position by R ⁸ ). Additionally, if R ^{is 7} in any of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa) is a ribooligonucleotide in which the OH group at the 2' position of at least ribose at the 5' end of the ribooligonucleotide is replaced by an R ^8' substituent selected from the group consisting of H , halogen and optionally substituted with alkoxy, and the ribose at the 3' end of the ribooligonucleotide has a free OH group at the 2' position, then the RNA (such as mRNA) that is modified with a 5' cap compound of the present invention preferably contains at its 5' -end in addition to the structure “1-(N ⁷ -(R ¹ )-guanin-9-yl)-pentoso-5-yl-(phosphothioate bond)-N (R ⁸ )-” of any of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa) ) also any nucleoside substituted at position 2' by an R ^8' group (together with any internucleotide linkage between the N(R ⁸ ) fragment and the N(R ^8' fragment), as well as any internucleotide linkage between each pair of N(R ^8' fragments ) if the ribooligonucleotide contains more than one fragment N(R ^8' )). For example, if the RNA is modified with a 5'-cap compound of the present invention of formula (I), where R ⁷ is a ribooligonucleotide of the formula [pN(R ^8' )] ₂ pN (i.e., only the nucleoside at the 3' end of the ribooligonucleotide has a free group OH at position 2', while the other two nucleosides are replaced by R ^8' at position 2'), the modified RNA will contain at its 5' end at least the structure "1-(N ⁷ -(R ¹ )-guanine- 9-yl)-pentoso-5-yl-(phosphothioate bond)-N(R ⁸ )-[pN(R ^8' )] ₂ ".

The term "increased RNA expression", preferably in connection with RNA modified with a 5'-cap compound of the present invention, preferably means a reduction or even inhibition of RNA recognition by cap-0 structure recognition proteins, such as IFIT proteins (particularly IFIT1).

According to the invention, a portion or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective nucleic acid binding. For example, a pharmacologically active peptide or protein fragment has at least one of the pharmacological activities of the peptide or protein from which the fragment was derived. The portion or fragment of the peptide or protein preferably contains a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50 consecutive amino acids of a peptide or protein. The portion or fragment of the peptide or protein preferably contains a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the peptide or protein.

According to the invention, a peptide or protein analog is a modified form of said peptide or protein from which it was derived and has at least one functional property of said peptide or protein. For example, a pharmacologically active peptide or protein analogue has at least one of the pharmacological activities of the peptide or protein from which the analogue was derived. Such modifications include any chemical modification and include single or multiple substitutions, deletions and/or additions of any molecules associated with a protein or peptide, such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, protein or peptide "analogues" include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting/blocking groups, proteolytic cleavage, or coupling to antibody or other cellular ligand. The term "analog" also covers all functional chemical equivalents of the specified proteins and peptides.

In the context of the present invention, the term "vaccine composition" refers to an antigen preparation that contains RNA. The vaccine composition is administered to an individual to stimulate the individual's humoral and/or cellular immune system against one or more antigens. In this context, RNA may encode an antigen, a protein, or a peptide containing said antigen, or an antigenic peptide. The vaccine composition in the context of the present invention may further include one or more adjuvants and/or excipients, and may be administered to an individual in any suitable manner to elicit a protective and/or therapeutic immune response against the antigen.

"Antigen" according to the invention covers any substance that will cause an immune response, and/or any substance against which an immune response or immune mechanism, such as a cellular response, is directed. This also includes situations where an antigen is processed into antigenic peptides and the immune response or immune mechanism is directed against one or more antigenic peptides, particularly if they are presented in the context of MHC molecules. In particular, "antigen" refers to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule that contains at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present invention is a molecule which, if necessary after processing, induces an immune response that is preferably specific for the antigen (including cells expressing the antigen).

According to the present invention, any suitable antigen that is a candidate for an immune response can be used, and the immune response can be either a humoral or a cellular immune response. In the context of some embodiments of the present invention, the antigen is preferably presented by a cell, preferably an antigen presenting cell, in the context of MHC molecules, resulting in an immune response against the antigen. The antigen is preferably a product that corresponds to or is derived from a naturally occurring antigen. Such natural antigens may include or may be derived from allergens, viruses (eg, influenza virus (A, B or C), CMV or RSV), bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a natural product, for example, a viral protein (for example, a protein of an influenza A virus, an influenza B virus or an influenza C virus, such as PB1, PB1-F2, PB2, PA, HA, NP, NA, M1, M2 , NS1 or NEP/NS2 from influenza A virus, influenza B virus or influenza C virus) or part thereof.

In a preferred embodiment, the antigen is a tumor antigen, that is, a portion of a tumor cell that may be derived from the cytoplasm, cell surface, or cell nucleus, particularly antigens that are primarily found intracellularly or as surface antigens of tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetal sulfoglycoprotein, α2-H-ferroprotein and γ-fetoprotein, as well as various viral tumor antigens. According to the present invention, a tumor antigen preferably includes any antigen that is characteristic of tumors or cancer, as well as tumor or cancer cells in terms of type and/or level of expression. In another embodiment, the antigen is a pathogen-associated antigen, i.e. an antigen derived from a pathogen, for example a virus (such as influenza virus (A, B or C), CMV or RSV), bacteria, single-celled organism or parasite, for example a viral antigen such as a viral ribonucleoprotein or envelope protein. In particular, the antigen must be presented by MHC molecules, which leads to modulation, in particular the activation of cells of the immune system, preferably CD4+ and CD8+ lymphocytes, in particular through modulation of T cell receptor activity.

In some embodiments, the antigen is a tumor antigen, and the present invention includes stimulating an antitumor CTL response against tumor cells expressing such tumor antigen and preferably presenting such tumor antigen to MHC class I.

The term "immunogenicity" refers to the relative effectiveness of an antigen in inducing an immune response.

The term "pathogen" refers to pathogenic microorganisms and includes viruses, bacteria, fungi, single-celled organisms and parasites. Examples of pathogenic viruses are human immunodeficiency virus (HIV), influenza virus (eg, influenza A virus, influenza B virus, or influenza C virus), respiratory syncytial virus (RSV), cytomegalovirus (CMV), herpes virus (HSV), hepatitis virus A (HAV), HBV, HCV, papilloma virus and human T-lymphotrophic virus (HTLV), such as HIV, CMV, HSV, HAV, HBV, HCV, papilloma virus and HTLV, preferably influenza virus, CMV, or RSV. Single-celled organisms include trypanosomes, plasmodium, amoeba, etc.

Thus, in another preferred embodiment, the antigen is one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) viral antigens, that is, one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) virus antigens, the virus preferably being an influenza virus (A, B or C), CMV or RSV. Therefore, in this embodiment, the present invention preferably includes generating an immune response against said virus. The one or more viral antigens are preferably selected from the group consisting of PB1, PB1-F2, PB2, PA, HA, NP, NA, M1, M2, NS1 and NEP/NS2 of influenza A, influenza B or influenza C.

Examples of antigens that can be used in the present invention are p53, ART-4, BAGE, ss-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN- 12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE -A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART -1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT, preferably WT-1.

The "part or fragment of an antigen" or "antigenic peptide" according to the invention is preferably a partial presentation of an antigen and is capable of inducing an immune response against the antigen or cells characterized by antigen expression and preferably antigen presentation.

In this context, the invention also uses peptides containing amino acid sequences derived from antigens, also referred to herein as "antigenic peptides". By "antigenic peptide" or "antigen-derived antigenic peptide" is meant an oligopeptide or polypeptide containing an amino acid sequence substantially corresponding to the amino acid sequence of an antigen fragment or peptide. The antigenic peptide can be of any length.

Preferably, the antigenic peptides are capable of stimulating an immune response, preferably a cellular response against an antigen or cells characterized by antigen expression and preferably antigen presentation. Preferably, the antigenic peptide is capable of stimulating a cellular response against a cell characterized by MHC class I antigen presentation, and is preferably capable of stimulating antigen-responsive CTLs. Preferably, according to the invention, the antigenic peptides are MHC class I and/or class II presented peptides, or can be processed to produce MHC class I and/or class II presented peptides. Preferably, the antigen peptides contain an amino acid sequence substantially corresponding to the amino acid sequence of the antigen fragment. Preferably, said antigen fragment is a peptide presented by MHC class I and/or class II. Preferably, the antigenic peptide of the invention contains an amino acid sequence substantially corresponding to the amino acid sequence of such fragment, and is processed to obtain such fragment, ie, an MHC class I and/or class II presented peptide derived from the antigen.

If the antigenic peptide is to be presented directly, that is, without processing, in particular without cleavage, it is of a length that is suitable for binding to an MHC molecule, in particular an MHC class I molecule, and is preferably 7-20 amino acids in length, more preferably 7-12 amino acids long, more preferably 8-11 amino acids long, in particular 9 or 10 amino acids long. Preferably, the sequence of the antigenic peptide to be presented directly is derived from the amino acid sequence of the antigen, that is, its sequence is substantially consistent with, and preferably completely identical to, a fragment of the antigen.

If the antigenic peptide is to be presented after processing, in particular after cleavage, the processed peptide is of a length that is suitable for binding to an MHC molecule, in particular an MHC class I molecule, and is preferably 7-20 amino acids in length, more preferably 7-12 amino acids long, more preferably 8-11 amino acids long, in particular 9 or 10 amino acids long. Preferably, the sequence of the peptide to be presented after processing is derived from the amino acid sequence of the antigen, that is, its sequence is substantially consistent with, and preferably completely identical to, a fragment of the antigen. Thus, the antigenic peptide of the invention in one embodiment comprises a sequence of 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-11 amino acids in length, in particular 9 or 10 amino acids in length , which essentially corresponds to and preferably is completely identical to the antigen fragment, and subsequent processing of the antigenic peptide constitutes the presented peptide. However, the antigenic peptide may also contain a sequence that is substantially consistent with, and preferably completely identical to, a fragment of the antigen that is even longer than the above sequence. In one embodiment, the antigenic peptide may comprise the entire sequence of an antigen.

Peptides having amino acid sequences substantially corresponding to the sequence of a peptide that is presented by MHC class I may differ at one or more residues that are not essential for TCR recognition of the MHC class I presented peptide or for binding of the peptide to MHC. Such substantially corresponding peptides are also capable of stimulating antigen-responsive CTLs. Peptides having amino acid sequences that differ from the presented peptide at residues that do not affect TCR recognition but improve the stability of MHC binding may improve the immunogenicity of the antigenic peptide and may be referred to herein as an “optimized peptide.” Using existing knowledge of which of these residues is more likely to affect binding to either the MHC or the TCR, a rational approach can be taken to design essentially relevant peptides. The resulting peptides, which are functional, are considered antigenic peptides.

In one embodiment, the antigenic peptide, when presented in the context of an MHC, such as the MHC of antigen presenting cells, is recognized by a T cell receptor. An antigenic peptide, if recognized by a T cell receptor, can induce, in the presence of appropriate co-stimulatory signals, the clonal expansion of a T cell bearing the T cell receptor specifically recognizing the antigen peptide. Preferably, antigenic peptides, in particular when presented in the context of MHC molecules, are capable of stimulating an immune response, preferably a cellular response against the antigen from which they are derived or cells characterized by antigen expression and preferably characterized by antigen presentation.

The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, that is, a part or fragment of a molecule that is recognized by the immune system, such as that recognized by a T cell, particularly when presented in the context of MHC molecules. The protein epitope preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length, for example the epitope may preferably be 9 in length. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. Particularly preferably, the epitope in the context of the present invention is a T cell epitope.

Terms such as "epitope", "T cell epitope", "antigen fragment", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and preferably refer to an incomplete presentation of an antigen that is preferably capable of inducing an immune response against the antigen or cell expressing or including, and preferably presenting, an antigen. Preferably, the terms refer to the immunogenic portion of the antigen. Preferably, it is the portion of the antigen that is recognized (ie specifically bound) by the T cell receptor, particularly if presented in the context of MHC molecules. Certain preferred immunogenic moieties bind to an MHC class I or class II molecule.

The term "target" means an agent, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigenic epitope, that is, a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses an antigen and preferably presents said antigen with MHC class I.

The term "part" refers to a share. With respect to a particular structure, such as an amino acid sequence or a protein, the term "part thereof" can refer to a continuous or discontinuous portion of the structure.

The terms "part" and "fragment" are used interchangeably in this application and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a contiguous element of said structure.

"Antigen processing" refers to the cleavage of an antigen into processing products that are fragments of said antigen (e.g., cleavage of a protein into peptides) and the association of one or more of these fragments (e.g., through binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells, specific T cells.

By “antigen-responsive CTL” is meant a CD8+ T cell that responds to an antigen or peptide derived from said antigen that is presented by MHC class I on the surface of antigen presenting cells.

According to the invention, CTL sensing may include sustained calcium influx, cell division, production of cytokines such as IFN-γ and TNF-α, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of a target cell expressing a tumor antigen. CTL sensitivity can also be determined using an artificial reporter that accurately indicates CTL sensitivity.

The terms "immune response" and "immune response" are used interchangeably herein in their generally accepted meaning and refer to the body's integrated response to an antigen and preferably refers to a cellular immune response, a humoral immune response, or both. According to the invention, the term "immune response to" or "immune response against" in relation to an agent, such as an antigen, cell or tissue, refers to an immune response, such as a cellular response, directed against that agent. The immune response may include one or more responses selected from the group consisting of the production of antibodies against one or more antigens and the proliferation of antigen-specific T lymphocytes, preferably CD4+ and CD8+ T lymphocytes, more preferably CD8+ T lymphocytes, which can be detected in various in vitro proliferation or cytokine production assays.

The terms “inducing an immune response” and “inducing an immune response” and similar terms as used herein refer to the induction of an immune response, preferably the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective/preventive/prophylactic and/or therapeutic. The immune response can be directed against any immunogen, antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (for example, a virus antigen (such as influenza virus (A, B or C), CMV or RSV) ). "Induction" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was a certain level of immune response against a particular antigen or pathogen before induction, and after induction, said immune response increased. Thus, “inducing an immune response” in this context also includes “enhancing an immune response.” Preferably, after inducing an immune response in an individual, said individual is protected from developing a disease, such as an infectious disease or cancer, or the pathological condition is alleviated by inducing an immune response.

The terms “cellular immune response,” “cellular response,” “cell-mediated immunity,” or similar terms are intended to include a cellular response directed to cells characterized by antigen expression and/or MHC class II antigen presentation. The cellular response refers to cells called T cells or T lymphocytes, which act as "helper" or "killer" cells. Helper T cells (also called CD4+ T cells) play a central role in regulating the immune response, and killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill cells such as diseased cells cells.

The term "humoral immune response" refers to the process in living organisms in which antibodies are produced in response to agents and organisms that ultimately neutralize and/or eliminate those agents/organisms. The specificity of the antibody response is mediated by T and/or B cells through membrane-associated receptors that specifically bind a single antigen. After binding the appropriate antigen and receiving various other activating signals, B cells divide to produce memory B cells as well as clones of antibody-secreting plasma cells, each producing antibodies that recognize the identical antigenic epitope that is recognized by its antigen receptor. Memory B cells remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis for memory and the resulting escalation of antibody response upon repeated exposure to a specific antigen.

As used herein, the term “antibody” refers to an immunoglobulin molecule that is capable of specifically binding to an epitope of an antigen. In particular, the term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the above. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH CDRs are designated HCDR1, HCDR2, and HCDR3; The VL CDRs are referred to as LCDR1, LCDR2, and LCDR3. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions include a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into the CH1 constant domain, the hinge region, and the CH2 and CH3 constant domains (located from the amino terminus to the carboxy terminus as follows order: CH1, CH2, CH3). Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. Antibodies can be intact immunoglobulins obtained from natural sources or from recombinant sources, and can be immunoactive portions of intact immunoglobulins. Antibodies are usually tetramers of immunoglobulin molecules. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) ₂ as well as single chain antibodies and humanized antibodies.

The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antigen receptors such as antibodies or B cell receptor (BCR). Immunoglobulins are characterized by a structural domain, that is, an immunoglobulin domain having a characteristic immunoglobulin (Ig) fold. This term covers membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are usually part of the BCR. Soluble immunoglobulins are usually called antibodies. Immunoglobulins usually contain multiple chains, usually two identical heavy chains and two identical light chains, which are linked by disulfide bonds. These chains are mainly composed of immunoglobulin domains such as VL (variable light chain) domain, CL (constant light chain) domain, VH (variable heavy chain) domain and CH (constant heavy chain) domains CH1, CH2, CH3 and CH4. There are five types of mammalian immunoglobulin heavy chains, i.e. α, δ, ε, γ and μ, which constitute the different classes of antibodies, i.e. IgA, IgD, IgE, IgG and IgM. Unlike the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy terminus. Mammals have two types of light chains: lambda and kappa. Immunoglobulin chains include a variable region and a constant region. The constant region is essentially conserved within different immunoglobulin isotypes, while the variable region is very diverse and is responsible for antigen recognition.

The terms "vaccination" and "immunization" describe the process of treating a person for therapeutic or prophylactic reasons and refer to the procedure of administering one or more immunogens or antigens or derivatives thereof, in particular in the form of RNA encoding them, as described herein, to an individual and stimulating an immune response against said one or more immunogens or antigen(s), or cells characterized by presenting said one or more immunogens or antigens.

By “antigen presenting cell” or “antigen presenting cell” or “MHC molecules that present antigen on the surface of an antigen presenting cell” or similar expressions is meant a cell such as a diseased cell, in particular a tumor cell or an antigen presenting cell presenting an antigen or antigenic peptide, either directly or after processing in the context of MHC molecules, preferably MHC class I molecules and/or MHC class II molecules, most preferably MHC class I molecules.

In the context of the present invention, terms such as “protect,” “prevent,” “prophylactic,” “preventative,” or “protective” refer to the prevention or treatment, or both, of the occurrence and/or spread of a disease in individual, and in particular to minimize the likelihood of the individual developing a disease, or to delay the development of a disease. For example, an individual at risk for a disease may be a candidate for therapy to prevent the disease. Prophylactic administration of an agent (e.g., RNA) or composition (such as a pharmaceutical composition, e.g., a vaccine composition) described herein may protect the recipient from developing a disease, such as infection by a pathogen (e.g., a virus such as influenza virus (A, B or C), CMV or RSV) or from the spread or metastasis of existing tumors. Therapeutic administration of an agent (eg, RNA) or composition (such as a pharmaceutical composition) described herein may result in inhibition of disease development/growth. This includes slowing the progression/growth of the disease, in particular interfering with the progression of the disease, preferably resulting in elimination of the disease.

The term "adjuvant" refers to compounds that, when administered in combination with an antigen, antigenic peptide or nucleic acid (such as RNA, preferably mRNA) encoding said antigen or antigenic peptide to an individual, prolong or enhance or accelerate the immune response. In the context of the present invention, RNA (preferably mRNA) can be administered with any adjuvants. Adjuvants are believed to exert their biological activity through one or more mechanisms, including increasing the surface area of the antigen, prolonging the retention of the antigen in the body, slowing the release of the antigen, targeting the antigen to macrophages, increasing the uptake of the antigen, enhancing the processing of the antigen, stimulating the release of cytokines, stimulation and activation immune cells such as B cells, macrophages, dendritic cells, T cells, and nonspecific activation of immune cells. For example, compounds that allow DC maturation, such as lipopolysaccharides or CD40 ligand, form a class of suitable adjuvants. In general, any agent that affects the immune system in a "danger signal" type (LPS, GP96, dsRNA, etc.) or cytokines such as GM-CSF can be used as an adjuvant that allows the immune response to be enhanced and/or or influences it in a controlled manner. CpG oligodeoxynucleotides (Krieg et al., 1995, Nature 374: 546-549) can also be used in this context if desired. Additional types of adjuvants include oil emulsions (eg, Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), liposomes, immunostimulatory complexes, cytokines (eg, monokines, lymphokines, interleukins, or chemokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN- γ, GM-CSF, LT-α or growth factors, e.g. hGH), lipopeptides (e.g. Pam3Cys). In the event that the RNA (preferably mRNA) of the invention, in one embodiment, may encode an immunostimulatory agent, and said immunostimulatory agent encoded by said RNA should act as a primary immunostimulator, however, only a relatively small amount of CpG DNA is needed (compared to immunostimulation alone CpG DNA). Examples of adjuvants are monophosphoryl lipid-A (MPL SmithKline Beecham, saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., 1997 , Mol. Cells 7: 178-186), incomplete Freund's adjuvant, complete Freund's adjuvant, vitamin E, montanide, alum, CpG oligonucleotides and various water-in-oil emulsions that are prepared from biodegradable oils such as squalene and /or tocopherol Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins or chemokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN-γ, GM-CSF, LT-α, growth factors such as hGH, or lipopeptides such as Pam3Cys, all of which are suitable for use as adjuvants in the pharmaceutical compositions of the present invention, or when the RNA of the present invention is used in therapy.

Terms such as "increase", "intensify" or "extend" preferably refer to an increase, intensification or extension of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably by at least 40%, more preferably by at least 50%, even more preferably by at least 80%, and most preferably by at least 100%. These terms can also refer to an increase, amplification, or extension from zero or an undetectable or undetectable level to a greater than zero or measurable or detectable level.

Terms such as "reduction", "reduction" or "inhibition" refer to the ability to cause an overall decrease in level, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. It also includes complete or substantially complete reduction, that is, reduction to zero or substantially zero.

Terms such as “transfer,” “transfection,” or “introduction into cells” are used interchangeably herein and refer to the introduction of nucleic acids, particularly exogenous or heterologous nucleic acids, preferably RNA (such as mRNA), into a cell. According to the present invention, a cell may form part of an organ, tissue and/or organism. The introduction of nucleic acids, in particular exogenous or heterologous nucleic acids, preferably RNA (such as mRNA), into a cell can be carried out in vivo or in vitro.

"Antigen presenting cells" (APCs) are cells that present antigen, particularly peptide fragments of protein antigens, in association with MHC molecules on their cell surface. T cells can recognize this complex using their T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells. The antigen presenting cell includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DC). In a preferred embodiment, the APCs of the present invention are mammalian cells, preferably human, mouse or rat.

Non-professional antigen-presenting cells do not constitutively express MHC class II proteins required for interaction with naïve T cells; they are expressed only when non-professional antigen-presenting cells are stimulated by certain cytokines such as IFNγ.

Professional antigen presenting cells are very efficient at internalizing antigen either through phagocytosis or receptor-mediated endocytosis and then presenting the antigen fragment bound to an MHC class II molecule on their membrane. T lymphocytes recognize and interact with the antigen-MHC class II complex on the membrane of the antigen-presenting cell. The antigen presenting cell then produces an additional co-stimulatory signal, which leads to activation of the T cell. Expression of costimulatory molecules is a defining feature of professional antigen-presenting cells.

The main types of professional antigen-presenting cells are dendritic cells, which have the widest range of antigen presentation and are probably the most important antigen-presenting cells, macrophages, B cells and some activated epithelial cells.

Dendritic cells (DCs) are populations of leukocytes that present antigens captured in peripheral tissues to T cells via both MHC class II and I antigen presentation pathways. It is well known that dendritic cells are potent inducers of immune responses, and the activation of these cells is a critical step for the induction of immunity.

Dendritic cells are conveniently divided into “immature” and “mature” cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted to exclude all possible intermediate stages of differentiation.

Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, which correlates with high expression of the Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by lower expression of these markers but high expression of cell surface molecules responsible for T cell activation, such as MHC class I and class II, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86 and 4-1 BB).

Dendritic cell maturation is referred to as a state of dendritic cell activation in which such antigen-presenting dendritic cells lead to T cell priming, while presentation by immature dendritic cells results in tolerance. Dendritic cell maturation is mainly triggered by biomolecules with microbial signatures detected by innate immune system receptors (bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL-1, IFN), CD40 ligation on the surface of dendritic cells by CD40L, and substances released from cells undergoing stress cell death. Dendritic cells can be produced by culturing bone marrow cells in vitro with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

The term "immunoreactive cell" or "effector cell" as used herein refers to a cell that performs effector functions during an immune response. An "immunoreactive cell" is preferably capable of binding an antigen or a cell characterized by the expression and/or presentation of an antigen or an antigenic peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, kill microbes, secrete antibodies, recognize infected or cancer cells and possibly eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, the “immunoreactive cells” are T cells, preferably CD4+ and/or CD8+ T cells.

Preferably, the "immunoreactive cell" recognizes an antigen or an antigen-derived peptide with some degree of specificity, particularly if it is presented in the context of MHC molecules, such as on the surface of antigen-presenting cells or pathological cells, such as tumor cells. Preferably, said recognition allows a cell that recognizes an antigen or an antigenic peptide derived from said antigen to be sensitive or reactive. If the cell is a helper T cell (CD4+ T cell) bearing receptors that recognize an antigen or an antigenic peptide derived from an antigen in the context of MHC class II molecules, such sensitivity or reactivity may include the release of cytokines and/or activation of CD8+ lymphocytes ( CTL) and/or B cells. If the cell is a CTL, such reaction or reactivity may involve the elimination of cells presented in the context of MHC class I molecules, that is, cells characterized by antigen presentation with MHC class I, for example, through apoptosis or perforin-mediated cell lysis. According to the invention, CTL sensing may include sustained calcium influx, cell division, production of cytokines such as IFN-α and TNF-α, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. CTL sensitivity can also be determined using an artificial reporter that accurately indicates CTL sensitivity. Such CTLs that recognize an antigen or an antigen-derived peptide and are sensitive or reactive are also referred to herein as “antigen-sensitive CTLs.” If the cell is a B cell, this response may involve the release of immunoglobulins.

The term "T cell" or "T lymphocyte" refers to thymus-derived cells that participate in various cell-mediated immune responses and includes T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), which include cytolytic T cells.

T cells belong to a group of white blood cells known as lymphocytes and play a central role in cellular immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface called the T-cell receptor (TCR). The thymus is the main organ responsible for the maturation of T cells. Several different populations of T cells have been discovered, each with a different function.

Helper T cells assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated by the presentation of peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they rapidly divide and release small proteins called cytokines that regulate or promote the active immune response.

Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to MHC class I-associated antigen, which is present on the surface of almost every cell in the body.

Most T cells have a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor consists of two separate peptide chains that are derived from independent T cell receptor alpha and beta genes (TCRα and TCRβ) and are called the α and β TCR chains. γδ T cells (gamma delta T cells) are a small subpopulation of T cells that possess a specific T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is much less common (2% of total T cells) than αβ T cells.

The structure of the T cell receptor is very similar to the Fab fragments of immunoglobulin, which are regions defined as the combined light and heavy chains of the antibody arm. Each TCR chain is a member of the immunoglobulin superfamily and has one N-terminal immunoglobulin (Ig) variable (V) domain, one Ig constant (C) domain, a transmembrane region, and a short cytoplasmic tail at the C terminus. The variable domain of both the TCR α chain and β chain has three hypervariable or complementarity determining regions (CDRs), whereas the β chain variable region has an additional hypervariable region (HV4) that does not normally contact antigen and is therefore not considered a CDR . CDR3 is the main CDR responsible for the recognition of processed antigen, although the α chain CDR1 has been shown to interact with the N-terminal part of the antigenic peptide, whereas the β chain CDR1 interacts with the C-terminal part of the peptide. CDR2 is thought to recognize MHC. The β chain CDR4 is not thought to be involved in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of short connecting sequences in which the cysteine residue forms disulfide bonds that form a link between the two chains.

The term "peripheral blood mononuclear cell" or "PBMC" refers to a peripheral blood cell having a round nucleus. These cells are composed of lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets have no nuclei and granulocytes (neutrophils, basophils and eosinophils) have multilobulated nuclei. These cells can be extracted from whole blood using Ficoll and gradient centrifugation, which separates the blood into an upper layer of plasma, followed by a layer of PBMCs and a lower fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and red blood cells.

The term "major histocompatibility complex" and the abbreviation "MHC" include the MHC class I and MHC class II molecules and refer to a complex of genes that is found in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or pathological cells in immune responses, where MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. MHC-encoded proteins are expressed on the surface of cells and display both self antigens (peptide fragments from the cell itself) and foreign antigens (such as fragments of invading microorganisms) to the T cell.

The MHC region is divided into three subgroups: class I, class II, and class III. MHC class I proteins contain the α-chain and β2-microglobulin (not the part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. On most cells of the immune system, especially antigen-presenting cells, MHC class II proteins contain α- and β-chains and present antigen fragments to T helper cells. The MHC class III region encodes other immune components, such as complement components and some components that encode cytokines.

In humans, genes in the MHC region that encode antigen-presenting proteins on the cell surface are called human leukocyte antigen (HLA) genes. However, the abbreviation MHC is often used to refer to HLA gene products. HLA genes include nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1.

In one preferred embodiment of all aspects of the invention related to immunotherapy or immune responses, the MHC molecule is an HLA molecule.

The term "immune effector functions" or "effector functions" in the context of the present invention includes any functions mediated by components of the immune system that lead, for example, to the destruction of cells. Preferably, the effector functions of the immune system in the context of the present invention are T cell-mediated effector functions. Such functions include, in the case of a helper T cell (CD4+ T cell), recognition of an antigen or an antigenic peptide derived from an antigen in the context of MHC class II molecules by T cell receptors, release of cytokines, and/or activation of CD8+ lymphocytes (CTL) and/or B -cells, and in the case of CTL, recognition of an antigen or antigenic peptide derived from an antigen in the context of MHC class I molecules by T cell receptors, elimination of cells presented in the context of MHC class I molecules, that is, cells characterized by antigen presentation with MHC class I, for example, through apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of antigen-expressing target cells.

The term "immune effector cells" in the context of the present invention refers to cells that exhibit effector functions during an immune response. "Immune effector cells" are preferably capable of binding an antigen or cell characterized by antigen presentation and mediated immune response. For example, such cells secrete cytokines and/or chemokines, kill microbes, secrete antibodies, recognize infected or cancer cells, and possibly remove such cells. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, the “immune effector cells” are T cells, preferably CD4+ and/or CD8+ cells.

Preferably, the "immune effector cell" recognizes an antigen or antigenic peptide derived from said antigen with some degree of specificity, particularly if it is presented in the context of MHC molecules such as the surface of antigen presenting cells or pathological cells such as tumor cells. Preferably, said recognition allows a cell that recognizes an antigen or an antigenic peptide derived from said antigen to be sensitive. If the cell is a helper T cell (CD4+ T cell) bearing receptors that recognize an antigen or an antigenic peptide derived from said antigen in the context of MHC class II molecules, such a response may include the release of cytokines and/or activation of CD8+ lymphocytes (CTL ) and/or B cells. If the cell is a CTL, such a response may involve the elimination of cells presented in the context of MHC class I molecules, that is, cells characterized by antigen presentation with MHC class I, for example, through apoptosis or perforin-mediated cell lysis. Such CTLs that recognize an antigen or an antigenic peptide derived from said antigen and are sensitive are also referred to herein as “antigen-sensitive CTLs.” If the cell is a B cell, this response may involve the release of immunoglobulins.

The term "half-life" refers to the period of time required to eliminate half the activity, quantity, or number of molecules. In the context of the present invention, the half-life of RNA (preferably mRNA) is an indicator of the stability of said RNA. The half-life of RNA can influence the "expression duration" of the RNA. RNA having a long half-life can be expected to be expressed over a long period of time.

Of course, if in accordance with the present invention it is necessary to reduce the stability and/or translation efficiency of RNA, it is possible to modify the RNA in such a way as to interfere with the function of elements that increase the stability and/or translation efficiency of RNA, as described above.

The terms "patient", "individual", "subject" or "animal" are used interchangeably and refer to vertebrates. For example, vertebrate animals in the context of the present invention are mammals, birds (e.g. poultry), reptiles, amphibians, bony fishes and cartilaginous fishes, in particular domesticated animals of any of the above, as well as captive animals such as zoo animals, and preferably mammals. Mammals in the context of the present invention include humans, non-human primates, domestic mammals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory mammals such as mice, rats, rabbits , guinea pigs, etc., as well as captive mammals such as but not limited to zoo mammals. The term "animal" as used herein also includes humans. The term "subject" may also include a patient, that is, an animal, preferably a human, suffering from a disease, preferably a disease described in this application.

According to the invention, the term "chronic patient" or "long-term ill patient" refers to a patient suffering from a chronic disease or disorder. "Chronic disease or disorder" is a disease or disorder that is persistent and/or whose effects (eg, symptoms) persist for at least 3 weeks, such as at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years , at least 5 years or at least 10 years, e.g. up to 4 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 12 months, up to 2 years , up to 3 years or up to 4 years, up to 5 years, up to 10 years or throughout the patient’s entire life.

In accordance with the invention, the term "tumor" or "tumor disease" refers to a swelling or lesion caused by abnormal growth of cells (called neoplastic cells or tumor cells). By "tumor cell" is meant an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after the stimulus that initiated new growth has ceased. Tumors exhibit partial or complete lack of structural organization and functional coordination with normal tissue and usually form a discrete mass of tissue that may be benign, precancerous, or malignant.

Preferably, the tumor disease according to the invention is a cancer disease, i.e. a malignant disease, and a tumor cell is a cancer cell. Preferably, the tumor disease is characterized by cells in which an antigen, ie tumor antigen, is expressed or abnormally expressed. Preferably, the tumor disease or tumor cells are characterized by MHC class I tumor antigen presentation.

"Abnormal expression" means, according to the invention, that the expression is altered, preferably increased, compared to the state in a healthy person. An increase in expression refers to an increase of at least 10%, in particular at least 20%, at least 50% or at least 100%. In one embodiment, expression is detected only in pathological tissue, while expression in healthy tissue is suppressed.

Preferably, the tumor disease according to the invention is cancer, and the term "cancer" according to the invention includes leukemia, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, hemoblastosis, skin cancer, malignant brain tumor, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, small intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, and their metastases. Examples of these are lung carcinomas, breast carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas or metastases of the types of cancer or tumors described above. The term "cancer" according to the invention also includes cancer metastases.

In one embodiment, the RNA of the invention is a (modified) RNA, in particular a (modified) mRNA encoding a peptide or protein. According to the invention, the term "RNA encoding a peptide or protein" means that the RNA, if present in a suitable environment, preferably inside a cell, can direct the assembly of amino acids to produce, i.e. expression, peptide or protein during translation. Preferably, the RNA (such as mRNA) of the invention is capable of interacting with the cellular translation machinery to enable translation of a peptide or protein.

"Coding" refers to the inherent property of specific nucleotide sequences in a nucleic acid to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence or a specific amino acid sequence. Thus, a nucleic acid encodes a protein if the expression (translation and optionally transcription) of the nucleic acid produces a protein in a cell or other biological system.

The term "expression" is used in accordance with the invention in its most general sense and includes the production of RNA and/or peptides or proteins, for example, by transcription and/or translation. With regard to RNA, the term "expression" or "translation" refers specifically to the production of peptides or proteins. It also involves partial expression of nucleic acids. Moreover, expression may be transient or stable.

In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can subsequently be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", where the term "in vitro transcription" refers to the process in which RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are used to generate transcripts. These cloning vectors are generally referred to as transcription vectors and are encompassed by the term “vector” in the present invention. According to the present invention, the RNA used in the present invention can be obtained by in vitro transcription of the corresponding DNA template. The promoter for transcription control can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. Preferably, in vitro transcription according to the invention is controlled by the T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be produced by reverse transcription of RNA.

A vector matrix containing cDNA may contain vectors carrying different cDNA inserts, which, after transcription, result in the formation of a population of different RNA molecules, optionally capable of expressing different peptides or proteins, or may contain vectors carrying only one type of cDNA insert, which, after transcription results in only a population of one RNA species capable of expressing only one peptide or protein. Thus, it is possible to obtain RNA capable of expressing only one peptide or protein, or to obtain compositions of different RNAs, such as RNA and whole cell RNA libraries capable of expressing more than one peptide or protein, such as compositions of peptides or proteins. The present invention provides for the introduction of all such RNA into cells.

As used herein, the term “vector” includes any vectors known to one skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, retro- or lentiviral vectors. , transposons or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). These vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors and typically contain the necessary coding sequence and corresponding DNA sequences necessary for expression of the operably linked coding sequence in a particular host organism (eg, bacteria, yeast, plants, insects or mammals) or in expression systems in vitro. Cloning vectors are typically used to construct and amplify a specific DNA fragment of interest and may not have the functional sequences required to express the desired DNA fragments.

A nucleic acid encoding a peptide or protein can be cloned into several types of vectors. However, the present invention should not be construed as limited to any particular vector. On the contrary, the present invention should be considered to cover a large number of vectors that are readily available and well known in the art. In specific embodiments, the vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. Many such systems are commercial and widely available.

The vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Preferably the virus is a helper-dependent adenovirus (HD-Ad). Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, restriction endonuclease convenience sites, and one or more selectable markers.

Molecular biologists typically know how to use promoters, enhancers, and combinations of cell types to express a protein. Promoters used may be constitutive, tissue specific, inducible, and/or useful under appropriate conditions to drive high levels of expression of the introduced peptide or protein encoding nucleic acid segment. The promoter may be heterologous or endogenous. Constitutive promoter sequences that can be used in accordance with the invention include the cytomegalovirus (CMV) immediate early promoter sequence, simian virus 40 (SV40) early promoter, murine mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (LTR) promoter (HIV), Moloney virus promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and muscle creatine promoter , but are not limited to them. Moreover, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothioneine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. In addition, the invention includes the use of a tissue-specific promoter that is active only in the desired tissue. Tissue-specific promoters are well known in the art, and include, but are not limited to, the HER-2 promoter and PSA-related promoter sequences.

To assess the expression of a peptide or protein, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells that are intended to transfection or infection with viral vectors. In other embodiments, the selectable marker may be located on a separate stretch of DNA and used in the cotransfection procedure. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to allow expression in cells. Suitable selectable markers are known in the art and include, for example, antibiotic resistance genes such as neo and the like. Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Reporter genes encoding easily analyzed proteins are well known in the art. In general, a reporter gene is a gene that is not present or expressed by the recipient organism or tissue and that encodes a protein whose expression exhibits some easily detectable property, such as enzymatic activity. Expression of the reporter gene is analyzed at an appropriate time after the nucleic acid has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein gene.

The vector can be easily introduced into a cell by any method in the art. For example, the expression vector can be transferred into a cell by physical, chemical or biological means. Physical methods for introducing a nucleic acid into a cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

Biological methods for introducing a nucleic acid of interest into a cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.

Chemical means for introducing nucleic acid into a cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (ie, an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

Regardless of the method used to introduce exogenous nucleic acids into a cell or otherwise increase the cellular level of a peptide or protein in a cell, various assays can be performed to confirm the presence and/or amount of the peptide or protein, or the nucleic acid encoding it, in the cell. Such assays include, for example, Southern and Northern blotting, RT-PCR and PCR, as well as assays to detect the presence or absence of a specific peptide, for example, by immunological methods (ELISA and Western blotting).

The term “cell” means any cell that can be transfected with RNA (preferably mRNA), with the transfected RNA preferably being exogenous or heterologous RNA. The cell can be obtained from any subject and, in one embodiment, can be obtained from a patient suffering from a disorder or disease. If the cell is obtained from a patient suffering from a disorder or disease, the cell may contain genetic material that is homologous to the input RNA, but the resulting peptide or protein has reduced activity. Reduced activity may result from (i) decreased expression of the peptide or protein (i.e., the peptide or protein is fully functional, but its quantity is reduced) or (ii) the presence of one or more mutations in the amino acid sequence of the expressed peptide or protein (i.e. peptide or protein is not fully functional). For example, such homologous genetic material that results in a peptide or protein having reduced activity may be a gene containing one or more mutations such that (i) the expression of said gene containing one or more mutations is reduced or suppressed, resulting in to a reduced amount of a fully functional peptide or protein and/or (ii) the amino acid sequence of the peptide or protein encoded by the gene contains one or more mutations, resulting in an incompletely functional (or non-functional) peptide or protein.

In the event that a cell or patient expresses a fully functional peptide or protein, but in an amount that is too low to support the function of the cell or patient (for example, resulting in the development of a disease or disorder in the patient that contains the cell), therapy will be beneficial to replace or supplement a specified peptide or protein (protein replacement therapy). Such protein replacement therapy may include the step of administering an RNA containing a nucleotide sequence encoding said peptide or protein (or a composition, such as a pharmaceutical composition containing such RNA) to a patient, or alternatively, the steps of (a) transferring the RNA containing the nucleotide sequence, encoding said peptide or protein into a cell (which may be obtained from a patient), and (b) administering said transfected cell to the patient.

In the event that the decrease in activity of a peptide or protein in a patient (and thus the development of a disease or disorder) is associated with the presence of one or more mutations in the amino acid sequence of said peptide or protein (i.e. the peptide or protein is not fully functional), may genomic engineering therapy may be useful. Such genetically engineered therapy may include the step of administering to the patient (i) RNA (in particular, the RNA of the present invention) containing a nucleotide sequence encoding the genetically engineered protein, and (ii) DNA containing a nucleotide sequence encoding the peptide or protein thereof. native (i.e. non-mutant) form.

Alternatively, genetic reprogramming therapy may be useful, particularly for patients suffering from a disease or disorder that causes the depletion or disappearance of cells that produce a desired peptide or protein (eg, a hormone such as insulin). For example, such genetic reprogramming therapy may include the steps of (a) introducing RNA (in particular, RNA of the present invention) containing a nucleotide sequence encoding one or more reprogramming factors into somatic cells; (b) creating conditions for the development of cells having the characteristics of stem cells; and (c) administering to the patient cells having the characteristics of stem cells. In a preferred embodiment, the somatic cells are autologous to the patient.

The term "translation" as used herein refers to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to form a peptide or protein. Translation can occur in vivo (eg, in a cell, tissue, or organism) or in vitro (eg, using a cell-free system).

Expression control sequences or regulatory sequences that can be operably linked to a nucleic acid according to the invention may be homologous or heterologous to the nucleic acid. A coding sequence and a regulatory sequence are linked together "functionally" if they are linked together covalently such that transcription or translation of the coding sequence is under the control or influence of the regulatory sequence. If the coding sequence is to be translated into a functional protein with the regulatory sequence functionally linked to the coding sequence, induction of the regulatory sequence results in transcription of the coding sequence without causing a frameshift in the coding sequence or failure to translate the coding sequence into the desired protein or peptide.

The term "expression control sequence" or "regulatory sequence" includes, in accordance with the invention, promoters, ribosome binding sequences and other control elements that control the transcription of a nucleic acid or the translation of an RNA derivative. In some embodiments of the invention, the regulatory sequences can be controlled. The exact structure of regulatory sequences may vary depending on the species or cell type, but typically include 5'-untranscribed and 5'- and 3'-untranslated sequences that are involved in the initiation of transcription or translation, such as the TATA box, capping sequence, CAAT sequence, etc. In particular, the 5' untranscribed regulatory sequences contain a promoter region that includes a promoter sequence to control transcription of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences.

According to the invention, it is preferred that a nucleic acid, such as RNA (preferably mRNA), encoding a peptide or protein, once taken up or introduced, that is, transfected or transduced, into a cell, which may be present in vitro or in a subject, results in the expression of said peptide or protein. The cell may express the encoded peptide or protein intracellularly (eg, in the cytoplasm and/or nucleus), may secrete the encoded peptide or protein, or may express it on the surface.

In accordance with the invention, terms such as “expressing nucleic acid” and “encoding nucleic acid” or similar terms are used interchangeably in this application and in relation to a particular peptide or polypeptide mean that the nucleic acid, if present in a suitable environment, preferably inside the cell, can be expressed to form the specified peptide or polypeptide.

According to the invention, the RNA must be transferred into cells either in vitro or in vivo, for example by introducing the RNA intraperitoneally, intramuscularly or intradermally, or, if the cells are immature antigen presenting cells, into the lymphatic system (such as lymph nodes). According to the present invention, any method that is suitable for transferring RNA into cells can be used to introduce RNA into cells. Preferably, the RNA is transfected into cells using standard methods. Such methods include transfection of nucleic acid precipitates with calcium phosphate, transfection of nucleic acids that are associated with DEAE, transfection or infection with viruses carrying the nucleic acids of interest, electroporation, lipofection, and microinjection. According to the present invention, the administration of the nucleic acid is carried out either in the form of a deproteinized nucleic acid or in combination with an administration reagent. Preferably, the administration of nucleic acids is in the form of deproteinized nucleic acids. Preferably, the RNA is administered in combination with stabilizing agents such as RNase inhibitors. In a particularly preferred embodiment, the RNA and/or compositions of the present invention are administered as deproteinized RNA, preferably intraperitoneally, intramuscularly, by intranodal injection or transdermal administration. In the case of antigen presenting cells (such as immature antigen presenting cells), preferably dendritic cells (such as immature dendritic cells), a conventional transfection method is not absolutely necessary to introduce deproteinized RNA into these cells, since, in particular, immature antigen presenting cells such as dendritic cells are able to take up deproteinized RNA through macropinocytosis. Preferably, introduction of RNA encoding a peptide or protein of interest into a cell results in expression of said peptide or protein of interest in the cell. In certain embodiments, targeting nucleic acids to specific cells is preferred. In such embodiments, the carrier that is used to introduce the nucleic acid into the cell (eg, a retrovirus or liposome) is a targeting molecule. For example, a molecule, such as an antibody specific for a surface membrane protein on a target cell, or a ligand for a receptor on a target cell, may be included in or linked to a nucleic acid carrier. In the case where the nucleic acid is administered via liposomes, proteins that bind to a surface membrane protein that is associated with endocytosis can be included in the liposome composition to provide targeting and/or uptake. Such proteins include capsid proteins or fragments thereof that are specific for a particular cell type, antibodies against proteins that are internalized, proteins that target subcellular locations, etc.

As used herein, the term "peptide" includes oligo- and polypeptides and refers to substances containing two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more, and preferably up to 8, 10, 20, 30, 40 or 50, in particular 100 amino acids covalently linked by peptide bonds. The term “protein” preferably refers to large peptides, preferably those with more than 100 amino acid residues, but in general the terms “peptide” and “protein” are synonymous and are used interchangeably herein.

The term “immunologically active compound” refers to any compound that modifies the immune response, preferably by inducing and/or inhibiting immune cell maturation, inducing and/or inhibiting cytokine biosynthesis, and/or altering humoral immunity by stimulating antibody production by B cells. The immunologically active compounds have potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can also suppress other aspects of the immune response, such as shifting the immune response away from the TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants.

In one embodiment, RNA (eg, mRNA) that encodes an antigen, such as a disease-associated antigen, is administered to a mammal, particularly if it is necessary to treat a mammal suffering from a disease comprising or expressing the antigen (disease-associated antigen). The RNA is preferentially taken up by antigen presenting cells of the mammal (monocytes, macrophages, dendritic cells or other cells). An antigenic RNA translation product is produced, and this product is exposed on the cell surface for recognition by T cells. In one embodiment, the antigen or product produced by optional processing is displayed on the cell surface in the context of MHC molecules for recognition by T cells through their T cell receptor, resulting in their activation.

The term "portion of MHC molecules that present the antigen of interest" refers to the fraction of MHC molecules on the surface of the antigen-presenting cell that are loaded, i.e. associated with a specific antigen or antigenic peptide derived from said antigen, relative to the total number of MHC molecules on the cell surface. In a preferred embodiment, the RNA modified with a 5' cap compound of the present invention is capable of increasing a portion of the MHC molecules that present the antigen of interest on the surface of the antigen presenting cell into which the RNA has been transferred. This can be compared to RNA that does not carry the 5'-cap compound structure of the present invention, in particular to RNA that carries a conventional RNA cap.

According to the invention, the terms "disease", "disorder" and "condition" are used interchangeably herein and refer to any pathological condition, including infectious diseases (ie diseases caused by a pathogen), neoplastic diseases and unwanted inflammation.

By “at risk” we mean an individual, i.e. a patient who is determined to be more likely than usual to develop a disease compared to the general population. In addition, an individual who has had or currently has the disease is a subject at increased risk of developing the disease, since such subject may continue to develop the disease.

The term "in vivo" refers to the situation in the subject.

The term "autologous" is used to describe everything that comes from the same entity. For example, "autologous cell" refers to a cell obtained from the same subject. Such procedures are beneficial because they overcome the immunological barrier that would otherwise lead to rejection.

The term “heterologous” is used to describe something that is made up of several different elements. For example, transferring bone marrow from one person to another is a heterologous transplant. A heterologous gene is a gene obtained from a source other than the subject.

As used herein, the term “non-nucleotide linker” means any two-nucleoside linker that is not a phosphate or a phosphate derivative (e.g., phosphorothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, alkylphosphonate, phosphotriester, or phosphoramidite). Preferably, the non-nucleotide linker is a peptide, amine, aliphatic hydrocarbon (e.g., alkyl), or aromatic hydrocarbon, where the hydrocarbons may optionally include one or more functional groups, including hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea or thiourea, but not limited to them. A specific example of such non-nucleotide linkers includes, but is not limited to, an alkyl linker. The alkyl linker may be branched or unbranched, cyclic or acyclic, substituted or unsubstituted, saturated or unsaturated, chiral, achiral, or a racemic mixture. Alkyl linkers can have from 2 to 18 carbon atoms, for example from 3 to 9 carbon atoms. Some alkyl linkers include one or more functional groups, including, but not limited to, hydroxy, amino, thiol, thioether, ether, amide, thioamide, ester, urea, and thioether. Such alkyl linkers may include 1-propanol, 1,2-propanediol, 1,3-propanediol, 1,2,3-propanetriol, triethylene glycol, hexaethylene glycol, polyethylene glycol linkers (for example, [-O-CH ₂ CH ₂ -] _c , (c = 1, 2, 3, 4, 5, 6, 7, 8 or 9)), but are not limited to methyl linkers, ethyl linkers, propyl linkers, butyl linkers or hexyl linkers. In some embodiments, the non-nucleotide linker is glycerol or a glycerol homolog of the formula HO-(CH ₂ ) _o -CH(OH)-(CH ₂ ) _p -OH, where o and p are independently integers from 1 to 6, for example, from 1 to 4 or from 1 to 3. In some other embodiments, the non-nucleotide linker is a 1,3-diamino-2-hydroxypropane derivative, for example having the formula HO-(CH ₂ ) _m -C(O)NH-CH ₂ -CH(OH)-CH ₂ -NHC(OMCH ₂ ) _m -OH, where each m independently represents an integer from 0 to 10, such as 0 to 6, 2 to 6, or 2 to 4.

The term "alkyl" refers to a monoradical of a saturated linear or branched hydrocarbon. Preferably the alkyl group contains from 1 to 20 carbon atoms, for example from 1 to 12 or from 1 to 10 carbon atoms, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 1 to 8 carbon atoms, for example 1 to 6 or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, sec-pentyl, neopentyl), 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, 2,2-dimethylbutyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, etc. "Substituted alkyl" means that one or more (for example, from 1 to the maximum number of hydrogen atoms associated with the alkyl group, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, for example from 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is substituted, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, such as halogen or optionally substituted aryl. Examples of substituted alkyl include trifluoromethyl, 2,2,2-trichloroethyl, arylalkyl (also called "aralkyl", e.g. benzyl, chloro(phenyl)methyl, 4-methylphenylmethyl, (2,4-dimethylphenyl)methyl, o-fluorophenylmethyl, 2 -phenylpropyl, 2-, 3- or 4-carboxyphenylalkyl) or heteroarylalkyl (also called "heteroaralkyl").

The term "alkenyl" refers to a monoradical of an unsaturated linear or branched hydrocarbon having at least one carbon-carbon double bond. Typically, the maximum number of carbon-carbon double bonds in an alkenyl group can be a whole number, which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is odd, rounding the result of the division to the next whole number . For example, for an alkenyl group containing 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has from 1 to 4, that is, 1, 2, 3 or 4 carbon-carbon double bonds. Preferably, the alkenyl group contains from 2 to 20 carbon atoms, for example from 2 to 12 or from 2 to 10 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 2 to 8 carbon atoms, for example 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group contains from 2 to 10 carbon atoms and 1, 2, 3, 4 or 5 carbon-carbon double bonds, more preferably it contains from 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, e.g. 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. Carbon-carbon double bond(s) can be in cis (Z) or trans (E) configuration. Examples of alkenyl groups include ethenyl (i.e. vinyl), 1-propenyl, 2-propenyl (i.e. allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6- nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, etc. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms associated with the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, such as halogen or optionally substituted aryl. An example of a substituted alkenyl is styryl (ie 2-phenylvinyl).

The term "alkynyl" refers to a monoradical of an unsaturated linear or branched hydrocarbon having at least one carbon-carbon triple bond. Typically, the maximum number of carbon-carbon triple bonds in an alkynyl group can be a whole number, which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is odd, rounding the result of the division to the next whole number . For example, for an alkynyl group containing 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has from 1 to 4, i.e. 1, 2, 3 or 4, more preferably 1 or 2 carbon-carbon triple bonds. Preferably, the alkynyl group contains from 2 to 20 carbon atoms, for example from 2 to 12 or from 2 to 10 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 2 to 8 carbon atoms, for example 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkynyl group contains from 2 to 10 carbon atoms and 1, 2, 3, 4 or 5 (preferably 1, 2 or 3) carbon-carbon triple bonds, more preferably it contains from 2 to 8 carbon atoms and 1, 2, 3 or 4 (preferably 1 or 2) carbon-carbon triple bonds, for example 2 to 6 carbon atoms and 1, 2 or 3 carbon-carbon triple bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Examples of alkynyl groups include ethynyl, 1-propynyl (i.e. -C≡CCH ₃ ), 2-propynyl (i.e. CH ₂ C≡CH or propargyl), 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3-heptynyl, 4- heptynyl, 5-heptynyl, 6-heptynyl, 1-octinyl, 2-octinyl, 3-octinyl, 4-octinyl, 5-octinyl, 6-octinyl, 7-octinyl, 1-nonylyl, 2-noninyl, 3-noninyl, 4-noninyl, 5-noninyl, 6-noninyl, 7-noninyl, 8-noninyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8- decynyl, 9-decynyl, etc. If an alkynyl group is attached to a nitrogen atom, the triple bond cannot be alpha to the nitrogen atom. "Substituted alkynyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms associated with the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , from 1 to 5, from 1 to 4, or from 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, such as halogen or optionally substituted aryl.

The term "aryl" or "aromatic ring" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains from 3 to 14 (for example, from 5 to 10, such as 5, 6 or 10) carbon atoms, which can be located in one ring (for example, phenyl) or two or more fused rings (for example, naphthyl) . Examples of aryl groups include cyclopropenyl, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl and phenanthryl. Preferably, "aryl" refers to a monocyclic ring system containing 6 carbon atoms, or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. "Substituted aryl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms associated with the aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, for example, halogen, CN, nitro, -OR ¹¹ (eg, -OH), -SR ¹¹ (for example, -SH), -N(R ¹² )(R ¹³ ) (for example, -NH ₂ ), =Z (for example, =O, =S or =NH), alkyl (for example, C _1-6 alkyl ), alkenyl (eg C _2-6 alkenyl) and alkynyl (eg C _2-6 alkynyl). Examples of substituted aryl include biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl, 4-hydroxyphenyl, methoxyphenyl (ie, 2-, 3-, or 4-methoxyphenyl), and 4-ethoxyphenyl.

The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms O, S or N. Preferably heteroaryl means a five- or six-membered aromatic monocyclic ring in which 1, 2 or 3 carbon atoms are replaced by the same or different O, N or S heteroatoms. Alternatively, it means an aromatic bicyclic or tricyclic ring system in which 1, 2, 3, 4 or 5 carbon atoms are replaced by the same or different O heteroatoms, N or S. Preferably, in each ring of a heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Examples of heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl , imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benz isothiazolyl, benzotriazolyl, quinolinyl , isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyri dinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl , perimidinyl, phenanthrolinyl and phenazinyl. Examples of 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl ), pyrimidinyl, pyrazinyl, triazinyl and pyridazinyl. “Substituted heteroaryl” means that one or more (e.g., 1 to the maximum number of hydrogen atoms associated with the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is substituted, the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, for example, halogen, -CN, nitro, -OR ¹¹ (for example, -OH), -SR ¹¹ (for example, -SH), -N(R ¹² )(R ¹³ ) (for example, -NH ₂ ), =Z (for example, =O, =S or =NH), alkyl (for example, C _{1- 6} alkyl), alkenyl (eg C _2-6 alkenyl) and alkynyl (eg C _2-6 alkynyl). Examples of substituted heteroaryl include 3-phenylpyrrolyl, 2,3'-bifuryl, 4-methylpyridyl, 2- or 3-ethylindolyl.

The term "cycloalkyl" or "cycloaliphatic" represents the cyclic non-aromatic versions of "alkyl" and "alkenyl", preferably containing from 3 to 14 carbon atoms, for example from 3 to 10 carbon atoms, i.e. 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, more preferably 3 to 7 carbon atoms. In one embodiment, the cycloalkyl group has 1, 2 or more (preferably 1 or 2) double bonds. Typical cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl and adamantyl. The term "cycloalkyl" also includes its bicyclic and tricyclic variants. If bicyclic rings are formed, it is preferable that the corresponding rings be connected to each other by two adjacent carbon atoms, but alternatively the two rings are connected through the same carbon atom, i.e. they form spiro ring systems, or they form "bridge" ring systems. Preferred examples of cycloalkyl include C ₃ -C ₈ -cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[ 4,3]octyl, spiro[4,5]decanyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl (i.e. norbornyl), bicyclo[2.2.2 ]octyl, bicyclo[5.1.0]octyl, bicyclo[4.2.0]octyl, bicyclo[4.3.0]nonyl, 1,2,3,4-tetrahydronaphthyl (i.e. tetralinyl) and bicyclo[4.4.0] decanil (i.e. decalinil). "Substituted cycloalkyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms associated with the cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, e.g., halogen, -CN, nitro, -OR ¹¹ (e.g., -OH) , -SR ¹¹ (for example, -SH), -N(R ¹² )(R ¹³ ) (for example, -NH ₂ ), =Z (for example, =O, =S or =NH), alkyl (for example, C _{1 -6} alkyl), alkenyl (eg C _2-6 alkenyl) and alkynyl (eg C _2-6 alkynyl). Examples of substituted cycloalkyl include oxocyclohexyl, oxocyclopentyl, fluorocyclohexyl and oxocyclohexenyl.

The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above, wherein 1, 2, 3 or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus or sulfur, preferably O, S or N The heterocyclyl group preferably has 1 or 2 rings containing from 3 to 10, for example 3, 4, 5, 6 or 7 ring atoms. Preferably, on each ring of a heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" also means partially or fully hydrogenated forms (such as dihydro-, tetrahydro- or perhydroforms) of the above heteroaryl groups. Typical heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. “Substituted heterocyclyl” means that one or more (e.g., 1 to the maximum number of hydrogen atoms associated with the heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced by a substituent other than hydrogen (when more than one hydrogen atom is substituted, the substituents may be the same or different). Preferably, the non-hydrogen substituent is a 1st level substituent, a 2nd level substituent, or a 3rd level substituent as defined herein, for example, halogen, -CN, nitro, -OR ¹¹ (for example, -OH) , -SR ¹¹ (for example, -SH), -N(R ¹² )(R ¹³ ) (for example, -NH ₂ ), =Z (for example, =O, =S or =NH), alkyl (for example, C _{1 -6} alkyl), alkenyl (eg C _2-6 alkenyl) and alkynyl (eg C _2-6 alkynyl).

The term "aromatic" when used in the context of hydrocarbons means that the entire molecule must be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or completely), the resulting hydrogenated cyclic structure is classified as cycloalkyl for purposes of the present invention. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated, the resulting hydrogenated bi- or polycyclic structure (such as 1,2-dihydronaphthyl) is classified as cycloalkyl for the purposes of the present invention (even if one ring, for example in 1, 2-dihydronaphthyl, still aromatic). This application makes a similar distinction between heteroaryl and heterocyclyl. For example, indolinyl, i.e. The dihydrovariant of indolyl is classified as a heterocyclyl for the purposes of the present invention because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

The term "halogen" or "halo" means fluorine, chlorine, bromine or iodine, preferably fluorine. The term "hydroxy" means OH. The term "alkoxy" means O-alkyl, where alkyl is as defined above and includes methoxy, ethoxy, propoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, hexyloxy, heptyloxy, octyloxy, nonyloxy and decyloxy. The term "substituted alkoxy" means O-(substituted alkyl), wherein substituted alkyl has the meaning given above and includes 2-methoxyethoxy. The term "nitro" means NO ₂ . The term "cyano" means a -CN group. The term "isocyano" means the -NC group. The term "cyanato" means the -OCN group. The term "isocyanato" means the -NCO group. The term "thiocyanato" means the -SCN group. The term "isothiocyanato" means the -NCS group. The term "azido" means N ₃ .

The term "amino" includes unsubstituted amino (ie, a -NH ₂ group) and substituted amino (ie, mono- or di-substituted amino in which one or two hydrogen atoms have been replaced by a group other than hydrogen). Thus, the term "amino" means the group N(R ¹² )(R ¹³ ), wherein R ¹² and R ¹³ are in each instance independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R ¹² and R ¹³ can combine together with the nitrogen atom to which they are attached to form the group -N=CR ¹⁵ R ¹⁶ , where each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optional substituted with one or more (e.g., 1 to the maximum number of hydrogen atoms attached to an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, for example from 1 to 5, from 1 to 4, or from 1 to 3, or 1, or 2) independently selected R ³⁰ ; R ¹⁵ and R ¹⁶ are independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and -NH _y R ²⁰ _2-y , or R ¹⁵ and R ¹⁶ may be combined together with an atom to which they are attached to form a ring which is optionally substituted with one or more (e.g. 1 to the maximum number of hydrogen atoms associated with the ring, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, for example from 1 to 5, from 1 to 4, or from 1 to 3, or 1 or 2) independently selected R ³⁰ , where each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups at optionally substituted with one or more (e.g., 1 to the maximum number of hydrogen atoms attached to an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9 or up to 10, for example 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R ³⁰ ; y is an integer from 0 to 2; R ²⁰ is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted by one or more (for example, from 1 up to the maximum number of hydrogen atoms associated with an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl or heterocyclyl group, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, for example 1 to 5, from 1 to 4 or from 1 to 3, or 1 or 2) independently selected R ³⁰ ; and R ³⁰ is a 1st (or 2nd or 3rd) level substituent.

The term "imino" means a group -N(R ¹⁴ )- in which both free valences of the nitrogen atom can bond to one other atom (eg C), resulting in a double bond (eg C=N(R ¹⁴ )) or with different atoms (eg two C atoms), resulting in two single bonds (eg CN(R ¹⁴ )-C). In each case, R ¹⁴ is selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted by one or more (e.g., from 1 to the maximum number of hydrogen atoms associated with an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 , for example from 1 to 5, from 1 to 4, or from 1 to 3, or 1 or 2) independently selected R ³⁰ ; and R ³⁰ is a 1st (or 2nd or 3rd) level substituent.

The term "optionally substituted" indicates that one or more (e.g., from 1 to the maximum number of hydrogen atoms associated with the moiety, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g. , 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms may be replaced by a group/substituent (i.e., 1st level substituent) other than hydrogen, such as alkyl ( preferably _C1-6 alkyl), alkenyl (preferably _C2-6 alkenyl), alkynyl (preferably _C2-6 alkynyl), aryl (preferably 3-14 membered aryl), heteroaryl (preferably 3-14 -membered heteroaryl), cycloalkyl (preferably 3-14 membered cycloalkyl), heterocyclyl (preferably 3-14 membered heterocyclyl), halogen, -CN, -NC, -NCO, -CNO, -SCN,
-NCS, -N ₃ , -NO ₂ , -OR ⁷¹ , -N(R ⁷² )(R ⁷³ ), -ON(R ⁷² )(R ⁷³ ), -N ⁺ (-O ^- )(R ⁷² )( R ⁷³ ), -S(O) _0-2 R ⁷¹ (i.e. -SR ⁷¹ , -S(O)R ⁷¹ , or -S(O) ₂ R ⁷¹ ), -S(O) _0-2 OR ⁷¹ (for example, -S(O) _1-2 OR ⁷¹ ), -OS(O) _0-2 OR ⁷¹ (for example, -OS(O) _1-2 OR ⁷¹ ), -S(O) _0-2 N (R ⁷² )(R ⁷³ ) (for example, -S(O) _1-2 N(R ⁷² )(R ⁷³ )), -OS(O) _0-2 N(R ⁷² )(R ⁷³ ) (for example, -OS(O) _1-2 N(R ⁷² )(R ⁷³ )), -N(R ⁷¹ )S(O) _0-2 R ⁷¹ (for example, -N(R ⁷¹ )S(O) _1-2 R ⁷¹ ), -NR ⁷¹ S(O) _0-2 OR ⁷¹ (for example, -NR ⁷¹ S(O) _1-2 OR ⁷¹ ), -NR ⁷¹ S(O) _0-2 N(R ⁷² )(R ⁷³ ) (for example, -NR ⁷¹ S(O) _1-2 N(R ⁷² )(R ⁷³ )), -C(=Z ¹ )R ⁷¹ , -C(=Z ¹ )Z ¹ R ⁷¹ , -Z ¹ C(=Z ¹ )R ⁷¹ , and -Z ¹ C(=Z ¹ )Z ¹ R ⁷¹ , and/or any two 1st level substituents that are bonded to the same carbon atom of a cycloalkyl or heterocyclyl group, can combine together to form =Z ¹ , where each of the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl group of the 1st level substituent may itself be substituted by one or more (for example, one, two or three) substituents (t .e. 2nd level substituents) selected from the group consisting of C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 3-14 membered aryl, 3-14 membered heteroaryl, 3-14 membered cycloalkyl, 3-14 membered heterocyclyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OR ⁸¹ , -N(R ⁸² )(R ⁸³ ), -ON(R ⁸² )(R ⁸³ ),
-N ⁺ (-O ^- )(R ⁸² )(R ⁸³ ), -S(O) _0-2 R ⁸¹ (i.e. -SR ⁸¹ , -S(O)R ⁸¹ , or -S(O) ₂ R ⁸¹ ), -S(O) _0-2 OR ⁸¹ (for example, -S(O) _1-2 OR ⁸¹ ), -OS(O) _0-2 R ⁸¹ (for example, -OS(O) _1-2 R ⁸¹ ), -OS(O) _0-2 OR ⁸¹ (for example, -OS(O) _1-2 OR ⁸¹ ), -S(O) _0-2 N(R ⁸² )(R ⁸³ ) (for example, - S(O) _1-2 N(R ⁸² )(R ⁸³ )), -OS(O) _0-2 N(R ⁸² )(R ⁸³ ) (for example, -OS(O) _1-2 N(R ⁸² )(R ⁸³ )), -N(R ⁸¹ )S(O) _0-2 R ⁸¹ (for example, -N(R ⁸¹ )S(O) _1-2 R ⁸¹ ), -NR ⁸¹ S(O) _{0 -2} OR ⁸¹ (for example, -NR ⁸¹ S(O) _1-2 OR ⁸¹ ), -NR ⁸¹ S(O) _0-2 N(R ⁸² )(R ⁸³ ) (for example, -NR ⁸¹ S(O) _1-2 N(R ⁸² )(R ⁸³ )), -C(=Z ² )R ⁸¹ , -C(=Z ² )Z ² R ⁸¹ , -Z ² C(=Z ² )R ⁸¹ , and - Z ² C(=Z ² )Z ² R ⁸¹ , and/or any two 2nd level substituents that are bonded to the same carbon atom of a cycloalkyl or heterocyclyl group can join together to form = Z ² , where each C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 3-14-membered aryl, 3-14-membered heteroaryl, 3-14-membered cycloalkyl, 3-14-membered heterocyclyl group of the 2nd substituent level is optionally substituted with one or more (for example, one, two or three) substituents (i.e. substituents of the 3rd level), independently selected from the group consisting of C _1-3 alkyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH, -O(C _1-3 alkyl), -OCF ₃ ,
-S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , -NHS(O) ₂ (C _1-3 alkyl), - S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)(C _1-3 alkyl), -C(=O)OH, -C(=O)O(C _{1 -3} alkyl), -C(=O)NH _2-z (C _1-3 alkyl) _z , -OC(=O)(C _1-3 alkyl), -OC(=O)O(C _1-3 alkyl), -OC(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=O)NH _z-2 (C _{1- 3} alkyl) _z , -NHC(=NH)NH _z-2 (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z is 0, 1 or 2, and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl, and/or any two 3rd level substituents that are bonded to the same carbon atom of a cycloalkyl or heterocyclyl groups can combine to form =O, =S, =NH or =N(C _1-3 alkyl);

Where

R ⁷¹ , R ⁷² and R ⁷³ are independently selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 3-7 membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl and 3-7-membered heterocyclyl, where each of C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 3-7-membered cycloalkyl, 5- or 6-membered aryl , 5- or 6-membered heteroaryl and 3-7-membered heterocyclyl group is optionally substituted with one, two or three substituents selected from the group consisting of C _1-3 alkyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH, -O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , -NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _{1- 3} alkyl) _z , -C(=O)(C _1-3 alkyl), -C(=O)OH, -C(=O)O(C _1-3 alkyl), -C(=O)NH _{2 -z} (C _1-3 alkyl) _z , -OC(=O)(C _1-3 alkyl), -OC(=O)O(C _1-3 alkyl), -OC(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=O)NH _z-2 (C _1-3 alkyl) _z , -NHC(=NH)NH _{z -2} (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z represents 0, 1 or 2, and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl,

or R ⁷² and R ⁷³ may combine together with the nitrogen atom to which they are attached to form a 5- or 6-membered ring which is optionally substituted with one, two or three substituents selected from the group consisting of C _1-3 alkyl , halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH,
-O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , - NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)(C _1-3 alkyl), -C(= O)OH, -C(=O)O(C _1-3 alkyl), -C(=O)NH _2-z (C _1-3 alkyl) _z , -OC(=O)(C _1-3 alkyl ), -OC(=O)O(C _1-3 alkyl), -OC(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=O)NH _z-2 (C _1-3 alkyl) _z , -NHC(=NH)NH _z-2 (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C( =NH)NH _2-z (C _1-3 alkyl) _z where z is 0, 1 or 2 and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl;

R ⁸¹ , R ⁸² and R ⁸³ are independently selected from the group consisting of H, C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 3-6 membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl and 3-6-membered heterocyclyl, where each of C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 3-6-membered cycloalkyl, 5- or 6-membered aryl , 5- or 6-membered heteroaryl and 3-6-membered heterocyclyl group is optionally substituted with one, two or three substituents selected from the group consisting of C _1-3 alkyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH, -O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , -NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _{1- 3} alkyl) _z , -C(=O)(C _1-3 alkyl), -C(=O)OH, -C(=O)O(C _1-3 alkyl), -C(=O)NH _{2 -z} (C _1-3 alkyl) _z , -OC(=O)(C _1-3 alkyl), -OC(=O)O(C _1-3 alkyl), -OC(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=O)NH _z-2 (C _1-3 alkyl) _z , -NHC(=NH)NH _{z -2} (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z is 0, 1 or 2, and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl,

or R ⁸² and R ⁸³ may combine together with the nitrogen atom to which they are attached to form a 5- or 6-membered ring which is optionally substituted with one, two or three substituents selected from the group consisting of C _1-3 alkyl , halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH,
-O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , - NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)(C _1-3 alkyl), -C(= O)OH, -C(=O)O(C _1-3 alkyl), -C(=O)NH _2-z (C _1-3 alkyl) _z , -OC(=O)(C _1-3 alkyl ), -OC(=O)O(C _1-3 alkyl), -OC(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=O)NH _z-2 (C _1-3 alkyl) _z , -NHC(=NH)NH _z-2 (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C( =NH)NH _2-z (C _1-3 alkyl) _z where z is 0, 1 or 2 and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl;

Z ¹ and Z ² are independently selected from O, S and N(R ⁸⁴ ), where R ⁸⁴ represents -H or C _1-3 alkyl.

Typical 1st level substituents are preferably selected from the group consisting of _C1-6 alkyl, _C2-6 alkenyl, _C2-6 alkynyl, 3-14 membered (eg 5 or 6 membered) aryl, 3- 14-membered (for example, 5- or 6-membered) heteroaryl, 3-14-membered (for example, 3-7-membered) cycloalkyl, 3-14-membered (for example, 3-7-membered) heterocyclyl, halogen, - CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ ,
-NO ₂ , -OR ⁷¹ , -N(R ⁷² )(R ⁷³ ), -S(O) _0-2 R ⁷¹ , -S(O) _0-2 OR ⁷¹ , -OS(O) _0-2 R ⁷¹ , -OS(O) _0-2 OR ⁷¹ , -S(O) _0-2 N(R ⁷² )(R ⁷³ ), -OS(O) _0-2 N(R ⁷² )(R ⁷³ ), - N(R ⁷¹ )S(O) _0-2 R ⁷¹ , -NR ⁷¹ S(O) _0-2 OR ⁷¹ , -C(=Z ¹ )R ⁷¹ , -C(=Z ¹ )Z ¹ R ⁷¹ , -Z ¹ C(=Z ¹ )R ⁷¹ , and -Z ¹ C(=Z ¹ )Z ¹ R ⁷¹ such as C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, 3-7-membered cycloalkyl, 3-7-membered heterocyclyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS , -N ₃ , -NO ₂ , -OH, -O(C _1-3 alkyl), -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , -NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)OH, -C (=O)O(C _1-3 alkyl), -C(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(= NH)NH _z-2 (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z is 0, 1 or 2, and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl; Z ¹ is independently selected from O, S, NH and N(CH ₃ ); and R ⁷¹ , R ⁷² and R ⁷³ are as defined above or are preferably independently selected from the group consisting of H, C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted by one, two or three substituents selected from the group consisting of C _1-3 alkyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH, -O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , - NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)OH, -C(=O)O(C _{1 -3} alkyl), -C(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=NH)NH _z-2 (C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z is 0, 1 or 2, and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl; or R ⁷² and R ⁷³ may combine together with the nitrogen atom to which they are attached to form a 5- or 6-membered ring which is optionally substituted with one, two or three substituents selected from the group consisting of C _1-3 alkyl , halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -NO ₂ , -OH, -O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _{1-3 alkyl), -N(C 1-3 alkyl} ) ₂ , -NHS(O) ₂ (C _1-3 alkyl), - S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)OH, -C(=O)O(C _1-3 alkyl), -C(=O)NH _{2- z} (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=NH)NH _z-2 (C _1-3 alkyl) _z , and -N(C _{1- 3} alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z is 0, 1 or 2 and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl.

Typical 2nd level substituents are preferably selected from the group consisting of C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, -CF ₃ , -CN, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ ,
-NO ₂ , -OH, -O(C _1-3 alkyl), -OCF ₃ , -S(C _1-3 alkyl), -NH ₂ , -NH(C _1-3 alkyl), -N(C _{1 -3} alkyl) ₂ ,
-NHS(O) ₂ (C _1-3 alkyl), -S(O) ₂ NH _2-z (C _1-3 alkyl) _z , -C(=O)OH, -C(=O)O(C _1-3 alkyl), -C(=O)NH _2-z (C _1-3 alkyl) _z , -NHC(=O)(C _1-3 alkyl), -NHC(=NH)NH _z-2 ( C _1-3 alkyl) _z , and -N(C _1-3 alkyl)C(=NH)NH _2-z (C _1-3 alkyl) _z , where z is 0, 1 or 2, and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl. Particularly preferred 2nd level substituents include 4-morpholinyl, homomorpholinyl, 4-piperidinyl, homopiperidinyl (i.e. azepanil, particularly 4-azepanyl), 4-piperazinyl, homopiperazinyl (i.e. diazepanil, particularly 2,4 -diazepanil), N-methyl-piperazin-4-yl, N-methyl-homopiperazinyl, -CH ₂ CH ₂ OCH ₃ , -OCH ₂ CH ₂ OCH ₃ , -CH ₂ CH ₂ NH _2-z (CH ₃ ) _z , -OCH ₂ CH ₂ NH _2-z (CH ₃ ) _z , -CF ₃ , and -OCF ₃ .

Exemplary 3rd level substituents are preferably selected from the group consisting of phenyl, furanyl, pyrrolyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, partially and fully hydrogenated forms of the above groups, morpholino, C _{1- 3} alkyl, halogen, -NC, -NCO, -CNO, -SCN, -NCS, -N ₃ , -CF ₃ , -OH, -OCH ₃ , -OCF _{3 , -SCH 3 ,} -NH _2-z (CH ₃ ) _z , -C(=O)OH, and -C(=O)OCH ₃ , where z is 0, 1 or 2.

The term “optional” or “optional” as used herein means that the event, circumstance, or condition described below may or may not occur, and that the description includes cases in which the event, circumstance, or condition occurs and cases in which it not happening.

“Isomers” are compounds that have the same molecular formula but differ in structure (“structural isomers”) or geometric arrangement of functional groups and/or atoms (“stereoisomers”). "Enantiomers" are a pair of stereoisomers that cannot superimpose each other in a mirror image. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is designated by the prefix (±). "Diastereomers" are stereoisomers that are not enantiomers. "Tautomers" are structural isomers of the same chemical substance that spontaneously transform into each other, even in their pure form.

The 5' cap compound of the present invention or RNA modified with the 5' cap compound of the present invention may be isotope labeled, i.e. one or more atoms are replaced by a corresponding atom having the same number of protons but a different number of neutrons. For example, a hydrogen atom can be replaced by a deuterium atom. Examples of isotopes that can be used in the 5' cap compound of the present invention or RNA modified with the 5' cap compound of the present invention include deuterium, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³² S, ³⁶ Cl, and ¹²⁵ I. The term “isotopically enriched” means that the presence of the isotope exceeds the limits of natural abundance. The 5'-cap compound of the present invention which is isotope-labeled, or RNAs modified with such an isotopically-labeled 5'-cap compound of the present application, can be prepared using suitably isotopically labeled nucleotides during in vitro transcription or by adding such suitably labeled nucleotides. isotopes of nucleotides after transcription.

The phrase “the stereochemical configuration of the P atom containing the R ⁵ substituent corresponds to that of the P _β atom of the D1 beta-S-ARCA diastereomer” means that the phosphorus atom containing the R ⁵ substituent has a chiral center and is therefore capable of existing in any of the two stereochemical configurations is present predominantly in one required stereochemical configuration, i.e. at the P _β atom of the D1 beta-S-ARCA diastereomer. In the case of the P _β atom of the D1 beta-S-ARCA diastereomer, it can be either the (R) configuration or the (S) configuration. Preferably, more than 50% of the group of interest has the required stereochemical configuration, preferably at least 75% of the group of interest has the required stereochemical configuration, more preferably at least 90% of the group of interest has the required stereochemical configuration, even more preferably at least 95% of the group of interest has the required stereochemical configuration. the group of interest has the required stereochemical configuration, and most preferably at least 99% of the group of interest has the required stereochemical configuration.

Diastereomer D1 beta-S-ARCA (β-S-ARCA) has the following structure:

"D1 beta-S-ARCA diastereomer" or "beta-S-ARCA(D1)" is the beta-S-ARCA diastereomer that elutes first on the HPLC column compared to the D2 beta-S-ARCA diastereomer (beta-S- ARCA(D2)) and thus has a shorter retention time. The HPLC is preferably analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column is used for separation, preferably format: 5 µm, 4.6 x 250 mm, and a flow rate of 1.3 ml/min can be used. In one embodiment, a gradient of methanol in ammonium acetate is used, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, over 15 minutes. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

The term "natural" as used herein in the context of an object refers to the fact that the object can be found in nature. For example, a protein, amino acid, or nucleic acid present in the body (including viruses) that can be isolated from a natural source and that has not been intentionally modified by humans in a laboratory is natural.

The present invention relates to modification of RNA, preferably mRNA, to increase the stability and/or expression of said RNA, preferably in immune cells, more preferably in immature immune cells, even more preferably in immature antigen presenting cells and most preferably in immature dendritic cells. The modified RNA described in the present invention is particularly useful for RNA vaccination.

5' cap connection

In a first aspect, the present application provides a 5' cap compound having a 5' cap structure according to formula (I):

Formula (I)

where R ¹ is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl;

R ² and R ³ are independently selected from the group consisting of H, halogen, OH and optionally substituted alkoxy, or R ² and R ³ together form OXO, where X is selected from the group consisting of optionally substituted CH ₂ , optionally substituted CH ₂ CH ₂ , optionally substituted CH ₂ CH ₂ CH ₂ , optionally substituted CH ₂ CH(CH ₃ ) and optionally substituted C(CH ₃ ) ₂ , or R ² combined with a hydrogen atom at the 4' position of the ring to which R ² is attached, with the formation of –O-CH ₂ - or -CH ₂ -O-;

R ⁴ and R ⁶ are independently selected from the group consisting of O, S, Se and BH ₃ ;

R ⁵ is selected from the group consisting of S, Se and BH ₃ ;

R ⁷ is a mononucleotide or oligonucleotide containing 2, 3, 4, 5, 6, 7, 8 or 9 (eg 2, 3, 4, 5 or 6) bases;

R ⁸ represents H, halogen or optionally substituted alkoxy;

n is 1, 2 or 3; And

B is a purine or pyrimidine base moiety.

In one embodiment, the 5'-cap compound has formula (Ia)

formula (Ia)

where R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , n and B are as defined above or below, and R ¹ is chosen such that the 5' cap compound does not inhibit RNA translation, containing the specified 5'-cap connection. In one embodiment, the 5' cap of the compound of formula (Ia) R ¹ is selected such that the capped RNA, in particular the 5' cap structure of the capped RNA, is recognized by the translation initiation machinery, preferably in vivo and in vitro, preferably the capped RNA, in In particular, the 5'-cap structure of capped RNA is recognized by the mechanism of eukaryotic translation initiation. For example, one skilled in the art can determine whether a capped RNA or a capped RNA 5' cap structure is recognized by the eukaryotic translation initiation machinery by determining the affinity of the eukaryotic translation initiation factor eIF4E for the capped RNA or the 5' cap structure.

In one embodiment, the 5'-cap of a compound of formula (Ia) R ¹ is selected from the group consisting of optionally substituted C ₁ -C ₄ alkyl (for example, methyl, ethyl, propyl, butyl, benzyl, phenylethyl and naphthylmethyl, any of which may be replaced if necessary); optionally substituted C ₂ -C ₄ alkenyl (eg ethenyl, propenyl or butenyl, any of which may be substituted if desired) and optionally substituted aryl. In a preferred embodiment, the 5'-cap of the compound of formula (Ia) R ¹ is selected from the group consisting of C ₁ -C ₄ alkyl and optionally substituted aryl. In a preferred embodiment, the 5'-cap of the compound of formula (Ia) R ¹ is selected from the group consisting of methyl, ethyl, optionally substituted benzyl, optionally substituted phenylethyl and optionally substituted naphthylmethyl. In a preferred embodiment, the 5'-cap of the compound of formula (Ia) R ¹ is methyl or ethyl, more preferably methyl.

In one embodiment, the 5'-cap compound has formula (Ib)

Formula (Ib)

wherein R ¹ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I) and (Ia)) or below, and the configuration R ² and R ³ are such that the 5' cap junction can be included in the RNA strand in only one orientation. Pasquinelli et al. (1995, RNA J. 1: 957-967) demonstrated that during in vitro transcription, bacteriophage RNA polymerases use a 7-methylguanosine unit to initiate transcription, with about 40-50% of capped transcripts having the cap dinucleotide in reverse orientation (i.e. the initial reaction product is Gpppm ⁷ GpN). Compared to RNAs containing the cap structure in the correct orientation, RNAs containing the cap structure in the reverse orientation (also called inverted cap RNAs) are not functional for the translation of encoded proteins. Thus, it is necessary to turn on the cap in the correct orientation, i.e. as a result, obtain RNA with a cap structure essentially corresponding to m ⁷ GpppGpN, etc. Inverted cap dinucleotide integration has been shown to be inhibited by substitution of either the 2'- or 3'-OH group of the methylated guanosine unit (Stepinski et al., 2001; RNA J. 7: 1486-1495; Peng et al., 2002; Org Lett. 24: 161-164). RNAs that are synthesized in the presence of such “anti-inverted cap analogues”, i.e. ARCAs are translated more efficiently than RNAs, which are transcribed in vitro in the presence of the conventional m ⁷ GpppG 5' cap. In addition, Kore et al. (J. Am. Chem. Soc. 2009, 131: 6364-6365) found that dinucleotide mRNA cap analogues modified with a locked nucleic acid (LNA) are also not incorporated in an inverted orientation into the RNA strand.

Therefore, in a preferred embodiment, the 5' cap of the compound of formula (Ib) R ¹ is selected such that the eukaryotic translation initiation apparatus is capable of recognizing RNA capped with the 5' cap compound of the present invention and at least one (or both) of R ² and R ³ are selected such that the 5' cap junction cannot be incorporated in an inverted orientation into the RNA strand.

In one embodiment, the 5'-cap compounds of formula (Ib) R ² and R ³ are independently selected from the group consisting of H, F, OH, methoxy, ethoxy, propoxy and 2-methoxyethoxy. In a preferred embodiment of the 5'-cap of the compound of formula (Ib), one of R ² and R ³ is OH and the other is not OH. In another preferred embodiment of the 5'-cap of the compound of formula (Ib) at least one of R ² and R ³ is not OH. For example, in one embodiment, the 5'-cap of the compound of formula (Ib) R ² is selected from the group consisting of H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy, preferably from the group consisting of H, F, methoxy, ethoxy and propoxy.

In any of the embodiments of the 5'-cap compound of formula (Ib) described above, the ring structure containing the R ² and R ³ substituents may have the stereochemical configuration of ribose. In this embodiment, preferably at least one of R ² and R ³ is not OH.

In those of the above embodiments where R ² (or R ³ ) is not OH, it is preferably selected from the group consisting of H, halogen and optionally substituted C ₁ -C ₁₀ alkoxy, more preferably from the group consisting of H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy, more preferably from the group consisting of H, F, methoxy, ethoxy and propoxy. More preferably it is methoxy.

In a preferred embodiment, the 5'-cap compound of formula (Ib), in particular when the ring structure containing the substituents R ² and R ³ has the stereochemical configuration of ribose, R ² is OH and R ³ is methoxy, or R ² is methoxy and R ³ is OH.

In one embodiment, the 5'-cap compounds of formula (Ib) R ² and R ³ together form OXO, where X is selected from the group consisting of CH ₂ and C(CH ₃ ) ₂ , both of which may be optionally substituted.

In one embodiment, the 5'-cap compound of formula (Ib) has a stereochemical configuration of the ring structure comprising the R ² and R ³ substituents that does not correspond to the stereochemical configuration of ribose. For example, the stereochemical configuration of the ring structure comprising the substituents R ² and R ³ may correspond to the stereochemical configuration of arabinose, xylose or lyxose, in particular when the stereochemical configuration of said ring structure corresponds to that of arabinose. In these embodiments, preferably both R ² and R ³ are OH. However, in these embodiments, it is also possible that R ² and R ³ are selected as described above.

In one embodiment, the 5'-cap compound has formula (Ic)

Formula (Ic)

where R ¹ , R ² , R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I), (Ia) and (Ib )) or lower, and R ⁵ is S or Se, preferably S. In one embodiment, the 5' cap of a compound of formula (Ic) R ⁵ is S or Se, preferably S, and n is 1 or 2. In one In an embodiment of the 5'-cap compound of formula (Ic), R ⁵ is S and n is 1 or 2, preferably 1. In any of the above embodiments, the 5'-cap compound of formula (Ic) is preferably R ⁴ and R ⁶ independently selected from the group consisting of O, Se and S, more preferably from the group consisting of O and S. In any of the above embodiments where n is 2 or 3, it should be understood that R ⁶ can be independently selected for each [ R ⁶ PO ₂ ] component. For example, if n is 2, the 5'-cap compound contains two [R ⁶ PO ₂ ] components, where the two R ⁶ residues may be the same (for example, the R ⁶ in both components [R ⁶ PO ₂ ] is O) or different (eg, R ⁶ in one component [R ⁶ PO ₂ ] is O, while R ⁶ in the other component [R ⁶ PO ₂ ] is S). In one embodiment, the 5' cap of a compound of formula (Ic) R ⁵ is S or Se, preferably S, n is 1 or 2, preferably 1, and R ⁴ and R ⁶ are independently selected from the group consisting of O and S, more preferably, R ⁴ and R ⁶ are O. In one embodiment, the 5' cap of the compound of formula (Ic) R ⁵ is S, n is 1 or 2, preferably 1, and R ⁴ and R ⁶ are O.

In one embodiment, the 5'-cap compound of formula (Ic) has a stereochemical configuration at the P atom containing the R ⁵ substituent that corresponds to that of the P _β atom of the D1 beta-S-ARCA diastereomer.

In one embodiment, the 5' cap compound has the formula (Id)

Formula (Id)

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I), (Ia), (Ib ) and (Ic)) or below, and R ⁷ is linked at its 5' end to the ring to which R ⁸ is attached. In one embodiment, the 5' cap compound of formula (Id) R ⁷ is a ribomononucleotide or ribooligonucleotide. In one preferred embodiment, the 5' cap of a compound of formula (Id) R ⁷ is a ribonucleotide having a free OH group at the 2' position. In another preferred embodiment, the 5' cap compound of formula (Id) R ⁷ is a ribooligonucleotide, wherein both the ribose moiety at the 3' end of the ribooligonucleotide and the ribose moiety at the 5' end of the ribooligonucleotide have a free OH group at the 2' position . In another preferred embodiment, the 5' cap of a compound of formula (Id) R ⁷ is a ribooligonucleotide in which the OH group at the 2' position of at least ribose at the 5' end of the ribooligonucleotide is replaced by a substituent selected from the group consisting of H, halogen and optionally substituted alkoxy (such as H, F, methoxy, ethoxy, propoxy or 2-methoxyethoxy, preferably H, F, methoxy, ethoxy or propoxy, most preferably methoxy), and the ribose at the 3' end of the ribooligonucleotide has a free OH group at position 2'. In any of the above embodiments of formula (Id), preferably the internucleotide linkage between the mononucleotide or oligonucleotide and the ring to which R ⁷ is attached is selected from the group consisting of phosphate, phosphorothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, alkylphosphonate, phosphotriester, phosphoamidite and a non-nucleotide linker, preferably from the group consisting of phosphate, phosphothioate and phosphodithioate (in one embodiment, the internucleotide linkage between the mononucleotide or oligonucleotide and the ring to which R ⁷ is attached is a phosphate). In any of the above embodiments of formula (Id), wherein R ⁷ is an oligonucleotide, in particular a ribooligonucleotide, preferably the internucleotide linkage(s) between nucleotides in the oligonucleotide is selected from the group consisting of phosphate, phosphothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, an alkylphosphonate, a phosphotriester, a phosphoamidite, and a non-nucleotide linker, preferably from the group consisting of phosphate, phosphothioate and phosphodithioate linkages (in one embodiment, the internucleotide linkage(s) between nucleotides in the oligonucleotide is a phosphate). In one embodiment, the 5' cap of a compound of formula (Id) R ⁷ is *[pN(R ^8' )] _a [pN] _b where * indicates the point of attachment of R ⁷ to the ring to which R ⁷ is attached; each N(R ^8' ) is a nucleoside (preferably adenosine, guanosine, uridine, 5-methyluridine or cytidine) which is substituted with R ^8' (selected from the group consisting of H, halogen and optionally substituted alkoxy, preferably from the group consisting of from H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy, more preferably from the group consisting of H, F, methoxy, ethoxy and propoxy, most preferably methoxy) at the 2'position; each N is a ribonucleoside (preferably adenosine, guanosine, uridine, 5-methyluridine or cytidine) having a free OH group at the 2'position; each p represents a phosphate group; a is equal to 0, 1, 2, 3, 4, 5, 6, 7 or 8; b is equal to 1, 2, 3, 4, 5, 6, 7, 8 or 9; and a+b is 1, 2, 3, 4, 5, 6, 7, 8 or 9 (preferably a is 0, 1 or 2; b is 1, 2, 3, 4, 5 or 6; and a + b equals 1, 2, 3, 4, 5 or 6). In one embodiment, the 5'-cap of a compound of formula (Id) R ⁷ is *pGpN or *pG, where N is adenosine, guanosine, uridine, 5-methyluridine or cytidine, and where * indicates the point of attachment of R ⁷ to the ring , to which R ⁷ is attached. In one embodiment, the 5'-cap of a compound of formula (Id) R ⁷ is *pm ^2'-O GpN, where N is adenosine, guanosine, uridine, 5-methyluridine or cytidine, and where * indicates the point of attachment of R ⁷ to ring to which R ⁷ is attached.

In one embodiment, the 5' cap compound has formula (Ie)

Formula (Ie)

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and n have the meanings given above (in particular with respect to one or more formulas (I), (Ia), ( Ib), (Ic), and (Id)) or lower, and B is a natural purine or pyrimidine base moiety or a modified form thereof. In one embodiment, the 5'-cap of the compound of formula (Ie) B is selected from the group consisting of guanine, adenine, cytosine, thymine, uracil and modified forms thereof, preferably from the group consisting of guanine, adenine, cytosine, uracil and modified forms thereof forms, more preferably from the group consisting of guanine, adenine, cytosine and modified forms thereof, more preferably from the group consisting of guanine, adenine and modified forms thereof. In one embodiment, the 5'-cap of a compound of formula (Ie) is modified with one or more alkyl groups, preferably one or more C _1-4 alkyl groups, more preferably one or more methyl groups. In a preferred embodiment of the 5'-cap compound of formula (Ie), the modified purine or pyrimidine base moiety is selected from the group consisting of N ⁷ -alkylguanine, N ⁶ -alkyladenine, 5-alkylcytosine, 5-alkyluracil and N(1)-alkyluracil , preferably from the group consisting of N ⁷ -C _1-4 alkylguanine, N ⁶ -C _1-4 alkyl adenine, 5-C _1-4 alkylcytosine, 5-C _1-4 alkyluracil and N(1)-C _1-4 alkyluracil, more preferably from the group consisting of N ⁷ -methylguanine, N ⁶ -methyladenine, 5-methylcytosine, 5-methyluracil and N(1)-methyluracil. In a preferred embodiment, the 5'-cap of a compound of formula (Ie) the natural purine or pyrimidine base moiety is G or A, preferably G. In a more preferred embodiment, the 5'-cap of a compound of formula (Ie) B is G or A, preferably G.

In any of the above embodiments, the 5'-cap compound of any of formulas (I), (Ia), (Ib), (Ic), (Id) and (Ie) preferably R ⁸ is selected from the group consisting of H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy, more preferably from the group consisting of H, F, methoxy, ethoxy and propoxy. Most preferably, in any of the above embodiments, the 5'-cap compound of any one of formulas (I), (Ia), (Ib), (Ic), (Id) and (Ie) R ⁸ is methoxy.

In one embodiment, the 5'-cap compound has formula (II)

Formula (II)

where R ¹ is selected from the group consisting of optionally substituted C ₁ -C ₄ alkyl and optionally substituted aryl;

R ² and R ³ are independently selected from the group consisting of H, F, OH, methoxy, ethoxy, propoxy and 2-methoxyethoxy;

R ⁴ and R ⁶ are independently selected from the group consisting of O and S;

R ⁵ represents S or Se;

R ⁷ is a ribomononucleotide or ribooligonucleotide containing 2, 3, 4, 5 or 6 (eg 2 or 3) bases;

R ⁸ is selected from the group consisting of H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy;

n is 1, 2 or 3; And

B is a purine or pyrimidine base moiety.

In one embodiment, the 5'-cap compound has formula (IIa)

Formula (IIa)

where R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I), (Ia), (Ib ), (Ic), (Id), (Ie) and (II)) or lower, and R ¹ is selected from the group consisting of methyl, ethyl, benzyl, phenylethyl and naphthylmethyl, more preferably from the group consisting of methyl and ethyl . In a preferred embodiment, the 5'-cap of the compound of formula (IIa) R ¹ is methyl or ethyl, more preferably methyl.

In one embodiment, the 5'-cap compound has formula (IIb)

formula (IIb)

where R ¹ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I), (Ia), (Ib), ( Ic), (Id), (Ie), (II) and (IIa)) or lower, and at least one of R ² and R ³ is not OH. In one embodiment of the 5' cap of a compound of formula (IIb), one of R ² and R ³ is OH and the other is not OH. In one embodiment, the 5'-cap of the compound of formula (IIb) ring structure containing R ² and R ³ substituents has the stereochemical configuration of ribose. In this embodiment, preferably at least one of R ² and R ³ is not OH. In those of the above embodiments where R ² (or R ³ ) is not OH, it is preferably selected from the group consisting of H, F, methoxy, ethoxy and propoxy. More preferably it is methoxy.

In a preferred embodiment, the 5'-cap compound of formula (IIb), in particular when the ring structure containing the substituents R ² and R ³ has the stereochemical configuration of ribose, R ² is OH and R ³ is methoxy, or R ² is methoxy and R ³ is OH.

In one embodiment, the 5'-cap compound of formula (IIb) has a stereochemical configuration of the ring structure containing R ² and R ³ substituents that does not correspond to the stereochemical configuration of ribose. For example, the stereochemical configuration of the ring structure comprising the substituents R ² and R ³ may correspond to the stereochemical configuration of arabinose, xylose or lyxose, in particular when the stereochemical configuration of said ring structure corresponds to that of arabinose. In these embodiments, preferably both R ² and R ³ are OH. However, in these embodiments, it is also possible that R ² and R ³ are selected as described above.

In one embodiment, the 5'-cap compound has formula (IIc)

formula (IIc)

where R ¹ , R ² , R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I), (Ia), (Ib ), (Ic), (Id), (Ie), (II), (IIa) and (IIb)) or below. In one embodiment, the 5' cap of a compound of formula (IIc) n is 1 or 2. In any of the above embodiments where n is 2 or 3, it is understood that R ⁶ can be independently selected for each [R ⁶ PO ₂ ] fragment. For example, if n is 2, the 5'-cap compound contains two [R ⁶ PO ₂ ] fragments, where the two R ⁶ residues can be the same (for example, the R ⁶ in both [R ⁶ PO ₂ ] fragments is O) or different (eg, R ⁶ in one fragment [R ⁶ PO ₂ ] represents O, while R ⁶ in another fragment [R ⁶ PO ₂ ] represents S). In one embodiment, the 5'-cap compound of formula (IIc) R ⁴ and R ⁶ are O. In one embodiment, the 5'-cap compound of formula (IIc) n is 1 or 2, preferably 1, and R ⁴ and R ⁶ represent O.

In one embodiment, the 5'-cap compound has formula (IId)

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁸ , n and B have the meanings given above (in particular with respect to one or more formulas (I), (Ia), (Ib ), (Ic), (Id), (Ie), (II), (IIa), (IIb) and (IIc)) or lower, and R ⁷ is connected at its 5' end to the ring to which R ⁸ is attached . In one preferred embodiment, the 5' cap of a compound of formula (IId) R ⁷ is a ribonucleotide having a free OH group at the 2' position. In another preferred embodiment, the 5' cap of the compound of formula (IId) R ⁷ is a ribooligonucleotide, wherein both the ribose moiety at the 3' end of the ribooligonucleotide and the ribose moiety at the 5' end of the ribooligonucleotide have a free OH group at the 2' position. In another preferred embodiment, the 5' cap of a compound of formula (IId) R ⁷ is a ribooligonucleotide wherein the OH group at the 2' position of at least ribose at the 5' end of the ribooligonucleotide is replaced by a substituent selected from the group consisting of H, halogen and optionally substituted alkoxy (such as H, F, methoxy, ethoxy, propoxy or 2-methoxyethoxy, preferably H, F, methoxy, ethoxy or propoxy, most preferably methoxy), and the ribose at the 3' end of the ribooligonucleotide has a free OH group at position 2'. In any of the above embodiments of formula (IId), preferably the internucleotide linkage between the mononucleotide or oligonucleotide and the ring to which R ⁷ is attached is selected from the group consisting of phosphate, phosphorothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, alkylphosphonate, phosphotriester, phosphoramidite and a non-nucleotide linker, more preferably an internucleotide linkage between the mononucleotide or oligonucleotide and the ring to which R ⁷ is attached is a phosphate. In any of the above embodiments of formula (IId), wherein R ⁷ is an oligonucleotide, in particular a ribooligonucleotide, preferably the internucleotide linkage(s) between nucleotides in the oligonucleotide is selected from the group consisting of phosphate, phosphothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, an alkylphosphonate, a phosphotriester, a phosphoamidite, and a non-nucleotide linker, more preferably the internucleotide linkage(s) between nucleotides in the oligonucleotide is a phosphate. In one embodiment, the 5' cap of a compound of formula (IId) R ⁷ is *[pN(R ^8' )] _a [pN] _b where * indicates the point of attachment of R ⁷ to the ring to which R ⁷ is attached; each N(R ^8' ) is a nucleoside (preferably adenosine, guanosine, uridine, 5-methyluridine or cytidine) which is substituted with R ^8' (selected from the group consisting of H, halogen and optionally substituted alkoxy, preferably from the group consisting of from H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy, more preferably from the group consisting of H, F, methoxy, ethoxy and propoxy, most preferably methoxy) at the 2'position; each N is a ribonucleoside (preferably adenosine, guanosine, uridine, 5-methyluridine or cytidine) having a free OH group at the 2'position; each p represents a phosphate group; a is equal to 0, 1, 2, 3, 4, 5, 6, 7 or 8; b is equal to 1, 2, 3, 4, 5, 6, 7, 8 or 9; and a + b is equal to 1, 2, 3, 4, 5, 6, 7, 8 or 9 (preferably a is equal to 0, 1 or 2; b is equal to 1, 2, 3, 4, 5 or 6; and a + b equals 1, 2, 3, 4, 5 or 6). In one embodiment, the 5'-cap of a compound of formula (IId) R ⁷ is *pGpN or *pG, where N is adenosine, guanosine, uridine, 5-methyluridine or cytidine, and where * indicates the point of attachment of R ⁷ to the ring, to which R ⁷ is attached. In one embodiment, the 5'-cap of a compound of formula (IId) R ⁷ is *pm ^2'-O GpN, where N is adenosine, guanosine, uridine, 5-methyluridine or cytidine, and where * indicates the point of attachment of R ⁷ to the ring to which R ⁷ is attached.

In one embodiment, the 5'-cap compound has formula (IIe)

formula (IIe)

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and n have the meanings given above (in particular with respect to one or more formulas (I), (Ia), ( Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), (IIc) and (IId)) or lower, and B is a moiety of a natural purine or pyrimidine base or its modified form. In one embodiment, the 5'-cap of the compound of formula (IIe) B is selected from the group consisting of guanine, adenine, cytosine, thymine, uracil and modified forms thereof, preferably from the group consisting of guanine, adenine, cytosine, uracil and modified forms thereof forms, more preferably from the group consisting of guanine, adenine, cytosine and modified forms thereof, more preferably from the group consisting of guanine, adenine and modified forms thereof. In one embodiment of the 5'-cap compound of formula (IIe), the modified purine or pyrimidine base moiety is modified with one or more alkyl groups, preferably one or more C _1-4 alkyl groups, more preferably one or more methyl groups. In a preferred embodiment of the 5'-cap of the compound of formula (IIe), the modified purine or pyrimidine base moiety is selected from the group consisting of N ⁷ -alkylguanine, N ⁶ -alkyladenine, 5-alkylcytosine, 5-alkyluracil and N(1)-alkyluracil , preferably from the group consisting of N ⁷ -C _1-4 alkylguanine, N ⁶ -C _1-4 alkyl adenine, 5-C _1-4 alkylcytosine, 5-C _1-4 alkyluracil and N(1)-C _1-4 alkyluracil, more preferably from the group consisting of N ⁷ -methylguanine, N ⁶ -methyladenine, 5-methylcytosine, 5-methyluracil and N(1)-methyluracil. In a preferred embodiment, the 5'-cap of a compound of formula (IIe) moiety from a natural purine or pyrimidine base is G or A, preferably G. In a more preferred embodiment, the 5'-cap of a compound of formula (IIe) B is G or A, preferably G.

In any of the above embodiments, the 5'-cap compound of any of formulas (II), (IIa), (IIb), (IIc), (IId) and (IIe) R ⁸ is preferably selected from the group consisting of H, F, methoxy, ethoxy and propoxy. Most preferably, in any of the above embodiments, the 5'-cap compound of any one of formulas (II), (IIa), (IIb), (IIc), (IId) and (IIe) R ⁸ is methoxy.

In one embodiment, the 5'-cap compound has formula (III)

formula (III)

where R ¹ represents methyl, ethyl, benzyl, phenylethyl or naphthylmethyl, more preferably methyl or ethyl;

R ² and R ³ are independently selected from the group consisting of H, F, OH and methoxy, where preferably at least one of R ² and R ³ is not OH;

R ⁷ is a ribomononucleotide, ribodinucleotide or ribotrinucleotide linked at its 5' end to a ring to which R ⁸ is attached, wherein the internucleotide bond between the ribomononucleotide, ribodinucleotide or ribotrinucleotide and the ring to which R ⁷ is attached is selected from the group consisting of phosphate , phosphothioate and phosphodithioate, and where, if R ⁷ is a ribodinucleotide or ribothrinucleotide, the internucleotide bond(s) between the nucleotides in the ribodinucleotide or ribothrinucleotide is selected from the group consisting of phosphate, phosphothioate and phosphodithioate;

R ⁸ is selected from the group consisting of H, F and methoxy;

n is 1 or 2; And

B is selected from the group consisting of guanine, adenine, cytosine, thymine and uracil, preferably from the group consisting of guanine, adenine, cytosine and uracil, more preferably from the group consisting of guanine, adenine and cytosine, more preferably from the group consisting of from guanine and adenine.

In one embodiment, the 5'-cap compound has formula (IIIa)

formula (IIIa)

where R ¹ represents methyl or ethyl; one of R ² and R ³ is OCH ₃ and the other is OH (for example, R ² is OCH ₃ and R ³ is OH, or R ² is OH and R ³ is OCH ₃ ); R ⁸ represents methoxy; n is 1; the internucleotide bond between a ribodinucleotide, ribodinucleotide or ribothrinucleotide and the ring to which R ⁷ is attached is a phosphate or phosphothioate, preferably phosphate, and where, if R ⁷ is a ribodinucleotide or ribotrinucleotide, the internucleotide bond(s) between the ribodinucleotide or ribotrinucleotide is ) is a phosphate or phosphorothioate, preferably phosphate; and B is guanine or adenine, preferably guanine. In one embodiment, the 5' cap of a compound of formula (IIIa) R ⁷ is a ribomononucleotide having a free OH group at the 2' position. In another preferred embodiment, the 5' cap of the compound of formula (IIIa) R ⁷ is a ribodinucleotide or ribothrinucleotide, wherein both the ribose moiety at the 3' end of the ribodinucleotide or ribotrinucleotide and the ribose moiety at the 5' end of the ribodinucleotide or ribothrinucleotide have a free group OH in position 2'. In another preferred embodiment, the 5' cap of the compound of formula (IIIa) R ⁷ is a ribodinucleotide or ribotrinucleotide, wherein the OH group at the 2' position of at least ribose at the 5' end of the ribodinucleotide or ribotrinucleotide is replaced by a substituent selected from the group consisting of of H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy (preferably from the group consisting of H, F, methoxy, ethoxy and propoxy, most preferably said substituent is methoxy), and the ribose at the 3' end of the ribodinucleotide or ribothrinucleotide has free OH group at position 2'. In one embodiment, the 5' cap of a compound of formula (IIIa) R ⁷ is *pGpN or *pG, where N is adenosine, guanosine, uridine, 5-methyluridine or cytidine, and where * indicates the point of attachment of R ⁷ to the ring, to which R ⁷ is attached. In one embodiment, the 5'-cap of a compound of formula (IIIa), R ⁷ is *pm ^2'-O GpN, where N is adenosine, guanosine, uridine, 5-methyluridine or cytidine, and where * indicates the point of attachment of R ⁷ to the ring to which R ⁷ is attached.

The 5'-cap compounds of the present invention can be synthesized starting from commercially available compounds (such as (pN) _2-4 ) using standard procedures. These oligonucleotides can be converted into the corresponding P-imidazolide derivatives by reacting them with imidazole in the presence of an activation system (e.g. 2,2'-dithiodipyridine/triphenylphosphine (2,2'-DTDP); see Fig. 1 and Mukaiyama and Hashimoto 1971 ( Bull. Chem. Soc. Jpn. 44, 2284 (1971).) A nucleotide subunit bearing a modified phosphate bridge (e.g. m2 ^7,2'-OMe GDPβS) can be synthesized as described in Kowalska et al. 2008 (RNA 14, 1119-1131 (2008). Further, two precursors can be coupled to produce the final 5'-cap compound of the present invention; see, for example, Figure 2. Diastereoisomers can be separated using RP-HPLC (e.g. using a Discovery Amide RP C16 column).

Preferably, when the 5' cap compound of the present invention is used to produce a suitably 5' cap RNA, the structure of the 5' cap when transporting the 5' cap RNA into cells is capable of increasing the stability of the RNA, reducing or inhibiting the recognition of the RNA by structure recognition proteins cap-0 proteins, such as IFIT proteins (particularly IFIT1), increase the efficiency of RNA translation, prolong RNA translation, increase overall RNA protein expression, and/or, if the RNA contains a nucleotide sequence encoding an antigen, increase the immune response against said antigen compared with the same RNA without the 5' cap structure. If the RNA contains a nucleotide sequence encoding an antigen, preferably the cells are immature antigen presenting cells, such as immature dendritic cells. One skilled in the art can readily determine whether the 5' cap structure of a 5' cap RNA is capable of performing the above functions, for example by generating two RNAs, for example by in vitro transcription, that differ only in the 5' cap structure, where one of the RNAs bears a 5'-cap structure according to any of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), (IIa), (IIb), ( IIc), (IId), (IIe), (III) and (IIIa), and the other RNA (control RNA) (i) does not contain a 5' cap structure; (ii) carries the normal 5'-cap of the mRNA, that is, the methyl-7-guanosine cap; or (iii) bears any other cap with which the 5'-cap structure function according to any of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (II), ( IIa), (IIb), (IIc), (IId), (IIe), (III), and (IIIa) will be comparable. For example, the reference RNA may carry a 5' cap structure that corresponds to the D2 diastereomer of beta S-ARCA. Particularly preferable 5'-cap structure of 5'-capped RNA, when transferring modified RNA into cells, is capable of increasing RNA stability, reducing or inhibiting RNA recognition by proteins that recognize the cap-0 structure, for example, IFIT proteins (in particular, IFIT1), and increasing translation efficiency RNA, prolong the translation of the RNA, increase the overall protein expression of the RNA and/or, if the RNA contains a nucleotide sequence encoding an antigen, enhance the immune response against said antigen compared to a reference RNA, such as the same RNA having a normal 5' cap of the mRNA.

Preferably, the stability and translation efficiency of RNA modified with a 5' cap compound of the present invention (in particular, a 5' cap compound according to any one of formulas (I), (Ia), (Ib), (Ic), (Id), ( Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) may be modified as necessary. For example, the RNA may be stabilized and its translation may be enhanced by one or more modifications that provide a stabilizing effect and/or an effect of increasing translation efficiency. Such modifications are, for example, described in WO 2007/036366, incorporated herein by reference.

For example, RNA having an unmasked poly-A sequence (unmasked poly-A tail) is translated more efficiently than RNA having a masked poly-A sequence. The term "poly-A sequence" refers to the sequence of adenyl (A) residues that is typically located at the 3' end of the RNA molecule, and "unmasked poly-A sequence" means that the poly-A sequence at the 3' end of the RNA molecule ends A is from the poly-A sequence, and is not followed by other nucleotides except A, located at the 3' end, that is, downstream in the poly-A sequence. In addition, the long poly-A sequence of approximately 120 nucleotides ensures optimal transcript stability and translation efficiency.

Thus, RNA, preferably mRNA, modified with a 5'-cap compound of the present invention (in particular, with a 5'-cap compound according to any of formulas (I), (Ia), (Ib), (Ic), (Id) , (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) may preferably further comprise a poly-A tail of length from 10 to 500 , preferably 30 to 300 nucleotides long, more preferably 65 to 200 nucleotides long, more preferably 100 to 150 nucleotides long, for example 100, 110, 120, 130, 140 or 150 nucleotides, preferably 120 nucleotides. Preferably, said poly-A sequence is an unmasked poly-A sequence. Thus, preferably RNA, preferably mRNA, modified with a 5'-cap compound of the present invention (in particular, a 5'-cap compound according to any of formulas (I), (Ia), (Ib), (Ic), (Id) , (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) is an unmasked poly-A tail ranging from 10 to 500 in length, preferably 30 to 300 nucleotides long, more preferably 65 to 200 nucleotides long, more preferably 100 to 150 nucleotides long, for example 100, 110, 120, 130, 140 or 150 nucleotides, preferably 120 nucleotides.

In addition, the inclusion of a 3' untranslated region (UTR) into the 3' untranslated region of an RNA molecule can lead to increased translation efficiency. A synergistic effect can be achieved by including two or more of these 3'UTRs. The 3'UTRs may be autologous or heterologous to the RNA into which they are inserted, for example the 3'UTR of beta globin mRNA. Thus, preferably RNA, preferably mRNA, modified with a 5' cap compound of the present invention (in particular, a 5' cap compound according to any one of formulas (I), (Ia), (Ib), (Ic), (Id) , (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) may additionally contain one or more copies, preferably two copies 3' -untranslated region (3'-UTR) of a beta globin gene, preferably the human beta globin gene.

In addition, replacement of uridine by pseudouridine or N(1)-methylpseudouridine or 5-methyluridine (m5U), resulting in Ψ-, m1Ψ-, or m5U-modified RNAs, can reduce the immunogenicity of the RNAs thus modified. Therefore, preferably in RNA, preferably mRNA modified with a 5'-cap compound of the present invention (in particular, a 5'-cap compound according to any of formulas (I), (Ia), (Ib), (Ic), (Id) , (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) pseudouridine or N(1)-methylpseudouridine or 5-methyluridine ( m5U) partially or completely, preferably completely, replaces uridine. That is, in one preferred embodiment, the RNA of the invention is Ψ- or m1Ψ- or m5U-modified, or any combination thereof (e.g., Ψ- and m1Ψ-modified, or Ψ- and m5U-modified, or m1Ψ- and m5U- modified, or Ψ- and m1Ψ- and m5U-modified).

In some embodiments, the modified nucleoside replacing one or more uridines in the RNA may be one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine , 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g. 5-iodo-uridine or 5-bromo-uridine), uridine-5-hydroxyacetic acid (cmo5U), uridine-5-hydroxyacetic acid methyl ester (mcmo5U), 5 -carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio -uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl -2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl -pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl 1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2- thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3 -carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U ), α-thiouridine, 2′-O-methyluridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O -methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl -uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F -ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in this field of technology.

Particularly preferably RNA, preferably mRNA, modified with a 5'-cap compound of the present invention (in particular, a 5'-cap compound according to any of formulas (I), (Ia), (Ib), (Ic), (Id), ( Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) is modified by a combination of the modifications described above, i.e. at least two (e.g., at least 3 or all 4) of the following modifications: inclusion of a poly-A sequence, unmasking of a poly-A sequence, inclusion of one or more 3'UTRs, and replacement of uridine with pseudouridine or N(1)- methylpseudouridine, or 5-methyluridine, or combinations thereof.

In a particularly preferred embodiment, RNA, preferably mRNA, modified with a 5'-cap compound of the present invention (in particular, a 5'-cap compound according to any of formulas (I), (Ia), (Ib), (Ic), (Id ), (Ie), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III) and (IIIa)) encodes a pharmaceutically active peptide or protein, for example selected from the group, consisting of cytokines such as erythropoietin; adhesion molecules such as integrin; immunoglobulins; immunologically active compounds, e.g., antigens, such as tumor-associated antigens, pathogen-associated antigens (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) viral antigens , such as one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) influenza virus antigens (A, B or C), CMV or RSV), allergens or autoantigens; hormones such as vasopressin, insulin or growth hormone; growth factors such as VEGFA; enzymes such as herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes or lactase; receptors such as growth factor receptors; protease inhibitors such as alpha-1-antitrypsin; apoptosis regulators such as BAX; transcription factors such as FOXP3; tumor suppressor proteins such as p53; structural proteins such as surfactant proteins; reprogramming factors such as OCT4, SOX2, c-MYC, KLF4, LIN28 or NANOG; genome-engineered proteins such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9); and blood proteins such as fibrinogen. For example, the pharmaceutically active peptide or protein may be a peptide or protein containing an immunogen, antigen, or antigenic peptide, wherein the peptide or protein may be processed after expression to produce said immunogen, antigen, or antigenic peptide. Alternatively, the peptide or protein itself may be an immunogen, an antigen, or an antigenic peptide.

Compositions and sets

In a further aspect, the present invention provides a composition or kit comprising a 5' cap compound of the present invention. Such a composition or kit can be used to provide RNA with the 5' cap structure of the present invention and/or to improve the stability of the RNA, for example in the corresponding methods disclosed herein. In one embodiment of this aspect, the kit may further contain reagents commonly used in in vitro transcription reactions (eg, NTP, RNA polymerase, one or more buffers, and/or DNA template) and/or instructions for use.

In a further aspect, the present invention provides a composition, preferably a pharmaceutical composition, comprising RNA (preferably mRNA) modified with a 5' cap compound of the present invention (such a composition comprising the RNA of the present invention is also referred to herein as an RNA composition of the present invention). The composition, in particular a pharmaceutical composition, in this aspect may contain RNA (preferably mRNA) modified with a 5'-cap compound of the present invention, in combination with one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition includes RNA (preferably mRNA) modified with a 5' cap compound of the present invention, one or more pharmaceutically acceptable excipients, and one or more additional/additional active compounds.

In an additional aspect, the present application provides a pharmaceutical composition, as defined herein, for use in therapy.

For example, particularly in those embodiments where the RNA modified by the 5' cap compound of the present invention includes a nucleotide sequence encoding a peptide or protein, the pharmaceutical compositions of the present invention can be used in protein replacement therapy, genomic engineering therapy, therapy genome reprogramming or immunotherapy.

Exemplary applications of protein replacement therapy for RNA or pharmaceutical compositions of the present invention include treatment (including prophylactic treatment) of a condition, disorder or disease caused by reduced activity of the peptide or protein, e.g., anemia (replacement protein: e.g., erythropoietin), diabetes (replacement protein: e.g. , vasopressin), congenital lung disease (replacement protein: e.g. surfactant protein B), asthma (replacement protein: e.g. FOXP3), myocardial infarction (replacement protein: e.g. VEGFA), melanoma (replacement protein: e.g. BAX), autoimmune diabetes (replacement protein: e.g. IL-4), autoimmune myocarditis (replacement protein: e.g. IL-10), inflammation (replacement proteins: e.g. P-selectin glycoprotein ligand-1 (PSGL-1)), Sialyl-Lewisx ( SLeX) and IL-10)), factor VII deficiency (replacement protein: e.g. factor VIIa), hemophilia A (replacement protein: e.g. factor VIII), hemophilia B (replacement protein: e.g. factor IX), factor X deficiency ( replacement protein: e.g. factor X), factor XI deficiency (replacement protein: e.g. factor XI), factor XIII deficiency (replacement protein: e.g. factor XIII), von Willebrand disease (replacement protein: e.g. von Willebrand factor), deficiency protein C (replacement protein: e.g. protein C), antithrombin deficiency (replacement protein: e.g. antithrombin III), fibrinogen deficiency (replacement protein: e.g. fibrinogen), hereditary angioedema (replacement protein: e.g. C1-esterase inhibitor), α1-PI deficiency (replacement protein: e.g. alpha-1 proteinase inhibitor), Gaucher disease (replacement protein: e.g. glucocerebrosidase), mucopolysaccharidosis I (replacement protein: e.g. alpha-L-iduronidase), mucopolysaccharidosis II (replacement protein: e.g. , iduronate sulfatase), mucopolysaccharidosis VI (replacement protein: e.g. N-acetylgalactosamine-4-sulfatase), mucopolysaccharidosis IVA (replacement protein: e.g. N-acetylgalactosamine-6-sulfatase), mucopolysaccharidosis IIIA (replacement protein: e.g. heparan sulfate sulfatase), diseases Fabry (replacement protein: e.g. alpha-galactosidase A), Pompe disease (replacement protein: e.g. alpha-glucosidase), Niemann-Pick disease type B (replacement protein: e.g. acid sphingomyelinase), alpha-mannosidosis (replacement protein: e.g. , alpha-mannosidase), metachromatic leukodystrophy (replacement protein: e.g. arylsulfatase A), LAL deficiency (replacement protein: e.g. lysosomal acid lipase (LAL)), sucrose-isomaltase deficiency (replacement protein: e.g. sucrose-isomaltase), ADA deficiency ( replacement protein: eg adenosine deaminase (ADA)), primary IGF-1 deficiency (replacement protein: eg insulin-like growth factor 1 (IGF-1)), hypophosphatasia (replacement protein: eg alkaline phosphatase) and acute intermittent porphyria (replacement protein : e.g. porphobilinogen deaminase).

Exemplary applications of genomic engineering therapy for RNA or pharmaceutical compositions of the present invention include treatment (including prophylactic treatment) of a condition, disorder or disease selected from the group consisting of X-linked severe combined immunodeficiency (X-SCID) (DNA corrected, encoding the common interleukin-2 receptor gamma chain (IL-2Rγ)), xeroderma pigmentosum (correction of native, i.e., non-mutant DNA), as well as the conditions, disorders and diseases noted above with respect to exemplary applications of protein replacement therapy. Additional genome engineering therapies for the RNA or pharmaceutical compositions of the present invention include genome editing using, for example, CRISPR/CAS.

Exemplary applications of genetic reprogramming therapy for RNA or pharmaceutical compositions of the present invention include treatment (including prophylactic treatment) of any of the conditions, disorders and diseases identified above with respect to exemplary applications of protein replacement therapy and/or exemplary applications of genomic engineering therapy.

Exemplary immunotherapeutic uses of the pharmaceutical compositions of the present invention include treatment (including prophylactic treatment) of a condition, disorder, or disease selected from the group consisting of infectious diseases (e.g., caused by a pathogen such as viruses (e.g., influenza virus (A, B, or C) , CMV or RSV), bacteria, fungi or other microorganisms); unwanted inflammation (eg, immune disorder); and cancer.

Cancer (medical term: malignancy) is a class of diseases in which a group of cells exhibit uncontrolled growth (dividing beyond normal limits), invasion (invasion and destruction of neighboring tissues), and sometimes metastasis (spread to other places in the body through lymph or blood). These three malignant properties of cancerous tumors distinguish them from benign tumors, which are self-limited, non-invasive and do not metastasize. Most cancers form a tumor, which is a tumor or lesion formed by abnormal growth of cells (called neoplastic cells or tumor cells), but some, such as leukemia, do not form tumors. The term “cancer” in accordance with the invention includes leukemia, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, hemoblastosis, skin cancer, malignant brain tumor, cervical cancer, colon cancer, liver cancer, colon cancer, stomach cancer, small intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose cancer and throat (ENT), breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, and their metastases. Examples of these are lung carcinomas, breast carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the types of cancer or tumors described above. The term "cancer" according to the invention also includes cancer metastases.

Examples of cancers that can be treated with the RNA and pharmaceutical compositions of the present invention include malignant melanoma, all types of carcinomas (colon, renal cell, bladder, prostate, non-small cell and small cell lung carcinoma, etc.), lymphomas , sarcomas, blastomas, gliomas, etc.

Malignant melanoma is a severe type of skin cancer. It is caused by the uncontrolled growth of pigment cells called melanocytes.

According to the invention, "carcinoma" is a malignant tumor originating from epithelial cells. This group represents the most common types of cancer, including common forms of breast, prostate, lung and colon cancer.

Lymphoma and leukemia are malignant neoplasms originating from hematopoietic (blood-forming) cells.

Sarcoma is a malignant tumor that arises from transformed cells in one of the many tissues that develop from the embryonic mesoderm. Thus, sarcomas include tumors of bone, cartilage, fat, muscle, vascular and hematopoietic tissue.

A blast tumor or blastoma is a tumor (usually malignant) that resembles immature or embryonic tissue. Many of these tumors most often occur in children.

A glioma is a type of tumor that develops in the brain or spinal cord. It is called glioma because it arises from glial cells. The most common site of appearance of gliomas is the brain.

“Metastasis” refers to the spread of cancer cells from the original site to another part of the body. The formation of metastases is a very complex process that depends on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration through the basement membranes of the endothelium into the body cavity and blood vessels, and then, after transport by blood, infiltration of target organs. Finally, the growth of a new tumor, that is, a secondary tumor or a metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis", which refers to metastasis distant from the primary tumor and the regional lymph node system.

Examples of immune disorders include autoimmune diseases (eg, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, sepsis and septic shock, inflammatory bowel disease, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug rashes, reversible leprosy reactions, erythema nodosum leprosy, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure erythrocyte anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, glomerulonephritis, idiopathic celiac disease, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, posterior uveitis and interstitial pulmonary fibrosis, graft-versus-host disease, transplant cases, and allergies such as atopic allergies; but are not limited to them.

Examples of viruses include human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) (eg, CMV5), human herpes viruses (HHV) (eg, HHV6, 7 or 8), herpes simplex viruses (HSV) , bovine herpes virus (BHV) (e.g. BHV4), equine herpes virus (EHV) (e.g. EHV2), human T-cell leukemia viruses (HTLV)5, varicella zoster virus (VZV), measles virus, papovaviruses (JC and BK), hepatitis viruses (eg, HBV or HCV), myxoma virus, adenovirus, parvoviruses, polyoma virus, influenza viruses (eg, influenza A virus, influenza B virus, or influenza C virus), respiratory syncytial virus (RSV), papillomaviruses and poxviruses such as, but not limited to, vaccinia virus and molluscum contagiosum virus (MCV), as well as lyssaviruses. Such a virus may or may not express an inhibitor of apoptosis. Examples of diseases caused by viral infection include, but are not limited to, chickenpox, cytomegalovirus infections, genital herpes, hepatitis B and C, influenza and shingles, and rabies.

Examples of bacteria include Campylobacter jejuni, Enterobacter species, Enterococcus faecium, Enterococcus faecalis, Escherichia coli (eg E. coli O157:H7), group A streptococci, Haemophilus influenzae, Helicobacter pylori, Listeria, Mycobacterium tuberculosis, Pseudomonas aeruginosa, S. pneumoniae, Salmonella, Shigella, Staphylococcus aureus and Staphylococcus epidermidis, as well as, but not limited to, Borrelia and Rickettsia. Examples of diseases caused by bacterial infection include anthrax, cholera, diphtheria, foodborne illnesses, leprosy, meningitis, peptic ulcers, pneumonia, sepsis, septic shock, syphilis, tetanus, tuberculosis, typhoid fever, urinary tract infections, Lyme disease, and spotted fever The Rocky Mountains, but not limited to them.

Specific examples of infectious diseases that can be treated with the RNA or pharmaceutical compositions of the present invention include viral infectious diseases such as AIDS (HIV), hepatitis A, B or C, herpes, herpes zoster (varicella), rubella (rubella virus ), yellow fever, dengue fever; infectious diseases caused by flaviviruses; flu; infectious diseases caused by RSV; infectious diseases caused by CMV; hemorrhagic infectious diseases (Marburg or Ebola viruses); bacterial infectious diseases (such as Legionnaires' disease (Legionella), stomach ulcers (Helicobacter), cholera (Vibrio), infections caused by E. coli, staphylococci, salmonella or streptococci (tetanus); infections caused by protozoan pathogens such as malaria, sleeping sickness , leishmaniasis, toxoplasmosis, that is, infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma, or fungal infections that are caused, for example, by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

For use according to the invention, particularly in the form of a pharmaceutical composition (eg a vaccine composition), the RNA may be deproteinized RNA or may be incorporated into a carrier, for example liposomes or other gene transfer particles, and preferably in the form of deproteinized RNA.

RNA (preferably mRNA) modified with a 5'-cap compound of the present invention, or pharmaceutical compositions of the present invention can be used alone or in combination with one or more additional/additional active compounds, which can be used before, simultaneously or after administration of the RNA or a pharmaceutical composition according to the present invention. Such one or more additional/additional active compounds include immunosuppressants (e.g., for applications in which the induction of an immune response should be avoided or minimized (e.g., protein replacement therapy, genome engineering therapy, and genetic reprogramming therapy, as described herein)) , nucleic acids (e.g. plasmids) containing a nucleotide sequence encoding a peptide or protein (particularly in genomic engineering therapy, where, for example, said nucleotide sequence must be inserted into the genome of a patient, for example, in order to replace the corresponding mutant nucleotide sequence in the patient's genome), cell differentiation compounds (e.g., compounds that induce the differentiation of cells having stem cell characteristics into cells expressing a peptide or protein (particularly a pharmaceutically active peptide or protein), particularly in genetic reprogramming therapy ), chemotherapy drugs for cancer patients (eg, gemcitabine, etopophos, cisplatin, carboplatin), antiviral agents, antiparasitic agents, antibacterial agents, immunotherapeutic agents (eg, antigens or fragments thereof (particularly immunogenic fragments thereof)) and adjuvants, and , if used simultaneously with the RNA of the present invention, may be present in the pharmaceutical composition of the present invention.

In particular, in the case of a vaccine composition, one or more additional/additional active compounds may include an immunotherapeutic agent, preferably an immunotherapeutic agent that induces or exhibits a targeted, ie specific, immune response. Thus, in one embodiment, the RNA and pharmaceutical compositions of the present invention can be used in combination with an immunotherapeutic agent, preferably an immunotherapeutic agent that induces or exhibits a targeted, that is, specific, immune response. Such immunotherapeutic agents include agents directed against a disease-associated antigen, such as therapeutic antibodies or agents that induce an immune response directed against a disease-associated antigen or cells expressing a disease-associated antigen. Suitable immunotherapeutic agents include proteins or peptides that induce a B cell or T cell response against a disease-associated antigen or cells expressing a disease-associated antigen. These proteins or peptides may contain a sequence substantially corresponding or identical to the sequence of a disease-associated antigen, or one or more fragments thereof. In one embodiment, the protein or peptide comprises an MHC presented peptide sequence derived from a disease associated antigen. Instead of introducing a protein or peptide, it is also possible to introduce a nucleic acid, preferably RNA, such as mRNA encoding the protein or peptide. The RNA encoding the protein or peptide may be RNA (preferably mRNA) modified with a 5' cap compound of the present invention. Alternatively or additionally, the RNA encoding the protein or peptide may be another RNA not in accordance with the present invention, which can be administered simultaneously (in which case the RNA may form part of the pharmaceutical composition of the invention) and/or before and/or after administration of the pharmaceutical composition according to invention. Accordingly, the pharmaceutical composition of the present invention can be used in genetic vaccination, where the immune response is stimulated by introducing into an individual a suitable nucleic acid molecule (DNA or mRNA) that encodes an antigen or a fragment thereof.

In one embodiment, the disease-associated antigen is a tumor-associated antigen. In this embodiment, the RNA (preferably mRNA) modified with the 5' cap compound of the present invention and the pharmaceutical compositions of the present invention may be useful in the treatment of cancer or cancer metastases. Preferably, the diseased organ or tissue is characterized by diseased cells, such as cancer cells, expressing a disease-associated antigen and/or having an association of a disease-associated antigen with their surface. Immunization with an intact or substantially intact tumor-associated antigen or fragments thereof, such as MHC class I and II peptides or nucleic acids, in particular the mRNA encoding such antigen or fragment, allows for the induction of an MHC class I and/or class II response and , thus stimulating T cells such as cytotoxic CD8+ T lymphocytes, which are capable of lysing cancer cells, and/or CD4+ T cells. Such immunization may also induce a humoral immune response (B cell response), resulting in the production of antibodies against the tumor associated antigen. In addition, antigen presenting cells (APCs), such as dendritic cells (DCs), can be loaded with MHC class I presented peptides directly or by transfection with nucleic acids encoding tumor antigens or tumor antigen peptides in vitro and administered to a patient.

According to the present invention, a tumor-associated antigen preferably includes any antigen that is characteristic of tumors or cancer, as well as tumor or cancer cells in terms of type and/or level of expression. In one embodiment, the term “tumor associated antigen” refers to proteins that under normal conditions, that is, in a healthy person, are specifically expressed in a limited number of organs and/or tissues or at certain stages of development, for example, a tumor associated antigen may in normal conditions specifically expressed in gastric tissue, preferably in the gastric mucosa, in reproductive organs, for example in the testes, in trophoblastic tissue, for example in the placenta or in germline cells, and is expressed or aberrantly expressed in one or more tumors or cancer tissues. In this context, "limited amount" preferably means no more than 3, more preferably no more than 2 or 1. Tumor associated antigens in the context of the present invention include, for example, differentiation antigens, preferably cell type specific differentiation antigens, i.e. e. proteins that are normally specifically expressed in a particular cell type at a particular stage of differentiation, cancer/testicular antigens, that is, proteins that are normally specifically expressed in the testes and sometimes in the placenta, and germline specific antigens. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell surface of the cancer cell and is preferably not expressed in normal tissues or expressed very rarely. Preferably, a tumor associated antigen or aberrant expression of a tumor associated antigen identifies cancer cells. In the context of the present invention, a tumor-associated antigen that is expressed by a cancer cell in an individual, for example a patient suffering from cancer, is preferably a self-protein in the individual. In preferred embodiments, the tumor-associated antigen in the context of the present invention is expressed under normal conditions, in particular, in a tissue or organ that is not essential, that is, in tissues or organs that, when damaged by the immune system, do not lead to the death of the individual, or in organs or structures of the body that are inaccessible or barely accessible to the immune system. In one embodiment, the amino acid sequence of the tumor-associated antigen is identical between a tumor-associated antigen that is expressed in normal tissues and a tumor-associated antigen that is expressed in cancerous tissues. Preferably, the tumor-associated antigen is presented in the context of MHC molecules by the cancer cell in which it is expressed.

Examples of differentiation antigens that ideally meet the criteria of tumor-associated antigens provided by the present invention as target structures in tumor immunotherapy, in particular in tumor vaccination, are cell surface proteins of the claudin family, such as CLDN6 and CLDN18.2. These differentiation antigens are expressed in tumors of various origins and are particularly suitable as target structures in connection with antibody-mediated cancer immunotherapy due to their selective expression (lack of expression in normal tissue where toxicity may occur) and plasma membrane localization.

Specific examples of antigens that can be used in the present invention are those that are explicitly mentioned herein, including p53 and WT-1.

The RNA or pharmaceutical compositions of the present invention are generally used in "pharmaceutically acceptable amounts" and in "pharmaceutically acceptable preparations." The term "pharmaceutically acceptable" refers to a non-toxic material that does not interfere with the action of the active agent(s) of the pharmaceutical composition.

A "therapeutically effective amount" refers to an amount that, alone or in combination with additional dosages, produces the desired response or effect. In the case of therapy for a specific disease or specific condition, the required response is associated with inhibition of the development of the disease. This includes slowing the progression of the disease, in particular stopping the progression of the disease. The desired response to therapy for a disease or condition may also be to delay the onset or inhibit the onset of the disease or condition. The effective amount of the composition of the present invention depends on the condition or disease, the severity of the disease, individual patient parameters, including age, physiological condition, height and weight, duration of treatment, type of optional concomitant therapy, specific route of administration and similar factors. If the patient's response to the initial dose is insufficient, higher dosages (or higher effective dosages, which can be achieved by a more localized route of administration) may be used. In general, for treatment of, or for inducing or enhancing an immune response in a human, dosages of RNA in the range of 1 ng to 700 μg, 1 ng to 500 μg, 1 ng to 300 μg, 1 ng to 200 μg are preferably prepared and administered. , or from 1 ng to 100 mcg.

According to the present invention, the use of RNA (such as mRNA) is either in the form of deproteinized nucleic acid or in combination with one or more pharmaceutically acceptable excipients. Preferably, the administration of nucleic acids is in the form of deproteinized nucleic acids. Preferably, the RNA is administered in combination with stabilizing agents such as RNase inhibitors. The present invention also provides for the reintroduction of nucleic acids into cells to ensure sustained expression over extended periods of time. However, due to the presence of the 5' cap structure of the present invention and possibly other stabilizing modifications, the RNAs of the present invention preferably exhibit the advantage that they can be administered less frequently than RNAs not containing the 5' cap structure of the present invention. inventions. Thus, the use of the RNA of the present invention preferably provides a benefit to the patient in that, for example, with respect to protein replacement therapy, fewer administrations (such as injections) of the RNA (or pharmaceutical compositions) of the present invention are required to achieve the desired effect (eg, expression of a desired peptide or protein in an amount sufficient to maintain patient function (eg, to maintain patient homeostasis)). Thus, in one embodiment, the RNA of the invention (e.g., an RNA composition or pharmaceutical composition of the invention) is administered to a patient (e.g., by injection, such as intraperitoneal, intramuscular, or intradermal injection) no more than once per day (i.e., the period of time between two administrations is at least 24 hours, for example at least 30 hours, at least 36 hours or at least 42 hours), preferably no more than once every two days (i.e. the period of time between two administrations is at least 48 hours, e.g. at least 54 hours, at least 60 hours or at least 66 hours), preferably no more than once every three days (i.e. the time period between two administrations is at least 72 hours , e.g., at least 78 hours, at least 84 hours, or 90 hours) or no more than once every four days (i.e., the time period between two administrations is at least 96 hours, e.g., at least 102 hours , at least 108 hours or at least 114 hours). Accordingly, the present invention is particularly useful for chronic patients and/or long-term ill patients, for example, patients who are being treated for a long period of time, for example, who receive the RNA of the invention (for example, an RNA composition or a pharmaceutical composition of the invention) for an extended period time, wherein the long period of time is preferably at least 1 week, for example at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years or at least 10 years, for example up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 12 months, up to 2 years, up to 3 years, or up to 4 years, up to 5 years, up to 10 years, or for the entire life of the patient. Thus, in one embodiment, the RNA of the present invention (e.g., an RNA composition or pharmaceutical composition of the present invention) is administered to a chronic or long-term ill patient (e.g., by injection, such as intraperitoneal, intramuscular, or intradermal injection) no more than once per day (i.e. the time period between two administrations is at least 24 hours, for example at least 30 hours, at least 36 hours or at least 42 hours), preferably no more than once every two days (i.e. i.e. the period between two administrations is at least 48 hours, for example at least 54 hours, at least 60 hours or at least 66 hours), preferably no more than once every three days (i.e. the period of time between two administrations is at least 72 hours, for example at least 78 hours, at least 84 hours or at least 90 hours) or no more than once every four days (that is, the period of time between two administrations is at least 96 hours, for example at least 102 hours, at least 108 hours or at least 114 hours) over a long period of time, in particular at least 1 week, for example at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least at least 2 years, at least 3 years, at least 4 years, at least 5 years or at least 10 years, for example up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 12 months, up to 2 years, up to 3 years or up to 4 years, up to 5 years, up to 10 years or throughout the life of a chronic or long-term ill patient.

Cells can be transfected with any excipients (particularly carriers) to which the RNA can be bound, for example by forming complexes with the RNA or the formation of vesicles in which the RNA is contained or encapsulated, resulting in increased stability of the RNA compared to deproteinized RNA . Excipients (in particular carriers) used according to the invention include, for example, lipid-containing carriers such as cationic lipids, liposomes, in particular cationic liposomes, micelles and nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. In accordance with the invention, any cationic lipid can be used. In addition, the cells can be taken from an individual, the cells can be transfected with RNA or a pharmaceutical composition of the invention, and the transfected cells can be administered to the individual.

Preferably, introduction of RNA encoding a peptide or polypeptide into a cell, in particular into a cell present in vivo, results in the expression of said peptide or polypeptide in the cell. In certain embodiments, targeting nucleic acids to specific cells is preferred. In such embodiments, the carrier that is used to introduce the nucleic acid into the cell (eg, a retrovirus or liposome) is a targeting molecule. For example, a molecule, such as an antibody, that is specific for a surface membrane protein on a target cell or a receptor ligand on a target cell may be included in or linked to a nucleic acid carrier. In the case where the nucleic acid is administered via liposomes, proteins that bind to surface membrane proteins associated with endocytosis may be included in the liposome composition to provide targeting and/or uptake. Such proteins include capsid proteins or fragments thereof that are specific for a particular cell type, antibodies against proteins that are internalized, proteins targeted to subcellular localization, etc.

In some embodiments of the present invention, the capped RNA described herein may be present in lipoplex RNA particles. Lipoplex RNA particles and compositions containing lipoplex RNA particles described herein are useful for delivering the capped RNA described herein to a target tissue after parenteral administration, particularly after intravenous administration. Lipoplex RNA particles can be prepared using liposomes, which can be prepared by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase contains acetic acid, for example, in an amount of about 5 mM. In one embodiment, the liposomes and lipoplex RNA particles contain at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid includes 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In one embodiment, the at least one additional lipid includes 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero -3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid includes 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid includes 1,2-di-(9Z-octadecenoyl)-sn - glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles contain 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) . Liposomes can be used to prepare lipoplex RNA particles by mixing liposomes with RNA. Specific lipoplex RNA particles targeting the spleen are described in WO 2013/143683, incorporated herein by reference. It has been discovered that lipoplex RNA particles having a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Accordingly, after administration of lipoplex RNA particles, RNA accumulation and/or RNA expression occurs in the spleen. Thus, the RNA lipoplex particles described herein can be used to express RNA in the spleen. In one embodiment, following administration of the lipoplex RNA particles, there is little or no accumulation of RNA and/or expression of RNA in the lungs and/or liver. In one embodiment, following administration of lipoplex RNA particles, RNA accumulation and/or RNA expression occurs in antigen presenting cells, such as professional antigen presenting cells in the spleen. Thus, the RNA lipoplex particles described herein can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

The term "excipient" as used herein is intended to refer to all substances in a pharmaceutical composition that are not active agents (e.g., which are therapeutically inactive ingredients that do not exhibit any therapeutic effect in the amount/concentration used), e.g., salts, carriers , binding agents, lubricants, thickeners, surfactants, dispersing agents, preservatives, emulsifiers, buffering agents, wetting agents, flavorings, coloring agents, stabilizing agents (such as RNase inhibitors) or antioxidants, all of which are preferably pharmaceutically acceptable.

"Pharmaceutically acceptable salts" include, for example, acid addition salts, which can, for example, be formed using a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid acid, tartaric acid, carbonic acid or phosphoric acid. In addition, suitable pharmaceutically acceptable salts may include alkali metal salts (eg, sodium or potassium salts); alkaline earth metal salts (eg calcium or magnesium salts); ammonium salts (NH ₄ ⁺ ); and salts formed with suitable organic ligands (eg, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkylsulfonate and arylsulfonate). Illustrative examples of pharmaceutically acceptable salts include acetate, adipate, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate , cyclopentane propionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisilate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptonate, hexano at, hexylresorcinate, hydrabamine, hydrobromide , hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalene sulfonate, napsylate, nicotinate, nitrate , N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, theoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66, pp. 1-19 (1977)). Salts that are not pharmaceutically acceptable can be used to prepare pharmaceutically acceptable salts and are included in the present invention.

The compositions of the present invention may contain a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, isotonic and absorption retarding agents, and the like that are physiologically compatible. The "pharmaceutically acceptable carrier" may be in the form of a solid, semi-solid, liquid, or combinations thereof.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, sterile non-aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active agents is known in the art. Unless any conventional vehicle or agent is incompatible with the active agent, it is intended for use in the pharmaceutical compositions of the present invention. Examples of pharmaceutically acceptable carriers for the injectable formulation include water, isotonic buffered saline (eg, Ringer's solution or Ringer-lactate), ethanol, polyols (eg, glycerol), polyalkylene glycols (eg, propylene glycol and liquid polyethylene glycol), hydrogenated naphthalenes, and in particular, biocompatible lactide polymers (for example, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers).

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Suitable buffering agents for use in the pharmaceutical compositions of the invention include acetic acid salt, citric acid salt, boric acid salt and phosphoric acid salt.

Suitable preservatives for use in the pharmaceutical compositions of the invention include various antibacterial and antifungal agents such as benzalkonium chloride, chlorobutanol, paraben, sorbic acid and merthiolate. Prevention of the presence of microorganisms can also be achieved by sterilization procedures (eg, sterilizing filtration), in particular sterilizing microfiltration).

The pharmaceutical composition of the present invention can be administered to an individual by any route, preferably parenterally. The expressions “parenteral administration” and “parenterally administered” as used herein mean methods of administration other than enteral administration (“enteral administration” and “enterally administered” as used herein mean that the administered drug enters the stomach and/or intestines). Parenteral administration is usually by injection and/or infusion and includes intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraosseous, intraorbital, intracardiac, intranodal, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intracerebral, intracerebroventricular , subarachnoid, intraspinal, epidural, intrasternal and local administration, but not limited to them. For applications other than immunotherapy (eg, protein replacement therapy, genome engineering therapy, or genetic reprogramming therapy), the pharmaceutical composition of the invention is administered intraperitoneally, intramuscularly, or intradermally. For immunotherapeutic applications, the pharmaceutical composition of the invention is preferably administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intralymphatically, intradermally or intranodally, more preferably intradermally or intranodally, for example, by intranodal injection.

The pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by one skilled in the art, the route and/or method of administration will vary depending on the desired results.

The active agents (i.e., the RNA of the invention and, if necessary, one or more additional/additional active compounds) can be formulated with carriers that will protect the compounds from rapid release, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

To administer the active agent (i.e., the RNA of the invention and, optionally, one or more additional/additional active compounds) by certain routes of administration, it may be necessary to coat the active agent or co-administer the compound with the material to prevent its inactivation and/or enhance the effectiveness of the active agent (in particular, RNA according to the invention) to be translated. For example, the active agent can be administered to the individual in a suitable vehicle, for example, lipid-containing carriers (particularly cationic lipids), liposomes (such as CGF water-in-oil-in-water emulsions, as well as conventional liposomes (Strejan et al. , J. Neuroimmunol. 7: 27 (1984)), in particular cationic liposomes), micelles, nanoparticles in which RNA is contained or encapsulated, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

Pharmaceutical compositions generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerin, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils such as olive oil, and organic esters for injections such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of a coating material such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases it will be preferable to include isotonic agents such as sugars, polyols such as mannitol, sorbitol, or sodium chloride in the pharmaceutical composition. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying agent in the composition, for example, monostearate salts and gelatin.

Typically, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains the basic dispersion medium and other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization (lyophilization), which provide the powder of the active agent plus any additional required ingredient from its solution, previously sterilized by filtration.

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the needs of the therapeutic situation. It is particularly preferable to formulate the pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit dosages for individuals to be treated; each unit contains a predetermined amount of active agent calculated to achieve the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification of unit dosage forms of the present invention is dictated by and directly depends on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the field of preparation of such an active agent for the treatment of sensitivity in individuals. The amount of active agent (particularly the amount of RNA) that can be combined with the carrier material to form a pharmaceutical composition (such as a single dose dosage form) will vary depending on the individual being treated and the particular route of administration. The amount of active agent that can be combined with the carrier material to form a single-dose dosage form will generally be that amount of the composition that produces a therapeutic effect.

Typically, from 100% (for pharmaceutical formulations/compositions), the amount of active agent (specifically, the amount of RNA of the present invention, optionally together with one or more additional/additional active compounds if present in the pharmaceutical formulations/compositions) will be from about 0.01% to 99%, preferably from about 0.1% to 70%, most preferably from about 1% to 30%, with the remainder preferably consisting of one or more pharmaceutically acceptable excipients.

The amount of active agent, e.g., RNA of the present invention, in unit dosage form and/or when administered to an individual or used in therapy may vary from about 0.001 mg to 1000 mg (e.g., from about 0.01 mg to 500 mg, from about 0.1 mg to 100 mg, for example about 1 mg to 50 mg) per unit, administration or therapy. In some embodiments, the appropriate amount of such active agent can be calculated using the weight or surface area of the human body, including amounts from about 0.1 mg/kg to 10 mg/kg (for example, from about 0.2 mg/kg to 5 mg /kg) or about 0.1 mg/m ² to 400 mg/m ² (for example, about 0.3 mg/m ² to 350 mg/m ² or about 1 mg/m ² to 200 mg/m ² ).

Regardless of the route of administration chosen, the active agents (i.e., RNA and possibly one or more additional/additional active compounds) that can be used in a suitable hydrated form and/or pharmaceutical compositions of the present invention are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art (see, for example, Remington, "The Science and Practice of Pharmacy" edited by Allen, Loyd V., Jr., 22nd edition, Pharmaceutical Sciences, September 2012; Ansel et al., "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7th edition, Lippincott Williams & Wilkins Publishers, 1999).

The actual dosage levels of the active agents in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active agent that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without toxicity to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the potency of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of elimination of the particular active agent employed, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular the compositions used, the age, sex, body weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe an effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may begin with doses of the active agents used in a pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, a suitable daily dose of a pharmaceutical composition of the invention will be that amount of active agent that is the lowest dose effective to produce a therapeutic effect. Such effective dose will generally depend on the factors described above. Preferably, administration is parenteral, such as intravenous, intramuscular, intraperitoneal or subcutaneous, preferably proximal to the target site. Administration can also be intratumoral. If necessary, an effective daily dose of the pharmaceutical composition can be administered in two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, if necessary in unit dosage forms. Although it is possible to administer the active agent (particularly RNA) of the present invention separately, it is preferable to administer the active agent in the form of a pharmaceutical formulation/composition.

In one embodiment, the RNA or pharmaceutical compositions of the present invention can be administered by infusion, preferably a slow continuous infusion over an extended period, for example, more than 24 hours, to reduce toxic side effects. Administration may also be by continuous infusion over a period of 2 to 24 hours, for example 2 to 12 hours. This regimen can be repeated one or more times as needed, for example after 6 or 12 months.

The pharmaceutical composition of the invention can be prepared for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations may be provided in unit dosage forms (eg, vial, multi-dose container) and with an added preservative. The pharmaceutical composition of the present invention may take forms such as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing or dispersing agents. Alternatively, the agent may be in powder form for mixing with a suitable vehicle (eg, sterile pyrogen-free water) prior to use. Typically, pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to relieve pain at the injection site. Typically, the ingredients are supplied either individually or as a mixture in a unit dosage form, such as a dry lyophilized powder or an anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. If the pharmaceutical composition is to be administered by infusion, it may be dispensed in the form of an infusion bottle containing sterile water or pharmaceutical grade saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

Pharmaceutical compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, the pharmaceutical composition of the invention can be administered using a needle-less hypodermic injection device, such as the devices described in US Pat. Nos. 5,399,163; US 5,383,851; US 5,312,335; US 5,064,413; US 4,941,880; US 4,790,824; or US 4,596,556. Examples of well-known implants and modules used in the present invention include those described in US Pat. No. 4,487,603, which discloses an implantable microinfusion pump for controlled rate drug dispensing; US 4,486,194, which discloses a therapeutic device for administering drugs through the skin; US 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; US 4,447,224, which discloses an implantable controlled-flow infusion device for continuous drug delivery; US Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system.

Many other such implants, delivery systems and modules are known to those skilled in the art. In some embodiments, the RNA or pharmaceutical compositions of the invention may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the RNA or pharmaceutical compositions of the invention cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, US 4,522,811; US 5,374,548; and US 5,399,331. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs and thus improve targeted drug delivery (see, for example, V. V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Examples of target groups include folate or biotin (see, for example, US 5,416,016, Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); and surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134).

In one embodiment of the invention, the RNA of the invention is formulated in liposomes. More preferably, the liposomes include a targeting moiety. In the most preferred embodiment, the RNA in the liposomes is delivered by bolus injection to a site proximal to the desired area. Such a liposome-based composition must be fluid to the extent that it can be easily drawn up through a syringe, must be stable under manufacturing and storage conditions, and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

A "therapeutically effective amount" for treatment can be measured by objective responses, which may be complete or partial. Complete response (CR) is defined as the absence of clinical, radiological or other evidence of a condition, disorder or disease. A partial response (PR) is the result of a reduction in incidence of more than 50%. The average time to progression is an indicator characterizing the durability of the objective response.

A "therapeutically effective amount" for a treatment can also be measured by its ability to stabilize the progression of a condition, disorder or disease, for example using appropriate animal model systems and/or in vitro assays known to one of ordinary skill in the art. A therapeutically effective amount of an active agent (in particular, RNA of the invention) refers to an amount that provides the desired response or effect, alone or together with additional doses. In the case of treating a particular disease or condition, the desired response preferably relates to inhibition of the progression of the disease. This includes slowing the progression of the disease and, in particular, interrupting or regressing the progression of the disease. The necessary response in treating a disease or condition may also be to delay the onset or prevent the onset of said disease or condition. Thus, a therapeutically effective amount of an active agent can treat, treat, alleviate, attenuate, modify, regulate, neutralize, ameliorate, or affect a condition, disorder, or disease, or symptoms of a condition, disorder, or disease, or a susceptibility to a condition, disorder, or disease, in individual. One of ordinary skill in the art will be able to determine such amounts based on factors such as the disease, disorder or condition being treated, the severity of the disease, disorder or condition, characteristics of the patient being treated (including age, physiological condition, body size and weight), duration of treatment, type of concomitant therapy (if any), specific route of administration and similar factors. Accordingly, the administered doses of the active agents described herein may depend on various such parameters. In the event that the individual/patient responds insufficiently at the initial dose, higher doses (or, in fact, higher doses achieved by another, more localized route of administration) can be used.

The pharmaceutical composition of the present invention may be in the form of a vaccine preparation containing an RNA of the present invention encoding at least one antigen, such as an antigen as described above, or a fragment thereof (in particular an immunogenic fragment thereof).

The pharmaceutical composition of the present invention may also, if desired, be presented in a package, kit or dosing device, which may contain one or more unit dosage forms comprising the active agent (i.e. RNA and optionally one or more additional/additional active compounds). The packaging may, for example, comprise metal or plastic foil, such as a blister pack. Instructions for use may be included with the package, kit or dispenser.

One or more additional/additional active compounds may include an immunomodulatory agent such as anti-CTL-A4 or anti-PD1 or anti-PDL1, or anti-regulatory T cell reagents such as anti-CD25 antibody or cyclophosphamide.

The pharmaceutical compositions of the invention may be administered together with additional immune enhancing substances, such as one or more adjuvants, and may contain one or more immune enhancing substances to further enhance their effectiveness, preferably to achieve a synergistic immunostimulating effect.

The term "adjuvant" refers to compounds that, when administered in combination with an antigen, antigenic peptide or nucleic acid (such as RNA, preferably mRNA) encoding said antigen or antigenic peptide to an individual, prolong or enhance or accelerate the immune response. In the context of the present invention, RNA (preferably mRNA) can be administered with any adjuvants. Adjuvants are believed to exert their biological activity through one or more mechanisms, including increasing the surface area of the antigen, prolonging the retention of the antigen in the body, delaying the release of the antigen, targeting the antigen to macrophages, increasing the uptake of the antigen, enhancing the processing of the antigen, stimulating the release of cytokines, stimulation and activation immune cells such as B cells, macrophages, dendritic cells, T cells, and nonspecific activation of immune cells. For example, compounds that mediate DC maturation, such as lipopolysaccharides or CD40 ligand, form a class of suitable adjuvants. In general, any agent that affects the immune system in a "danger signal" type (LPS, GP96, dsRNA, etc.) or cytokines such as GM-CSF can be used as an adjuvant that allows the immune response to be enhanced, and/or influences it in a controlled manner. CpG oligodeoxynucleotides (Krieg et al., 1995, Nature 374: 546-549) can also be used in this context if necessary. Additional types of adjuvants include oil emulsions (eg, Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), liposomes, immunostimulatory complexes, cytokines (eg, monokines, lymphokines, interleukins, or chemokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN- γ, GM-CSF, LT-α or growth factors, e.g. hGH), lipopeptides (e.g. Pam3Cys). However, in the event that the RNA (preferably mRNA) of the invention in one embodiment may encode an immunostimulatory agent, and said immunostimulatory agent encoded by said RNA is to act as a primary immunostimulator, only a relatively small amount of CpG DNA is needed (compared to immunostimulation by CpG alone DNA). Examples of adjuvants are monophosphoryl lipid-A (MPL SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants, complete Freund's adjuvants, vitamin E, montanide, alum, CpG oligonucleotides and various water-in-oil emulsions that are prepared from biodegradable oils such as squalene and/or tocopherol. Particularly preferred adjuvants are cytokines such as monokines, lymphokines, interleukins or chemokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN-γ, GM-CSF, LT-α, growth factors such as hGH or lipopeptides such as Pam3Cys, all of which are suitable for use as adjuvants in the pharmaceutical compositions of the present invention, or when the RNA (in particular, mRNA) of the present invention is used in therapy.

Treatment can be done at home, in a doctor's office, clinic, clinic, or hospital. Typically, treatment begins under the supervision of a physician so that medical personnel can closely monitor the results of treatment and make necessary adjustments. The duration of treatment depends on the age and condition of the patient, as well as how the patient responds to treatment.

An individual who is at higher risk of developing a condition, disorder, or disease may receive prophylactic treatment to suppress or delay the onset of symptoms of the condition, disorder, or disease.

The term “treatment” is known to one of ordinary skill in the art and includes the use or administration of an active agent (e.g., a pharmaceutical composition containing said active agent) as described herein (e.g., RNA, such as mRNA, or a pharmaceutical composition containing RNA , such as mRNA) or a procedure for an individual/patient, or the use or administration of an active agent (e.g., a pharmaceutical composition containing said active agent) as described herein (e.g., RNA, such as mRNA, or a pharmaceutical composition containing RNA , such as mRNA), or a procedure with a cell, cell culture, cell line, sample, tissue or organ isolated from a person who has a condition, disorder or disease, a symptom of a condition, disorder or disease, or a predisposition to a condition, disorder or disease disease, for the purpose of treating, curing, ameliorating, mitigating, modifying, adjusting, neutralizing, improving or influencing or preventing a condition, disorder or disease, symptoms of a condition, disorder or disease, or a predisposition to a condition, disorder or disease (for example, to prevent or eliminating a disease, including reducing the size of a tumor or the number of tumors in an individual; stopping or slowing the progression of a disease in an individual; suppressing or slowing the development of a new disease in an individual; reducing the frequency or severity of symptoms and/or relapses in an individual who currently or previously had the disease; and/or extension, i.e. increasing the life expectancy of an individual). In particular, the term “treating a disease” includes treating, shortening the duration of, alleviating, preventing, slowing, or inhibiting the progression or worsening of, or preventing or delaying the onset of a disease or its symptoms. Therefore, the term “treatment” may include prophylactic treatment of a condition, disorder or disease, or a symptom of a condition, disorder or disease. The active agent, when used in treatment, includes the RNA of the invention, as well as one or more additional/additional active compounds described herein, and includes other therapeutically active compounds, which may be small molecules, peptides, peptidomimetics, polypeptides/proteins, antibodies, other polynucleotides such as DNA or dsRNA, cells, viruses, ribozymes and antisense oligonucleotides, but not limited to.

In a preferred embodiment, the pharmaceutical composition of the present invention contains substantially no dsRNA, preferably substantially free of dsRNA and DNA.

The term "substantially free of dsRNA" as used herein in connection with an RNA preparation containing RNA modified with a 5' cap compound according to the present application, in particular a pharmaceutical composition, especially a pharmaceutical composition containing RNA modified with a 5' cap compound according to the present application of the present invention, means that the amount of dsRNA in the RNA preparation or pharmaceutical composition is such that the RNA preparation or pharmaceutical composition, when administered to an individual, does not substantially cause an undesirable response (such as undesirable induction of inflammatory cytokines (for example, IFN-α) and/or undesirable activation of an effector enzyme leading to inhibition of protein synthesis from RNA according to the invention) in the specified individual. Preferably, the terms “substantially free of dsRNA” and “substantially free of an adverse response” mean that, when administered to an individual, the RNA preparation or pharmaceutical composition results in translation of the RNA into a peptide or protein within at least 10 hours (e.g., at least at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 78 hours, at least 84 hours, at least 90 hours or at least 96 hours) after application. For example, the content of dsRNA in the RNA preparation or pharmaceutical composition may be at most 5 wt. % (preferably at most 4 wt.%, at most 3 wt.%, at most 2 wt.%, at most 1 wt.%, at most 0.5 wt.%, at most 0.1 wt.%, at most at most 0.05 wt%, at most 0.01 wt%, at most 0.005 wt%, at most 0.001 wt%), based on the total weight of said RNA preparation or pharmaceutical composition.

The term "substantially free of DNA" as used herein in connection with an RNA preparation containing RNA modified with a 5'-cap compound of the present invention, in particular a pharmaceutical composition, especially a pharmaceutical composition containing RNA modified with 5 The '-cap compound of the present invention means that the amount of DNA in the RNA preparation or pharmaceutical composition can be at most 5 wt. % (preferably at most 4 wt.%, at most 3 wt.%, at most 2 wt.%, at most 1 wt.%, at most 0.5 wt.%, at most 0.1 wt.%, at most at most 0.05 wt%, at most 0.01 wt%, at most 0.005 wt%, at most 0.001 wt%), based on the total weight of said RNA preparation or pharmaceutical composition.

The term "substantially free of dsRNA and DNA" as used herein in combination with an RNA preparation containing RNA modified with a 5'-cap compound of the present invention, in particular a pharmaceutical composition, especially a pharmaceutical composition containing RNA modified with a 5'-cap cap compound of the present invention means that said RNA preparation or pharmaceutical composition is substantially free of dsRNA as defined above (e.g., translation lasts at least 10 hours after administration and/or dsRNA content is at most 5 wt.%) and contains virtually no DNA as stated above (eg DNA content is at most 5% by weight).

In one embodiment, the pharmaceutical composition of the invention is a vaccine composition.

The vaccine composition of the present invention can be considered as a pharmaceutical composition of the present invention for a specific use (eg, vaccination). Thus, one or more of the features and embodiments described above in connection with the pharmaceutical composition of the present invention (e.g., route of administration; presence of other components (such as one or more pharmaceutically acceptable carriers, excipients and/or diluents and/or adjuvants and/or one or more additional/additional active compounds); amount of active agent(s); pharmaceutically acceptable salts, etc.) may also be applied to the vaccine composition of the present invention.

In a preferred embodiment, said vaccine composition further contains one or more pharmaceutically acceptable carriers, excipients and/or diluents. Said vaccine composition may further contain compounds or substances that are capable of enhancing and/or maintaining the immune response in humans. For example, the vaccine composition of the present invention may further contain an adjuvant, as described above, or cytokines, such as interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-18 (IL-18). In addition, the vaccine composition of the present invention may further contain RNA stabilizing agents such as RNase inhibitors, pharmaceutically acceptable salts or buffers, preservatives (such as benzalkonium chloride, chlorobutanol, paraben or merthiolate), wetting agents, emulsifying agents and/or additional drugs or active agents.

In a particularly preferred embodiment, the RNA is present in the vaccine composition of the present invention in the form of "deproteinized" RNA.

Particularly preferably, the vaccine composition of the present invention is formulated for parenteral administration, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, intralymphatic, intradermal or intranodal administration, more preferably for intradermal or intranodal administration, such as intranodal injection. The vaccine compositions of the present invention are most preferably formulated for injection into the lymph nodes, preferably the inguinal lymph nodes, for injection into the lymphatic vessels and/or the spleen.

Preferably, the vaccine composition is in the form of an aqueous or non-aqueous solution that is isotonic with the recipient's blood, i.e. the individual who should be vaccinated. For example, Ringer's solution, isotonic sodium chloride solution, or phosphate buffered saline (PBS) can be used. In particular, the vaccine composition is preferably sterile and contains the above RNA in a therapeutically effective amount.

In a preferred embodiment, the vaccine composition is substantially free of dsRNA, preferably substantially free of dsRNA and DNA.

Cells

In another aspect, the present invention provides a cell containing RNA that is modified with a 5' cap compound of the present application, wherein the RNA preferably contains a nucleotide sequence encoding a peptide or protein. In this preferred embodiment, a cell where the RNA of the invention contains a nucleotide sequence encoding a peptide or protein, the cell can be used to produce said peptide or protein, for example in a corresponding peptide or protein production method described herein, or to express said peptide or protein in an individual by introducing said cell into the individual, for example, in the appropriate peptide or protein expression method described herein.

In a preferred embodiment, the cell is an antigen presenting cell, such as a mature antigen presenting cell, and may be selected from the group consisting of macrophages, monocytes, B cells and dendritic cells.

In a particularly preferred embodiment, the cell of the present invention is formulated into a pharmaceutical composition as described above, wherein said pharmaceutical composition is preferably suitable for expressing a peptide or protein, such as a pharmaceutically active peptide or protein. In an alternative embodiment, the cell of the present invention is formulated as a pharmaceutical composition as described above, wherein said pharmaceutical composition is preferably suitable for inducing an immune response when administered to an individual, the immune response being preferably directed against a protein or peptide encoded by the RNA or antigen, and/or an immunogen consisting of a protein or peptide encoded by RNA present in an immature antigen presenting cell of the present invention. Thus, the present invention provides a pharmaceutical composition containing an immature antigen presenting cell in accordance with the third aspect of the present invention.

Methods and use

In one aspect, the present invention provides a method for producing RNA with a 5' cap structure, wherein the method comprises performing a transcription reaction using a template nucleic acid in the presence of a 5' cap compound from the first aspect. In one embodiment, the template nucleic acid is DNA. The transcription reaction can be carried out in vivo or in vitro, but is preferably carried out in vitro. In one embodiment, the transcription reaction is carried out using an RNA polymerase selected from the group consisting of T3, T7 and SP6 RNA polymerases. The RNA may contain a nucleotide sequence encoding a peptide or protein, where the peptide or protein is preferably a pharmaceutically active peptide or protein as described herein. In one embodiment, the method is carried out in the absence of 2'-O-ribose methyltransferase. In an alternative embodiment, the method is carried out in the presence of 2'-O-ribose methyltransferase.

In another aspect, the present invention provides a method of increasing the stability of RNA in cells and/or increasing the expression of RNA in cells, wherein said method comprises providing said RNA with a structure according to formula (I) as defined in the first aspect; and transferring said RNA modified with the structure according to formula (I) into cells. Preferably, said cells are antigen presenting cells, such as immature antigen presenting cells, preferably selected from the group consisting of monocytes, macrophages, glial cells, B cells and dendritic cells. To assess the stability of RNA in an immature antigen-presenting cell, one can detect the presence of the RNA of interest or quantify the RNA in the cell at specific time points after administration of the RNA, for example using real-time RT-PCR. RNA expression in cells can be determined using RNA encoding a marker protein such as luciferase or green fluorescent protein, preferably d2EGFP, but any other marker protein known to one skilled in the art can be used and determining the expression of said marker protein at specific times. time points after RNA injection. In one embodiment, the step of providing said RNA structure according to formula (I) is carried out in the absence of 2'-O-ribose methyltransferase. In an alternative embodiment, the method is carried out in the presence of 2'-O-ribose methyltransferase.

In a further aspect, the present invention provides a method for producing a peptide or protein of interest, comprising the step of using an RNA, RNA composition or cell of the invention, wherein in each case the RNA contains a nucleotide sequence encoding the peptide or protein. In one embodiment, the peptide or protein is a pharmaceutically active protein, preferably selected from the group consisting of cytokines such as erythropoietin; adhesion molecules such as integrin; immunoglobulins; immunologically active compounds, e.g. antigens, such as tumor-associated antigens, pathogen-associated antigens (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) viral antigens, such as one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) influenza virus antigens (A, B or C), CMV or RSV), allergens or autoantigens; hormones such as vasopressin, insulin or growth hormone; growth factors such as VEGFA; enzymes such as herpes simplex virus thymidine kinase type 1 (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes or lactase; receptors such as growth factor receptors; protease inhibitors such as alpha-1-antitrypsin; apoptosis regulators such as BAX; transcription factors such as FOXP3; tumor suppressor proteins such as p53; structural proteins such as surfactant proteins; reprogramming factors such as OCT4, SOX2, c-MYC, KLF4, LIN28 or NANOG; genome-engineered proteins such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9); and blood proteins such as fibrinogen. In one embodiment of a method using RNA or an RNA composition, the method may include the step of transferring said RNA or RNA composition into a cell. In this regard, any method that is suitable for transferring RNA into cells can be used. Preferably, the RNA is transfected into cells by standard methods as described herein, for example, calcium phosphate precipitation, DEAE transfection, electroporation, lipofection or microinjection. The cell can be any cell that can be transfected with RNA, and is preferably an antigen presenting cell, such as an immature antigen presenting cell, more preferably selected from the group consisting of macrophages, monocytes, B cells and dendritic cells. The method of obtaining a peptide or protein of interest can be carried out in vivo or in vitro, but is preferably carried out in vitro.

In a further aspect, the present invention provides a method of expressing a peptide or protein in an individual, comprising the step of administering to said individual an RNA, RNA composition, or cell of the invention, wherein in each case the RNA contains a nucleotide sequence encoding the peptide or protein. In one embodiment, the peptide or protein is a pharmaceutically active protein, preferably selected from the group consisting of cytokines such as erythropoietin; adhesion molecules such as integrin; immunoglobulins; immunologically active compounds, for example antigens, such as tumor-associated antigens, pathogen-associated antigens (for example, one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) virus antigens, such as one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) influenza virus antigens (A, B or C), CMV or RSV), allergens or autoantigens; hormones such as vasopressin, insulin or growth hormone; growth factors such as VEGFA; enzymes such as herpes simplex virus thymidine kinase type 1 (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes or lactase; receptors such as growth factor receptors; protease inhibitors such as alpha-1-antitrypsin; apoptosis regulators such as BAX; transcription factors such as FOXP3; tumor suppressor proteins such as p53; structural proteins such as surfactant proteins; reprogramming factors such as OCT4, SOX2, c-MYC, KLF4, LIN28 or NANOG; genome-engineered proteins such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9); and blood proteins such as fibrinogen. The RNA, RNA composition or cell of the invention can be administered by any route, for example, as described above with respect to the pharmaceutical compositions of the invention.

In additional aspects, the present invention provides (i) an RNA, RNA composition, or cell of the invention for use in therapy, particularly for use in a method of treating a disease or disorder in a subject; (ii) a method of treating a disease or disorder in a subject, comprising the step of administering to said subject an RNA, RNA composition, or cell of the invention; and (iii) using the RNA, RNA composition or cell of the invention to prepare a medicament for treating a disease or disorder in a subject, wherein each of (i) to (iii) the RNA comprises a nucleotide sequence encoding a peptide or protein, which preferably is a peptide or protein associated with a disease. In one embodiment of these aspects (i) to (iii), the treatment of the disease or disorder is selected from the group consisting of protein replacement therapy, genomic engineering, genetic reprogramming, and immunotherapy, as described herein. Preferably the peptide or protein is a pharmaceutically active protein, more preferably selected from the group consisting of cytokines such as erythropoietin; adhesion molecules such as integrin; immunoglobulins; immunologically active compounds, e.g. antigens, such as tumor-associated antigens, pathogen-associated antigens (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) viral antigens, such as one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) influenza virus antigens (A, B or C), CMV or RSV), allergens or autoantigens; hormones such as vasopressin, insulin or growth hormone; growth factors such as VEGFA; enzymes such as herpes simplex virus thymidine kinase type 1 (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes or lactase; receptors such as growth factor receptors; protease inhibitors such as alpha-1-antitrypsin; apoptosis regulators such as BAX; transcription factors such as FOXP3; tumor suppressor proteins such as p53; structural proteins such as surfactant proteins; reprogramming factors such as OCT4, SOX2, c-MYC, KLF4, LIN28 or NANOG; genome-engineered proteins such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9); and blood proteins such as fibrinogen. In one embodiment, the disease or disorder is selected from the diseases and disorders disclosed herein, for example, the exemplary diseases and disorders described herein with respect to protein replacement therapy, genome engineering therapy, genetic reprogramming therapy, and/or immunotherapy. The RNA, RNA composition or cell of the invention can be administered in any route and/or in any mode or amount, for example those routes and/or modes and/or amounts described above with respect to the pharmaceutical compositions of the invention. In one embodiment, the RNA, RNA composition, or cell of the invention is administered to a subject (e.g., by injection, such as intraperitoneal, intramuscular, or intradermal injection) no more than once per day, preferably no more than once every two days, preferably no more than once every three days or no more than once every four days. In one embodiment, the RNA, RNA composition, or cell of the invention is administered to a chronic or long-term ill patient (e.g., by injection, such as intraperitoneal, intramuscular, or intradermal injection) over an extended period of time, in particular at least 1 week, e.g. at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years or at least 10 years, e.g. up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 12 months, up to 2 years, up to 3 years or up to 4 years, up to 5 years, up to 10 years or throughout the patient's life. In one embodiment, the RNA of the present invention (e.g., an RNA composition or pharmaceutical composition of the present invention) is administered to a chronic or long-term disease patient (e.g., by injection, such as intraperitoneal, intramuscular, or intradermal injection) at most once per day ( i.e. the time period between two administrations is at least 24 hours, for example at least 30 hours, at least 36 hours or at least 42 hours), preferably no more than once every two days (i.e. the period of time between two administrations is at least 48 hours, for example at least 54 hours, at least 60 hours or at least 66 hours), preferably no more than once every three days (i.e. the period of time between two administrations is at least 72 hours, for example at least 78 hours, at least 84 hours or at least 90 hours) or no more than once every four days (that is, the time period between two administrations is at least 96 hours, e.g. at least 102 hours, at least 108 hours, or at least 114 hours) over a long period of time, i.e. at least 1 week, e.g. at least 2 weeks, at least 3 weeks, at least at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years or at least 10 years, for example up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2 months, up to 3 months , up to 4 months, up to 5 months, up to 6 months, up to 12 months, up to 2 years, up to 3 years or up to 4 years, up to 5 years, up to 10 years or for the entire life of the patient.

In additional aspects, the present invention provides the following:

(I) A method of inducing an immune response in an individual is provided, comprising the step of administering to said individual a vaccine composition of a second aspect or an immature antigen presenting cell of a third aspect. Preferably, said immune response is specifically directed against a protein or peptide encoded by an RNA included in the vaccine composition or an immature antigen-presenting cell of the present invention, or is specifically directed against an antigen contained in said protein or peptide. Said immune response may be therapeutic and/or protective. Particularly preferably, said vaccine composition and said immature antigen presenting cells, preferably immature dendritic cells, are administered parenterally as indicated above for the second aspect of the present invention, preferably by intranodal injection, preferably by injection into the inguinal lymph nodes. In one embodiment, the method is designed to induce an immune response against a virus, such as a response against influenza virus (A, B or C), CMV or RSV.

(II) A method is provided for increasing the proportion of MHC molecules that represent an antigen of interest (e.g., an antigen of a virus such as influenza virus (A, B, or C), CMV, or RSV) on the surface of an antigen-presenting cell, wherein the method includes providing an RNA containing a nucleotide sequence encoding a peptide or protein containing said antigen of interest (for example, an antigen of a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA is modified with a structure according to formula (I) , as defined in the first aspect; and transferring said RNA modified with a structure corresponding to formula (I) into an immature antigen-presenting cell. In one embodiment, the step of providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (for example, an antigen virus such as influenza virus (A, B or C), CMV or RSV), or an antigenic peptide thereof, wherein said RNA modified with a structure according to formula (I), is performed in the absence of 2'-O-ribose methyltransferase. In an alternative embodiment, this step is carried out in the presence of 2'-O-ribose methyltransferase. Without being limited to any theory, it is believed that modification of RNA with the 5' cap compound of the present invention increases the stability and/or expression of said RNA, particularly in immature antigen presenting cells, for example, immature dendritic cells. This increased stability and/or expression results in the accumulation of the protein or peptide encoded by said RNA. Said protein or peptide may comprise an antigen or an antigenic peptide. Thus, after processing of the specified protein, antigens or antigenic peptides can be loaded onto MHC molecules on the surface of the antigen presenting cell. Alternatively, the protein or peptide may itself be an antigen or antigenic peptide and may be loaded onto MHC molecules without processing. An RNA encoding a specific protein or peptide containing an antigen or an antigenic peptide that has been modified with a 5' cap compound of the present invention is believed to increase the proportion/fraction of MHC molecules on the cell surface of an antigen presenting cell that present a peptide derived from the protein or peptide encoded by the RNA, compared to the same RNA having a conventional 5' cap structure, preferably compared to a reference RNA, such as the same RNA having an ARCA 5' cap structure. Since the density of MHC molecules presenting a particular antigen on the surface of an antigen-presenting cell is critical to the intensity of the induced immune response specific for that particular antigen, it is hypothesized that increasing the stability of the antigen-coding RNA that has been introduced into the antigen-presenting cells results in an enhanced immune response against specified specific antigen.

(III) A method is provided for stimulating and/or activating effector immune cells, comprising producing RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (for example, an antigen of a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA is modified in structure according to formula (I) as defined in the first aspect; transferring said RNA modified by the structure of formula (I) into immature antigen-presenting cells; and bringing antigen-presenting cells into contact with immune effector cells. Preferably, said immune effector cells are activated and/or stimulated in an antigen-specific manner. Preferably, by this method the number of antigen-specific effector cells, preferably T cells, is increased. Preferably, the immature antigen presenting cells are immature dendritic cells. In one embodiment, the step of producing RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (for example, an antigen of a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA modified with a structure corresponding to formula (I) is carried out in the absence of 2'-O-ribose methyltransferase. In an alternative embodiment, this step is carried out in the presence of 2'-O-ribose methyltransferase. In a preferred embodiment, the effector immune cells are T cells, preferably CD4+ and/or CD8+ cells. In one embodiment, the step of transferring said RNA into immature antigen presenting cells is performed in vitro by any nucleic acid transfer method, such as a transfection method known to those skilled in the art, such as lipofection, electroporation, or microinjection as described above. In another embodiment, the step of transferring said RNA into immature antigen presenting cells is performed in vivo, for example by administering the RNA to the individual, preferably by parenteral administration, preferably by intralymphatic administration, preferably by injection into the lymph node(s), i.e. by intranodal injection, by injection into the lymphatic vessels or by injection into the spleen. Preferably, said administration is an intranodal injection of RNA, which is preferably taken up by immature dendritic cells in the lymph node(s). The RNA input is preferably in the form of "deproteinized" RNA. In one embodiment, the step of contacting antigen presenting cells with immune effector cells is performed in vitro, for example in a tissue culture dish. In another embodiment, the step of contacting antigen presenting cells with immune effector cells is carried out in vivo. In this embodiment, the step of transferring the RNA into immature antigen presenting cells can be performed in vitro or in vivo as described above. To contact antigen presenting cells into which the RNA has been transferred in vitro with immune effector cells in vivo, the antigen presenting cells are administered to the individual, preferably parenterally, for example by intravenous, intramuscular, subcutaneous, intranodal, intralymphatic or intraperitoneal injection, preferably by injection into the lymphatic system, for example by injection into the lymphatic vessel(s), spleen and/or lymph node(s), preferably the inguinal lymph node(s). In one embodiment, the method may further include the step of differentiating immature antigen presenting cells into mature antigen presenting cells after transfer of RNA to the immature antigen presenting cells and before contacting the antigen presenting cells with immune effector cells. The differentiation step can be performed in vitro or in vivo. For example, the RNA may be transferred to immature antigen presenting cells, preferably immature dendritic cells, the immature antigen presenting cells are differentiated in vitro, and the differentiated mature antigen presenting cells, preferably mature dendritic cells, are contacted with immune effector cells in vitro or in vivo, as described above, preferably in vivo. The immature antigen presenting cells into which the RNA is transferred in vitro can be isolated from an individual, for example from a patient to be immunized, or they can be differentiated from hematopoietic stem cells.

Stimulation and/or activation of immune effector cells, in particular T cells, is generally associated with proliferation, cytotoxic reactivity and/or secretion of cytokines by the immune effector cells. Thus, one skilled in the art can determine whether immune effector cells are stimulated and/or activated by simple in vitro tests typically performed using T cells. Such T cells can be provided by transformed T cell lines, such as T cell hybridomas or T cells that have been isolated from a mammal, such as a rodent, such as a mouse or rat. Suitable T cell hybridomas are commercially available or can be prepared by known methods. T cells can be isolated from a mammal by known methods (see Shimonkevitz et al., 1983, J. Exp. Med. 158: 303-316). Suitable experimental conditions for testing T cell activation and/or stimulation are described below for steps (1) - (4). The T cells express a suitable marker that can be tested and which indicates T cell activation or modulation of T cell activity. The DO11.10 murine T cell hybridoma can be used for this purpose since the hybridoma expresses interleukin-2 (IL-2) upon activation. IL-2 concentrations can be measured to test T cell activation/stimulation or to determine whether the composition is able to modulate the activity of the T cell hybridoma. This test is performed with the following steps: (1) obtaining T cells from a T cell hybridoma or by isolation from mammals; (2) culturing T cells under conditions that allow proliferation; (3) bringing the proliferating T cells into contact with an antigen-presenting cell that has been in contact with the antigen or the nucleic acid encoding it; and (4) testing T cells for a marker, such as IL-2 production. The cells used for the test are cultured under conditions that allow proliferation. For example, T cell hybridoma DO11.10 is adequately cultured at 37°C and 5% CO ₂ in complete medium (RPMI 1640, supplemented with 10% FBS, penicillin/streptomycin, L-glutamine and 5x10 ^-5 M 2-mercaptoethanol). T cell activation signals are transmitted by antigen presenting cells that have been loaded with the appropriate antigenic peptide.

Alternatively, modulation of T cell activity can be confirmed by determining changes or proliferation of antigen-specific T cells, which can be measured, for example, by known radiolabeling techniques. For example, the labeled nucleotide can be added to the test culture medium. The incorporation of such labeled nucleotides into DNA may serve as an indicator of T cell proliferation. This test is not applicable to T cells that do not require antigen presentation for their proliferation, such as T cell hybridomas. This test is useful for determining the modulation of T cell activity in the case of non-transformed T cells that have been isolated from a mammal.

(IV) A method is provided for inducing an immune response in an individual, comprising providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (e.g., an antigen from a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA is modified with a structure according to formula (I) as defined in the first aspect; and administering said RNA modified in structure according to formula (I) to said individual. In one embodiment, the RNA is administered by intranodal injection or intradermally. The antigen of interest can be any antigen (eg, a viral (A, B or C) antigen such as influenza virus, CMV or RSV) and is preferably as defined above. In a preferred embodiment, said RNA is administered in the form of deproteinized RNA, preferably parenterally, for example by intravenous, intramuscular, subcutaneous, intranodal, intradermal, intralymphatic or intraperitoneal injection, preferably by injection into the lymphatic system, for example by injection into the lymphatic vessel(s), spleen and/or lymph node(s), preferably inguinal lymph node(s). Preferably, the administered RNA is taken up by immature dendritic cells of the individual. Preferably, the immune response is protective and/or therapeutic, for example useful for the treatment and/or prevention of diseases such as cancer or infectious diseases. In one embodiment, the step of providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (for example, an antigen of a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA modified with a structure corresponding to formula (I) is carried out in the absence of 2'-O-ribose methyltransferase. In an alternative embodiment, this step is carried out in the presence of 2'-O-ribose methyltransferase.

(V) A method is provided for inducing an immune response in an individual, comprising providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (e.g., an antigen from a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA is modified with a structure according to formula (I) as defined in the first aspect; transfer of said RNA, modified with the structure according to formula (I), into immature antigen-presenting cells; and administering antigen presenting cells to said individual. In one embodiment, the step of providing an RNA containing a nucleotide sequence encoding a peptide or protein containing an antigen of interest (for example, an antigen of a virus such as influenza virus (A, B or C), CMV or RSV) or an antigenic peptide thereof, wherein said RNA modified structure according to formula (I), carried out in the absence of 2'-O-ribose methyltransferase. In an alternative embodiment, this step is performed in the presence of 2'-O-ribose methyltransferase. In this aspect of the present invention, the RNA is transferred into immature antigen presenting cells in vitro by any nucleic acid transfer method, for example transfection such as lipofection, electroporation or microinjection, known to one skilled in the art, as described above. Preferably, the immature antigen presenting cells are immature dendritic cells. The immature antigen presenting cells into which the RNA is transferred in vitro can be isolated from an individual, for example from a patient who is to be immunized, or they can be differentiated from hematopoietic stem cells, wherein the hematopoietic stem cells can be isolated from the individual. Immature antigen presenting cells or hematopoietic stem cells can be isolated from an individual by leukapheresis. Preferably, the immature antigen presenting cells are immature dendritic cells. Preferably, immature antigen presenting cells are isolated from the individual to be immunized, the RNA is transferred into said isolated cells, and the cells are transferred back to said individual, preferably by parenteral administration, for example by intravenous, intramuscular, subcutaneous, intranodal, intralymphatic or intraperitoneal injection, preferably by lymphatic injection system, for example by injection into the lymphatic vessel(s), spleen and/or lymph node(s), preferably the inguinal lymph node(s).

The ability to induce an immune response, including suitability for vaccination against the target disease, can be readily determined using in vivo tests. For example, a composition, such as a vaccine composition or a pharmaceutical composition, can be administered to a mammal, such as a laboratory animal, such as a mouse, rat, rabbit, etc., and blood samples can be taken from said animal before administration of the composition and at certain times after administration compositions, for example 1, 2, 3, 4, 5, 6, 7 and 8 weeks after administration. Serum can be obtained from blood samples and the formation of antibodies generated upon administration/immunization can be determined. For example, the concentration of antibodies can be determined. In addition, T cells can be isolated from the blood and/or lymphatic system of a mammal and tested for their reactivity against the antigen used for immunization. Any reading system known to one skilled in the art may be used, for example proliferation assays, cytokine secretion assays, cytotoxic activity assays or tetramer assays, etc. may be used. In addition, increased immune reactivity can also be determined by analyzing the number of antigen-specific T cells, their cytotoxic potential, or their cytokine secretion pattern, as outlined above.

As demonstrated in the examples of the present application, the present inventors have unexpectedly discovered that using the 5'-cap compounds of the present invention, it is possible to incorporate cap-1 of the beta-S-ARCA structure into RNA in a single step, thereby combining the beneficial effects of thio-substitution with the defining cap -1 2'-O-methylation.

The present invention is illustrated by the following examples, which demonstrate preferred embodiments of the invention, and should not be interpreted as limiting the scope of the present invention as defined in the claims. Those examples not covered by the appended claims are given for comparison purposes only.

Examples

Abbreviations

h: hour(s)

mM: millimolar (10 ^-3 mol/l)

NTP: nucleoside triphosphate

Example 1 - Synthesis of cap analogues

To obtain co-transcriptionally capped mRNA transcribed in vitro, a 5' cap compound of the invention (Compound 1, compound of formula (I)) was constructed as shown in FIG. 2 (OR = OCH ₃ ). Compound 1 contains a phosphorothioate substitution at the beta position of the 5'-5'-triphosphate bridge, a terminal 2'-O methylation to avoid inclusion in the inverted orientation, an internal 2'-O methylation reflecting the cap-1 structure, plus an additional guanosine moiety, which ensures incorporation by phage RNA polymerase. The synthesis and use of the modified cap trinucleotide is described below.

The 5'-cap compounds of the present invention and other cap analogues can be synthesized starting from commercially available oligonucleotides such as (pN) _2-4 using standard procedures. For m ₂ ^7,2-O Gpp _S pm ^2'-O GpG (Compound 1) 5'-phosphorylated 2'-O-methylated diguanosine 5',3'-dinucleotide (pm ^2'-O GpG) was prepared commercially and used as starting material without additional processing. The dinucleotide was converted to the corresponding P-imidazolide derivative (Im-pm ^2'-O GpG) by reaction with 10 equiv. imidazole in DMF in the presence of 3 equiv. 2,2'-dithiodipyridine/triphenylphosphine activation system (see Fig. 1; Mukaiyama and Hashimoto 1971). The nucleotide subunit, 2'-O-methylguanosine 5'-O-(2-thiodiphosphate) (m2 ^7,2'-OMe GDPβS), was synthesized as described previously (Kowalska et al 2008). Then m2 ^7,2'-OMe GDPβS and P-imidazolide Im-pm ^2'-O GpG were coupled in DMF in the presence of excess ZnCl ₂ (8 equiv.) with a cap analog ( _m2 ^7,2-O Gpp _S pm ^2'-O GpG, 38% HPLC yield, Figure 2) as a mixture of two diastereoisomers (D1 and D2, named according to the order of elution from the RP-HPLC column). Diastereoisomers were separated by RP-HPLC (Discovery Amide RP C16 column) into pure diastereoisomers D1 and D2.

Spectroscopic data:

m ₂ ^7,2'-O Gpp _S p ^2'-O GpG (Compound 1)

(D1): ¹ H NMR (400 MHz; D ₂ O) δ 9.07 (s; 1H); 8.16(s;1H); 8.07(s;1H); 5.97 (d; J = 2.24 Hz; 1H); 5.87 (d; J = 5.98 Hz; 1H); 5.82 (d; J = 5.48 Hz; 1H); 4.85 - 5.00 (m; 1H); 4.74 - 4.87 (m; 2H; partially overlaps with the HDO signal); 4.61 (t; J = 5.35 Hz; 1H); 4.51 (t; J = 4.36 Hz; 2H); 4.12 - 4.48 (m; 9H); 4.07(s;3H); 3.58(s;3H); 3.43(s;3H); ³¹ P NMR (162 MHz; D ₂ O) δ 30.93 (t; J=26.9 Hz; 1 P) -0.59 (br. s; 1 P) -11.94 (d; J=26 .9 Hz; 1 P) -12.08 (d; J=26.9 Hz; 1 P);

(D2): ^1H NMR (400 MHz; _D2O ) δ 9.08 (s; 1H); 8.16(s;1H); 8.10(s;1H); 5.89 (d; J = 2.49 Hz; 1H); 5.87 (d; J = 5.98 Hz; 1H); 5.82 (d; J = 5.98 Hz; 1H); 4.97 (m; 1H); 4.58 – 4.55 (~t; 1H) 4.85 -4.73 (m; 2H; overlaps with the HDO signal); 4.54 - 4.47 (m; 2H); 4.13 - 4.46 (m; 9H); 4.07(s;3H); 3.57(s;3H); 3.44(s;3H); ³¹ P NMR (162 MHz; D ₂ O) δ 30.40 (dt; J=26.9 Hz; 1 P) -0.60 (br. s; 1 P) -11.98 (d; J=26 .9 Hz; 1 P) -12.43 - 12.06 (d; J=26.9 Hz; 1 P)

MSHR calculation, m/z for C ₃₃ H ₄₄ N ₁₅ O ₂₄ P ₄ S ^- [MH] ^- 1190.1360; registered, 1190.13936.

Example 2 - In vitro synthesis of capped mRNA by in vitro transcription using cap analogues

To incorporate different cap analogues during in vitro transcription, the same protocol can be used as for the incorporation of conventional beta-S-ARCA dinucleotide caps. In the example shown here, 3 mM cap analog and 7.5 mM NTP were added to the transcription reaction. The yield (in mg RNA per ml reaction volume) and integrity of RNA produced by one of the diastereomers (D1/D2) of Compound 1 or one of the diastereomers (D1/D2) of beta-S-ARCA were comparable to the data shown in the following table.

Reaction output
(mg RNA/ml reaction volume) RNA integrity
(BIOANALYZER) beta-S-ARCA (D1) 6.54 82% Connection 1 (D1) 6.39 87% beta-S-ARCA (D2) 6.42 82% Connection 1 (D2) 5.81 80%

Example 3 - Protein expression from differently capped mRNA in cell culture

As a functional assay for cap analog incorporation and as a test for the translatability of the resulting cap structure, differently capped mRNA encoding a luciferase reporter was transfected into human immature dendritic cells (hiDCs). To do this, 700 ng of capped RNA per well was prepared with liposomes as described in Kranz et al., Nature 534, 396-401 (2016) and added to a 96-well plate containing 5E04 hiDC. Reporter activity was then recorded for 72 hours. The results are presented in Fig. 3.

mRNA co-transcriptionally capped with the D1 or D2 diastereomer of Compound 1 was functional and translated into hiDCs at comparable levels to mRNA capped with the corresponding D1 or D2 diastereomer of beta-S-ARCA, indicating that with compounds of formula (I) in accordance with the present invention, the benefits of the beta-S-ARCA modification were still active (Figure 3).

Example 4 - mRNA modified with a 5' cap compound of formula (I) combines improved stability and translational efficiency of mRNA with immune evasion via IFIT1

Luciferase mRNAs were co-transcriptionally capped with the D1 or D2 diastereomer of Compound 1 or the corresponding D1 or D2 diastereomer of beta-S-ARCA as described above. In addition, the triphosphate Luc mRNA (ie, transcribed in the absence of any cap analogue) was enzymatically capped using the vaccinia NEB capping enzyme kit (enzymatic Cap-0/Cap-1 RNA). To obtain enzymatic cap-0 structures, the vaccinia capping enzyme was used as such; for enzymatic cap-1 structures, vaccinia methyltransferase was also added. The resulting capped mRNA preparations were then purified to reduce or eliminate double-stranded RNA. In addition, the small amount of uncapped RNA present in co-transcriptionally capped mRNA preparations, which has been shown to interfere with the analysis in previous experiments, was enzymatically converted to cap-0 structures (for dinucleotide-capped mRNAs) or cap-1 structures (for trinucleotide-capped mRNAs). The resulting mRNA preparations were then prepared with F12 and administered intravenously to Balb/c mice. Five mice were tested in each group with a dose of 10 μg of RNA per mouse. The strength and kinetics of luciferase expression were monitored by in vivo bioluminescence imaging at 6, 24 and 48 hours after application.

Although in this case, the beta-S substitution has only a minor effect (if any), as observed by the similar expression profiles of enzymatically capped cap-0 RNA compared to beta-S-ARCA (D1) and (D2) cap-0 RNA , the main factor driving expression in vivo is 2'-O-methylation of the cap-1 structure. Accordingly, enzymatically capped cap-1 mRNA preparations yield the highest protein expression at any time point measured. However, Compound 1-capped RNAs exhibited similar protein levels at 20 hours postapplication and only slightly lower levels at other time points (and always higher levels compared to all cap-0 RNAs). Thus, the 5'-cap compounds of the present invention allow the incorporation of beta-S-ARCA structures into cap-1 RNA, which combine the beneficial effect of thio-substitution with cap-1 defining 2'-O-methylation.

Example 5 - Protein Expression from Differently Capped mRNAs in Vivo

Murine erythropoietin (mEPO) mRNAs containing 1-methylpseudouridine (m1Ψ) were co-transcriptionally capped with ARCA G or the D1 diastereomer beta S-ARCA (designated D1) as described above. In addition, triphosphate EPO mRNA (ie, transcribed in the absence of any cap analogue) was enzymatically capped using the vaccinia NEB capping enzyme kit (enzymatic Cap-0/Cap-1 RNA). To obtain cap-0 enzymatic structures (designated as Esar-0), vaccinia capping enzyme with RNA triphosphatase and guanylyltransferase activities was used as such, for cap-1 enzymatic structures (designated as Esar-1), vaccinia methyltransferase with 2'- activity was also added O-methyltransferases. The resulting mRNA preparations with different caps were then purified to reduce or eliminate double-stranded RNA. In addition, the small amount of uncapped RNA present in co-transcribed mRNA preparations, which had been shown to interfere with the analysis in previous experiments, was enzymatically converted into cap-0 structures and then into cap-1 structures. In the mRNA preparation using D1 structures, the resulting enzymatic product was designated D1+Esar-1, whereas in the case of ARCA G, the resulting enzymatic product was designated ARCA G+Esar-1. mRNA preparations with TransIT® were then prepared and administered intraperitoneally to Balb/c mice. Five mice were tested in each group with a dose of 3 μg of RNA per mouse. mEPO mRNA translation was monitored by ELISA in plasma collected 6, 24, 48 and 72 hours after application.

As can be seen in FIG. 5, the presence of the cap-1 structure of the invention in RNA results in much higher levels of mEPO expression compared to RNA having the cap-0 structure, particularly 24 hours after injection. In addition, in FIG. 5 shows that using RNA containing the cap-1 structure of the present invention, high plasma mEPO levels can be maintained for at least 72 hours. Thus, this example demonstrates that it is not necessary to administer RNA containing a nucleotide sequence encoding a peptide or protein at least twice daily to maintain high levels of expression of the peptide or protein. Rather, using the present invention, it is possible to administer RNA no more than once per day, preferably no more than once every two days, preferably no more than once every three days, or no more than once every four days while maintaining high levels of peptide or protein expression. This provides the patient with the advantage that the number of administrations (eg, injections) can be significantly reduced, which is particularly beneficial for patients who receive treatment (eg, a pharmaceutical composition) over a long period of time, such as chronic or long-term ill patients.

Claims (58)

1. 5'-cap compound having the structure according to formula (I): formula (I) where R ¹ is selected from the group consisting of C _1-4 alkyl, C _2-4 alkenyl and aryl; R ² and R ³ are independently selected from the group consisting of H, F, OH, methoxy, ethoxy, propoxy and 2-methoxyethoxy; R ⁴ and R ⁶ are O; R ⁵ represents S; R ⁷ is a mononucleotide or di- or tri-oligonucleotide, wherein the nucleotide closest to the ring to which R ⁷ is attached is a nucleoside guanosine; R ⁸ is selected from the group consisting of methoxy, ethoxy, propoxy and 2-methoxyethoxy; n is 1, 2 or 3; And B is a purine or pyrimidine base moiety. 2. The 5'-cap compound according to claim 1, wherein R ² is selected from the group consisting of H, F, methoxy, ethoxy, propoxy and 2-methoxyethoxy. 3. A 5'-cap compound according to claim 1 or 2, wherein B is a fragment of a natural purine or pyrimidine base, or a modified form thereof. 4. 5'-cap connection according to any one of paragraphs. 1-3, wherein B is selected from the group consisting of guanine, adenine, cytosine, thymine, uracil and modified forms thereof, preferably from the group consisting of guanine, adenine, cytosine, uracil and modified forms thereof, more preferably from the group consisting of of guanine, adenine, cytosine and modified forms thereof, more preferably from the group consisting of guanine, adenine and modified forms thereof. 5. The 5'-cap compound according to claim 4, wherein the modified purine or pyrimidine base moiety is modified with one or more alkyl groups, preferably one or more C _1-4 alkyl groups, more preferably one or more methyl groups. 6. 5'-cap compound according to claim 4 or 5, where the modified purine or pyrimidine base fragment is selected from the group consisting of N ⁷ -alkyl-guanine, N ⁶ -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl -uracil and N(1)-alkyl-uracil, preferably from the group consisting of N ⁷ -C _1-4 alkyl guanine, N ⁶ -C _1-4 alkyl adenine, 5-C _1-4 alkyl cytosine, 5-C _1-4 alkyl-uracil and N(1)-C _1-4 alkyl-uracil, more preferably from the group consisting of N ⁷ -methyl-guanine, N ⁶ -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil. 7. 5'-cap connection according to any one of paragraphs. 3-6, wherein the natural purine or pyrimidine base moiety is G or A, preferably G. 8. 5'-cap connection according to any one of paragraphs. 1-4, where B is G or A, preferably G. 9. 5'-cap connection according to any one of paragraphs. 1-8, where R ⁷ is connected at its 5' end to the ring to which R ⁸ is attached. 10. 5'-cap connection according to any one of paragraphs. 1-9, where R ⁷ represents a ribomononucleotide or ribooligonucleotide having 2 or 3 bases. 11. The 5'-cap compound according to claim 10, where R ⁷ is a ribonucleotide having a free OH group at position 2'. 12. The 5'-cap compound according to claim 10, where R ⁷ is a ribooligonucleotide, and both the ribose fragment at the 3' end of the ribooligonucleotide and the ribose fragment at the 5' end of the ribooligonucleotide have a free OH group at position 2'. 13. The 5' cap compound of claim 10, wherein R ⁷ is a ribooligonucleotide, wherein the OH group at position 2' of at least ribose at the 5' end of the ribooligonucleotide is replaced by a substituent selected from the group consisting of H, halogen, and optionally substituted alkoxy, and the ribose at the 3' end of the ribooligonucleotide has a free OH group at position 2'. 14. 5'-cap connection according to any one of paragraphs. 1-13, wherein the internucleotide linkage between the mononucleotide or oligonucleotide and the ring to which R ⁷ is attached is selected from the group consisting of phosphate, phosphothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, alkylphosphonate, phosphotriester, phosphoamidite and a non-nucleotide linker. 15. 5'-cap connection according to any one of paragraphs. 1-14, wherein the internucleotide bond(s) between nucleotides in the oligonucleotide is selected from the group consisting of phosphate, phosphorothioate, boranophosphate, imidophosphate, alkylene phosphate, phosphodithioate, alkylphosphonate, phosphotriester, phosphoamidite, and a non-nucleotide linker. 16. 5'-cap connection according to any one of paragraphs. 1-15, where the stereochemical configuration of the P atom containing the R ⁵ substituent corresponds to the configuration of the P _β atom of the D1 beta-S-ARCA diastereomer. 17. 5'-cap compound having the structure according to formula (I): formula (I) where R ¹ is methyl or ethyl; one of R ² and R ³ is methoxy and the other is OH; R ⁴ and R ⁶ are O; R ⁵ represents S; R ⁷ is a mononucleotide or di- or tri-oligonucleotide, wherein the nucleotide closest to the ring to which R ⁷ is attached is a nucleoside guanosine; R ⁸ is methoxy; n is 1 and B is a purine or pyrimidine base moiety. 18. Composition for providing RNA with a 5'-cap structure, containing an effective amount of a 5'-cap compound according to any one of paragraphs. 1-17. 19. A kit for providing RNA with a 5'-cap structure, containing a 5'-cap compound according to any one of claims. 1-17. 20. RNA encoding a peptide or protein of interest, modified with a 5'-cap compound according to any one of claims. 1-17. 21. RNA for expression of a peptide or protein of interest, modified with a 5'-cap compound according to any one of claims. 1-17 and containing a nucleotide sequence encoding a peptide or protein of interest, operably linked to expression control sequences or regulatory sequences. 22. A cell for producing a peptide or protein of interest, containing RNA according to claim 21. 23. A method for producing a peptide or protein of interest, comprising the step of transferring the RNA of claim 21 into a cell. 24. A method of expressing a peptide or protein of interest in an individual, comprising the step of administering to said individual the RNA of claim 21 or the cell of claim 22. 25. A method for increasing the stability of RNA in cells, including the stages at which: - provide said RNA with a structure according to formula (I), as defined in any of paragraphs. 1-17; And - transfer the specified RNA, modified by the structure according to formula (I), into cells. 26. A method for increasing RNA expression in cells, including the stages of: - provide said RNA with a structure according to formula (I), as defined in any of paragraphs. 1-17; And - transferring said RNA, modified with a structure according to formula (I), into cells where said RNA contains expression control sequences or regulatory sequences. 27. The method according to claim 25 or 26, where the step of providing said RNA with the structure according to formula (I) is carried out in the absence of 2'-O-ribose methyltransferase. 28. A method for providing RNA with a 5' cap structure, comprising: carrying out a transcription reaction using a template nucleic acid in the presence of a 5'-cap compound according to any one of claims. 1-17, where the template nucleic acid is DNA complementary to the target RNA. 29. The method according to claim 28, where the transcription reaction is carried out in vitro. 30. The method according to claim 28 or 29, where the transcription reaction is carried out using an RNA polymerase selected from the group consisting of T3, T7 and SP6 RNA polymerases. 31. Method according to any one of paragraphs. 28-30, where the RNA contains a nucleotide sequence encoding a peptide or protein of interest. 32. Method according to any one of paragraphs. 28-31, which is carried out in the absence of 2'-O-ribose methyltransferase. 33. The RNA of claim 21, wherein the peptide or protein of interest is selected from the group consisting of cytokines such as erythropoietin; adhesion molecules such as integrin; immunoglobulins; immunologically active compounds, for example antigens, such as tumor-associated antigens, pathogen-associated antigens (such as viral antigens, for example one or more influenza virus (influenza virus A, B or C), cytomegalovirus (CMV) or respiratory syncytial virus (RSV)), allergens or autoantigens; hormones such as vasopressin, insulin or growth hormone; growth factors such as VEGFA; enzymes such as herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes and lactase; receptors such as growth factor receptors; protease inhibitors such as alpha-1-antitrypsin; apoptosis regulators such as BAX; transcription factors such as FOXP3; tumor suppressor proteins such as p53; structural proteins such as surfactant proteins; reprogramming factors such as OCT4, SOX2, c-MYC, KLF4, LIN28 or NANOG; genome-engineered proteins such as CRISPR clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR-Cas9); and blood proteins such as fibrinogen.