RU2772349C2 - Artificial nucleic acid molecules - Google Patents

Abstract

'FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to an artificial nucleic acid molecule containing at least one open reading frame and at least one 3'-UTR element and optionally at least one 5'-UTR element, where the specified artificial nucleic acid molecule differs in a high translation efficiency compared to a translation efficiency of a reference nucleic acid molecule that is identical to the artificial nucleic acid molecule except for 3'-UTR element. The invention also relates to an expression vector, a cell for the production of polypeptide or peptide, pharmaceutical compositions for the vaccination or gene therapy, their use as a vaccine or gene therapy, a method for the production of an artificial molecule, a method for ensuring an increased translation efficiency, a vaccination kit, a gene therapy kit and the use of 3'-UTR element.

EFFECT: obtaining an artificial nucleic acid molecule.

31 cl, 9 dwg, 8 tbl, 3 ex

Description

The present invention relates to artificial nucleic acid molecules that contain an open reading frame, a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element) and optionally poly( A) sequence and/or polyadenylation signal. The invention also relates to a vector containing a 3'-UTR element and/or a 5'-UTR element, to a cell containing an artificial nucleic acid molecule or a vector, to a pharmaceutical composition containing an artificial nucleic acid molecule or a vector, and to a kit, containing an artificial nucleic acid molecule, a vector and/or a pharmaceutical composition, preferably intended for use in the field of gene therapy and/or genetic vaccination.

Gene therapy and genetic vaccination are among the most promising and rapidly developing methods of modern medicine. They can be applied in the development of highly specific and individual approaches for the treatment of a wide variety of diseases. Such treatment approaches can primarily target congenital genetic diseases, as well as malignant or tumor-related diseases and also inflammatory diseases. It is understood that these approaches can prevent the early onset of such diseases.

The main conceptual idea of gene therapy is the implementation of appropriate modulation of impaired gene expression associated with pathological conditions in specific diseases. Pathologically altered gene expression may result in a deficiency or overproduction of important gene products, for example, signaling factors such as hormones, housekeeping gene factors, metabolic enzymes, structural proteins, or the like. Altered gene expression may be due not only to dysregulation of transcription and/or translation, but also to mutations in the ORF encoding a particular protein. Pathological mutations can be caused, for example, by a chromosomal aberration or by more specific mutations such as point or frameshift mutations, all of which result in limited functionality and possibly complete loss of function of the gene product. However, dysregulation of transcription or translation can also occur if mutations affect genes encoding proteins that are involved in the mechanism of transcription or translation in the cell. Such mutations can lead to abnormal up- or down-regulation of genes that are themselves functional. Genes encoding gene products that have such regulatory functions may be, for example, genes for transcription factors, signaling receptors, messenger proteins, or the like. However, the loss of function of such genes encoding regulatory proteins can, under certain circumstances, be restored by the artificial introduction of other factors functioning downstream of the disrupted gene product. Such gene defects can also be compensated by gene therapy by replacing the damaged gene itself.

Genetic vaccination makes it possible to induce the desired immune response to selected antigens, such as characteristic components of bacterial surfaces, viral particles, tumor antigens, or the like. In general, vaccination represents one of the most important achievements of modern medicine. However, effective vaccines are currently only available for a limited number of diseases. Therefore, infections that cannot be prevented by vaccination still affect millions of people every year.

In general, vaccines can be classified into "first", "second" and "third" generation vaccines. "First generation" vaccines are generally vaccines containing whole organisms. They are based either on live and attenuated or killed pathogens such as viruses, bacteria or the like. The main disadvantage of live and attenuated vaccines is the risk of reversion to form life-threatening variants. Thus, these pathogens, although attenuated, may still carry inherent unpredictable risks. Killed pathogens may not be effective enough to create a specific immune response. In order to minimize these risks, "second generation" vaccines have been developed. As a rule, they are subunit vaccines, consisting of components of certain antigens or recombinant proteins derived from pathogens.

Genetic vaccines, i.e. vaccines for genetic vaccination are generally referred to as "third generation" vaccines. They typically consist of genetically engineered nucleic acid molecules that provide in vivo expression of peptide or protein (antigen) fragments characteristic of a pathogen or tumor antigen. When administered to a patient, expression of genetic vaccines occurs after uptake by target cells. Expression of the introduced nucleic acids results in the production of encoded proteins. When these proteins are recognized by the patient's immune system as foreign, an immune response is triggered.

As follows from the above, both methods, and gene therapy, and genetic vaccination, essentially based on the introduction of nucleic acid molecules to the patient and subsequent transcription and/or translation of encoded genetic information. Alternatively, genetic vaccination or gene therapy may also include methods that involve isolating specific cells from the body of the patient to be treated, then ex vivo transfecting said cells, and re-introducing the treated cells to the patient.

In the context of gene therapy or genetic vaccination, both DNA and RNA can be administered as nucleic acid molecules. DNA is known to be relatively stable and easier to manipulate. However, the use of DNA carries the risk of undesirably inserting the introduced DNA fragments into the patient's genome, which can lead to mutagenic effects such as loss of function of the disrupted genes. Another risk is that an undesirable occurrence of antibodies to DNA occurs. Another disadvantage is the limited level of expression of the encoded peptide or protein achievable with the introduction of DNA, since the DNA must enter the nucleus in order for it to be transcribed before the resulting mRNA can be translated. The level of expression of the introduced DNA may depend, inter alia, on the presence of specific transcription factors that regulate DNA transcription. In the absence of such factors, DNA transcription does not result in DNA in sufficient quantities. As a result, the level of translated peptide or protein achievable is limited.

When using RNA instead of DNA for gene therapy or genetic vaccination, the risk of unwanted integration into the genome and the formation of antibodies to DNA is minimized or eliminated. However, RNA is considered to be a relatively unstable molecule that can be easily cleaved by the ubiquitous RNases.

Cleavage of RNA in vivo contributes to the regulation of RNA half-life. This effect has been studied and found to strongly regulate eukaryotic gene expression (Friedel et al., Conserved principles of mammalian transcriptional regulation revealed by RNA half-life, Nucleic Acid Research 37(17), 2009, pp. 1-12 ). Thus, each naturally occurring mRNA has its own individual half-life, depending on the gene from which the mRNA originates and on the type of cell in which expression occurs. It contributes to the regulation of the level of expression of the gene in question. Unstable RNAs are important for transient gene expression at specific time points. However, long-lived RNAs may be associated with the accumulation of various proteins or with continuous gene expression. The in vivo half-life of mRNAs may also depend on environmental factors such as hormone treatment, as demonstrated for example for mRNAs of insulin-like growth factor I, actin, and albumin (Johnson et al. Newly synthesized RNA: Simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes, Proc. Natl. Acad. Sci., vol. 88, 1991, pp. 5287-5291).

Gene therapy and genetic vaccination generally require stable RNA. This is due, on the one hand, to the fact that, as a rule, it is required that in vivo accumulation of the product encoded by the RNA sequence takes place. On the other hand, RNA must maintain its structural and functional integrity during the preparation of a suitable dosage form, during its storage and administration. Therefore, efforts have been made to create stable RNA molecules for gene therapy or genetic vaccination in order to prevent them from early degradation or cleavage.

Data have been published that the content of G/C in nucleic acid molecules can affect its stability. Thus, nucleic acids containing a greater amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than nucleic acids containing a large number of adenine (A) and thymine (T) or uracil (U) nucleotides. . In this context, WO 02/098443 proposes a pharmaceutical composition containing mRNA that is stabilized by sequence modifications in the coding region. Such sequence modification is possible due to the degeneracy of the genetic code. Accordingly, codons containing a less preferred combination of nucleotides (less preferred in terms of RNA stability) can be changed to alternative codons without changing the encoded amino acid sequence. This method of RNA stabilization is limited by the fact that for each individual RNA molecule it is necessary to obtain a specific nucleotide sequence that does not lead to an amino acid sequence that deviates from the required one. This approach is also limited in that it is applied to RNA coding regions.

In the search for an alternative approach to mRNA stabilization, it was found that naturally occurring eukaryotic mRNA molecules contain characteristic stabilizing elements. For example, they may contain so-called untranslated regions (UTRs) at their 5' end (5'-UTR) and/or at their 3' end (3'-UTR), as well as other structural features such as 5' -cap-structure or 3'-poly(A)-tail. Both the 5'-UTR and the 3'-UTR are typically transcribed from genomic DNA and are therefore an immature mRNA element. Structural features of the mature mRNA, such as a 5' cap and a 3' poly(A) tail (which are also referred to as the poly(A) tail or poly(A) sequence), are typically added to the transcribed (immature) mRNA during mRNA processing.

The 3'-poly(A) tail is typically a monotonic stretch of sequence that consists of adenosine nucleotides added to the 3' end of the transcribed mRNA. It may contain up to about 400 adenosine nucleotides. It has been found that the length of such a 3'-poly(A)-tail may be an element of decisive importance for the stability of an individual mRNA.

It has also been demonstrated that the 3'-UTR of α-globin mRNA may be an important contributor to the well-known stability of α-globin mRNA (Rodgers et al., Regulated α-globin mRNA decay is a cytoplasmic still t proceeding through 3'-to- 5' exosome-dependent decapping, RNA, 8, 2002, pp. 1526-1537). The 3'-UTR of α-globin mRNA appears to be involved in the formation of a specific ribonucleoprotein complex, the α-complex, whose presence correlates with in vitro mRNA stability (Wang et al., An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro Molecular and Cellular Biology, Vol. 19, No. 7, July 1999, pp. 4552-4560).

In addition, an important regulatory function has been identified for the UTR in ribosomal protein mRNA: although the 5'-UTR of ribosomal protein mRNA controls growth-associated mRNA translation, the rigor of this regulation is provided by the corresponding 3'-UTR in ribosomal protein mRNA (Ledda et al.. Effect of the 3'-UTR length on the translational regulation of 5'-terminal oligopyrimidine mRNAs, Gene, vol. 344, 2005, pp. 213-220). This mechanism affects the specific expression pattern of ribosomal proteins, which are usually transcribed in a constant manner, as a result of which the mRNA of some ribosomal proteins, such as the S9 ribosomal protein or the L32 ribosomal protein, are called housekeeping genes (Janovick-Guretzky et al. Housekeeping Gene Expression in Bovine Liver is Affected by Physiological State, Feed Intake, and Dietary Treatment, J. Dairy Sci., vol. 90, 2007, pp. 2246-2252). Thus, the expression pattern of ribosomal proteins associated with growth is mainly due to regulation at the level of translation.

WO 2014/164253 A1 describes some specific nucleic acid molecules having a 5'-UTR and/or 3'-UTR, but does not provide details regarding the efficiency of translation of such molecules.

Regardless of the factors affecting mRNA stability, the efficiency of translation of the introduced nucleic acid molecules by target cells or tissue is critical to any approach using nucleic acid molecules for gene therapy or genetic vaccination. As follows from the examples cited above, structural features such as UTR, 5'-cap and 3'-poly(A)-tail, along with the regulation of stability, also regulate the translation of most mRNA. In this context, published data suggest that the length of the poly(A) tail may also play an important role in the efficiency of translation. However, 3' stabilizing elements can also have a translational dampening effect.

The present invention was based on the task of creating nucleic acid molecules that can be used for gene therapy and/or genetic vaccination. The objective of the invention was primarily to create mRNA species that are stabilized to protect against premature degradation or cleavage, but which do not suffer from a significant functional loss in terms of translation efficiency. It was also an object of the invention to provide an artificial nucleic acid molecule, preferably mRNA, which is characterized by high translation efficiency. One of the specific objectives of the invention was to create an mRNA that allows you to increase the efficiency of translation (production by ribosomes of the corresponding encoded protein), for example, compared with a reference nucleic acid molecule (reference mRNA). Another object of the present invention was to provide nucleic acid molecules encoding advantageous mRNA species that may be suitable for use in gene therapy and/or genetic vaccination. A further object of the present invention was to provide a pharmaceutical composition for use in gene therapy and/or genetic vaccination. In general, the aim of the present invention was to provide improved nucleic acid species that overcome the above disadvantages of the prior art using an economical and simple approach.

The problem underlying the present invention is solved with the help of the objects of the invention presented in the claims. In particular, the present invention has identified UTR elements (5'-UTR elements and 3'-UTR elements) that provide high translation efficiency. A common feature of the preferred nucleic acid molecules of the present invention is that they all have translation efficiencies that exceed those of previously known nucleic acid molecules. An assay is also provided to determine high translation efficiency relative to a reference nucleic acid molecule.

The present invention was made with the support of the US government under agreement No. HR 0011-11-3-0001 signed by DARPA (Defense Advanced Research Projects Agency). The US government has certain rights to the invention.

For purposes of clarity and readability, the following definitions are given below. Any specifications specified in these definitions apply to each and all embodiments of the invention. In the context of these embodiments of the invention, further definitions and explanations may be specifically given.

Adaptive immune response: Typically, adaptive immune response refers to an antigen-specific response of the immune system. Antigen specificity allows for responses tailored to specific pathogens or pathogen-infected cells. The ability to create such adapted responses is usually maintained in the body by "memory cells". If the pathogen infects the body more than once, then these specific memory cells are used for its rapid elimination. In this context, the first stage of an adaptive immune response is the activation of naïve antigen-specific T cells or other immune cells capable of inducing an antigen-specific immune response by antigen-presenting cells. This occurs in lymphoid tissues and organs, through which naive T cells constantly pass. Three types of cells that can serve as antigen presenting cells are dendritic cells, macrophages, and B cells. Each of these cells performs a separate function in inducing immune responses. Dendritic cells can take up antigens via phagocytosis and macropinocytosis, and they can be stimulated upon contact with, for example, a foreign antigen, to migrate to the local lymphoid tissue where they can differentiate into mature dendritic cells. Macrophages engulf particulate antigens such as bacteria and can be induced by infectious agents or other appropriate stimuli and thereby express MHC molecules. The unique ability of B cells to bind and internalize soluble protein antigens through their receptors may also be important for T cell induction. MHC molecules are generally responsible for antigen presentation to T cells. At the same time, antigen presentation on MHC molecules leads to the activation of T cells, which induces their proliferation and differentiation into “armed” effector T cells. The most important function of effector T cells is the destruction (cytolysis) of infected cells by cytotoxic CD8 ⁺ T cells and the activation of macrophages by Th1 cells, which together create cell-mediated (cellular) immunity, and the activation of B cells by both Th2 and Th1 cells, leading to the formation of various classes of antibodies, triggers a humoral immune response. T cells recognize an antigen through their T cell receptors, which do not recognize the antigen and do not bind to it directly, but instead recognize short peptide fragments, such as pathogen-derived protein antigens, such as so-called epitopes, which bind to MHC molecules on the surfaces of other cells.

Adaptive immune system: The adaptive immune system is primarily designed to eliminate or prevent the growth of pathogens. Typically, it regulates the adaptive immune response, giving the vertebrate immune system the ability to recognize and remember specific pathogens (to build immunity) and mount a stronger attack each time a pathogen is detected. The system is highly adaptable due to somatic hypermutation (a process of accelerated somatic mutations) and V(D)J recombination (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to create a huge number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because the rearrangement of a gene results in an irreversible change in the DNA of each cell, all progeny of that cell must then inherit genes encoding the same receptor specificity, including memory B cells and memory T cells, which are critical for long-term specific immunity.

Adjuvant/Adjuvant Component: An adjuvant or adjuvant component in the broadest sense is generally a pharmacological and/or immunological agent that can modify, for example enhance, the effect of other agents such as a drug or vaccine. This concept should be considered in a broad sense, and it refers to a wide range of substances. As a rule, these substances have the ability to increase the immunogenicity of antigens. For example, adjuvants may be recognized by the innate immune systems and, for example, may elicit an innate immune response. "Adjuvants" generally do not elicit an adaptive immune response. Thus, "adjuvants" cannot be classified as antigens. Their mechanism of action is different from that triggered by antigens that lead to an adaptive immune response.

Antigen: In the context of the present invention, the term "antigen" refers generally to a substance that can be recognized by the immune system, preferably the adaptive immune system, and which has the ability to trigger an antigen-specific immune response, for example, through the formation of antibodies and/or antigen-specific T cells , as a component of the adaptive immune response. Typically, an antigen may be or contain a peptide or protein that can be presented by MHC to T cells. In the context of the present invention, the antigen may be a translation product of the proposed nucleic acid molecule, preferably mRNA, presented in the present description. In this context, antigens also include fragments, variants and derivatives of peptides and proteins containing at least one epitope. In the context of the present invention, tumor antigens and pathogenic antigens are most preferred.

In other words, an artificial nucleic acid molecule can be considered as a nucleic acid molecule that does not occur naturally. Such a nucleic acid molecule may not occur naturally due to its individual sequence (which does not occur naturally) and/or due to other modifications, such as structural modifications of nucleotides, which do not occur naturally. The artificial nucleic acid molecule may be a DNA molecule, an RNA molecule, or a hybrid molecule containing portions of DNA and RNA. Generally, artificial nucleic acid molecules can be designed and/or genetically engineered to match the desired artificial nucleotide sequence (heterologous sequence). In this context, an artificial sequence is generally one that cannot occur naturally, i.e. differs from the wild-type sequence by at least one nucleotide. We can consider that the concept of "wild type" refers to a sequence found in nature. In addition, it should not be understood that the term "artificial nucleic acid molecule" means "one single molecule", as a rule, it includes a group of identical molecules. Therefore, it may refer to a plurality of identical molecules contained in an aliquot.

bicistronic RNA. polycistronic RNA: A bicistronic or polycistronic RNA is typically an RNA, preferably an mRNA, which can typically have two (bicistronic) or more (polycistronic) open reading frames (ORFs). In this context, an open reading frame is a sequence of multiple codons that can be translated into a peptide or protein.

Carrier/Polymer Carrier: In the context of the invention, a carrier can generally be a compound that facilitates the transport and/or incorporation of another compound (cargo, payload). The polymeric carrier, as a rule, is a carrier that forms a polymer. The carrier may be associated with its payload through a covalent or non-covalent interaction. The carrier can transport nucleic acids, such as RNA or DNA, to target cells. The carrier may (in some embodiments of the invention) be a cationic component.

Cationic component: The term "cationic component" generally refers to a charged molecule that is positively charged (cation) at a pH value typically between 1 and 9, preferably at or below pH 9 (e.g., 5 to 9) equal to or less than 8 (eg 5 to 8), equal to or less than 7 (eg 5 to 7), most preferably at physiological pH, eg 7.3 to 7.4. Thus, the cationic component may be any positively charged compound or polymer, preferably a cationic peptide or protein, which is positively charged under physiological conditions, especially in vivo physiological conditions. A "cationic peptide or protein" may contain at least one positively charged amino acid, or more than one positively charged amino acid, for example selected from Arg, His, Lys, or Orn. Therefore, "polycationic" compounds also fall under the specified definition if they carry more than one positive charge under the specified conditions.

5' Cap: The 5' cap is a structure, typically a modified nucleotide structure, that typically "caps" the 5' end of a mature mRNA. The 5'-cap can generally be formed by a modified nucleotide, primarily a derivative of a guanine nucleotide. Preferably the 5' cap is linked to the 5' end via a 5'-5' triphosphate bond. The 5' cap may be methylated, eg m7GpppN, where N is the 5' terminal nucleotide of the nucleic acid carrying the 5' cap, typically the 5' end of the RNA. Other examples of 5'-cap structures are glyceryl, inverted deoxy group (fragment) devoid of a nitrogenous base, 4',5'-methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1, 5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, base-modified nucleotide, treopentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3 '-Inverted Nucleotide Fragment, 3'-3'-Inverted Nucleotide Fragment, 3'-2'-Inverted Nucleotide Fragment, 3'-2'-Inverted Nucleotide Fragment, 1,4-Butanediol Phosphate, 3'-Phosphoramidate , hexylphosphate, aminohexylphosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, or a bridged or unbridged methylphosphonate moiety.

Cellular Immunity/Cellular Immune Response: Cellular immunity generally refers to the activation of macrophages, natural killer (NK) cells, antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to an antigen. In a more general sense, cellular immunity is not based on antibodies, but on the activation of cells of the immune system. Typically, a cellular immune response may be characterized, for example, by the activation of antigen-specific cytotoxic T-lymphocytes that are capable of inducing cell apoptosis, for example, specific immune cells such as dendritic cells or other cells displaying foreign gene epitopes on their surface. Such cells may be virus-infected or intracellular bacteria-infected, or cancer cells displaying tumor antigens. Other characteristics may be the activation of macrophages and natural killer cells, allowing them to destroy pathogens, and the stimulation of cells to secrete various cytokines that affect the function of other cells involved in adaptive immune responses and innate immune responses.

DNA: DNA is the common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e. a polymer made up of nucleotides. These nucleotides are typically deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate monomers, which themselves consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and form polymers with a characteristic backbone structure. The framework structure, as a rule, is formed by phosphodiester bonds between the sugar fragment of the nucleotide, i.e. deoxyribose, the first monomer and a phosphate fragment of the second adjacent monomer. The specific order of the monomers, i.e. the order of bases linked to the sugar/phosphate backbone is called the DNA sequence. DNA can be single stranded or double stranded. In the double stranded form, first strand nucleotides typically hybridize to second strand nucleotides, for example, by A/T base pairing and G/C base pairing.

Epitope: Epitopes (also referred to as "antigenic determinants") can be divided into T-cell epitopes and B-cell epitopes. In the context of the present invention, T-cell epitopes or portions of proteins may contain fragments, preferably having a length of from about 6 to about 20 or even more amino acids, for example, fragments processed and presented by MHC class I molecules, preferably have a length of from about 8 to about 10 amino acids, e.g. 8, 9 or 10, (or even 11 or 12 amino acids), or fragments processed and presented by MHC class II molecules are preferably about 13 amino acids or more in length, e.g. 13, 14, 15, 16, 17 , 18, 19, 20, or even more amino acids, and these fragments can be selected from any part of the amino acid sequence. These fragments are usually recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, i.e. fragments are generally not recognized in their native form. B-cell epitopes are typically fragments located on the outer surface of the (native) protein or peptide antigens described herein, preferably consisting of 5-15 amino acids, more preferably consisting of 5-12 amino acids, even more preferably consisting of of 6-9 amino acids that can be recognized by antibodies, i.e. in their native form.

Such protein or peptide epitopes may furthermore be selected from any of the recited variants of such proteins or peptides. In this context, antigenic determinants may be conformational or discontinuous epitopes consisting of segments of proteins or peptides as defined herein that are located at intervals in the amino acid sequence of proteins or peptides as described herein, but which are close to each other in a three-dimensional structure. , or continuous or linear epitopes consisting of a single polypeptide chain.

Sequence Fragment: A sequence fragment is typically a shortened portion of a full-length sequence, such as a nucleic acid molecule or an amino acid sequence. Therefore, a fragment typically consists of a sequence that is identical to the corresponding segment of the full-length sequence. In the context of the present invention, a preferred sequence fragment consists of a contiguous segment containing elements such as nucleotides or amino acids, which corresponds to a contiguous segment of these elements of the molecule from which the fragment is derived, the length of which is at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the total (i.e. full length) molecule from which fragment occurs.

G/C-modified: G/C-modified nucleic acid, as a rule, can be a nucleic acid, preferably an artificial nucleic acid molecule, specified in the present description, created on the basis of a modified wild-type sequence, which preferably contains an increased amount of guanosine and/ or cytosine nucleotides compared to the wild-type sequence. To obtain this increased number, it is possible to carry out the replacement of codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. With an increased content of G / C in the coding region of DNA or RNA, the degeneracy of the genetic code can be used for substitutions. Thus, codon changes preferably do not change the encoded amino acid residues, but only increase the G/C content of the nucleic acid molecule.

Gene therapy: Gene therapy can generally be the treatment of a patient's body or isolated elements of the patient's body, eg, isolated tissues/cells, with nucleic acids encoding a peptide or protein. Typically, it may include at least one of the following steps: a) administering a nucleic acid, preferably an artificial nucleic acid molecule as defined herein, directly to a patient - by any route of administration - or in vitro into isolated patient cells/tissues, resulting in transfection of patient cells, either in vivo/ex vivo or in vitro; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administering the isolated transfected cells to the patient if the nucleic acid was not administered directly to the patient.

Genetic vaccination: Genetic vaccination, as a rule, can be a vaccination carried out by introducing a nucleic acid molecule encoding an antigen or immunogen or fragments thereof. The nucleic acid molecule can be introduced into the body of an individual or into isolated cells of an individual. After transfection of certain cells of the body or after transfection of isolated cells, the antigen or immunogen can be expressed by these cells and then presented to the immune system, causing an adaptive, i. antigen-specific immune response. Thus, genetic vaccination typically comprises at least one of the following steps: a) introducing a nucleic acid, preferably an artificial nucleic acid molecule as defined herein, into an individual, preferably a patient, or into isolated cells of an individual, preferably a patient, which usually results in transfection of the individual's cells in vivo or in vitro; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administering the isolated transfected cells to an individual, preferably a patient, if the nucleic acid was not administered directly to the patient.

Heterologous sequence: Generally, two sequences are considered to be "heterologous" if they do not come from the same gene. This means that although heterologous sequences may originate from the same organism, they do not naturally occur in the same molecule, for example, in the same nucleic acid molecule.

Humoral Immunity/Humoral Immune Response: Humoral immunity generally refers to the production of antibodies and not necessarily the ancillary processes that may accompany it. The humoral immune response can typically be characterized by, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell formation. Humoral immunity, as a rule, can also refer to the effector functions of antibodies, including the neutralization of pathogens and toxins, classical complement activation, and the stimulation of phagocytosis and elimination of pathogens by opsonins.

Immunogen: In the context of the present invention, an immunogen is generally understood to mean a compound that has the ability to stimulate an immune response. Preferably the immunogen is a peptide, polypeptide or protein. In the most preferred embodiment of the invention, the immunogen in the context of the present invention is a translation product of the nucleic acid molecule used, preferably the artificial nucleic acid molecule referred to in the present description. Typically, the immunogen elicits at least an adaptive immune response.

Immunostimulatory composition: In the context of the invention, an immunostimulatory composition can generally be a composition containing at least one component that has the ability to induce an immune response or from which a component that has the ability to induce an immune response can be derived. Such an immune response may preferably be an innate immune response or a combination of an adaptive and an innate immune response. In the context of the present description, the immunostimulatory composition preferably contains at least one artificial nucleic acid molecule, more preferably RNA, for example, an mRNA molecule. An immunostimulatory component, such as mRNA, may be complexed with a suitable carrier. Thus, the immunostimulatory composition may comprise an mRNA/carrier complex. In addition, the immunostimulatory composition may contain an adjuvant and/or a suitable excipient for the immunostimulatory component, such as mRNA.

Immune response: An immune response can generally be either a specific reaction of the adaptive immune system to a particular antigen (so-called specific or adaptive immune response), or a non-specific reaction of the innate immune system (so-called non-specific or innate immune response), or a combination of both. .

Immune System: The immune system can protect organisms from infection. If the pathogen has managed to overcome the physical barrier of the organism and enter the specified organism, then the innate immune system provides an immediate, but non-specific response. If the pathogen eludes this innate response, vertebrates have a second layer of defense, which is the adaptive immune system. In this case, the immune system adapts its response during infection to improve recognition of the pathogen. This improved response is then stored after elimination of the pathogen in the form of an immunological memory and allows the adaptive immune system to mount faster and stronger attacks each time that pathogen enters. Thus, the immune system includes the innate and adaptive immune systems. Each of these two parts, as a rule, includes the so-called humoral and cellular components.

Immunostimulatory RNA: Immunostimulatory RNA (isRNA) in the context of the invention, as a rule, can be an RNA that has the ability to induce an innate immune response. It usually does not have an open reading frame and thus does not encode an antigen peptide or immunogen, but elicits an immune response, for example by binding to a specific type of Toll-like receptor (TLR) or other suitable receptors. However, of course, also mRNAs having an open reading frame and encoding a peptide/protein can induce an innate immune response and can therefore be immunostimulatory RNAs.

Innate immune system: The innate immune system, also referred to as the non-specific immune system, typically includes cells and mechanisms that non-specifically protect the host from infection by other organisms. This means that the cells of the innate system can recognize and respond to pathogens in the usual way, but, unlike the adaptive immune system, it does not provide long-term or protective immunity to the host. The innate immune system can, for example, be activated by ligands of pathogen-associated molecular pattern recognition (PAMP) receptors, such as Toll-like receptors (TLRs), or other accessory substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors and hGH, human Toll-like receptor ligand TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 , mouse Toll-like receptor ligand TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13, NOD-like receptor ligand, RIG-1-like receptor ligand, Immunostimulatory nucleic acid that, Immunostimulatory RNA (isPHK), CpG-DNA, antibacterial agent or antiviral agent. Typically, the response of the innate immune system involves the recruitment of immune cells to areas of infection through the production of chemical factors, including specialized chemical mediators called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, blood and lymph by specialized leukocytes; activation of the adaptive immune system through a process known as antigen presentation; and/or functioning as a physical and chemical barrier to infectious agents.

Cloning site: A cloning site is generally understood to mean a segment of a nucleic acid molecule suitable for insertion of a nucleotide sequence, for example a nucleotide sequence containing an open reading frame. Insertion can be accomplished by any molecular biology technique known to one of skill in the art, such as restriction and ligation. The cloning site typically contains one or more sites recognized by restriction enzymes (restriction sites). These one or more restriction sites can be recognized by restriction enzymes that cleave DNA at these sites. A cloning site that contains more than one restriction site may also be referred to as a multiple cloning site (MCS) or polylinker.

Nucleic acid molecule: A nucleic acid molecule is a molecule that contains, preferably consists of nucleic acid components. The term "nucleic acid molecule" preferably refers to DNA or RNA molecules. It is preferably used as a synonym for the term "polynucleotide". Preferably, the nucleic acid molecule is a polymer containing or consisting of nucleotide monomers that are covalently linked to each other by sugar/phosphate backbone phosphodiester bonds. The term "nucleic acid" also includes modified nucleic acid molecules, such as DNA or RNA molecules with a modified base, a modified sugar moiety, or a modified backbone.

Open Reading Frame: An Open Reading Frame (ORF) in the context of the invention can typically be a multiple nucleotide triplet sequence that can be translated into a peptide or protein. The open reading frame preferably contains a start codon at its 5' end, i. e. a combination of three consecutive nucleotides encoding, as a rule, the amino acid methionine (ATG or AUG), and the region following it, the length of which, as a rule, is a multiple of 3 nucleotides. The ORF preferably ends with a stop codon (eg TAA, TAG, TGA). Typically, there is only one stop codon in an open reading frame. Thus, an open reading frame in the context of the present invention is preferably a nucleotide sequence consisting of a multiple of three nucleotides, which begins with a start codon (for example, ATG or AUG), and which preferably ends with a stop codon (for example, TAA, TGA or TAG , or UAA, UAG, UGA respectively). The open reading frame may be isolated or inserted into a longer nucleotide sequence, such as a vector or mRNA. An open reading frame may also be referred to as a "protein coding region".

Peptide: A peptide or polypeptide is typically a polymer of amino acid monomers linked by peptide bonds. It usually contains less than 50 monomeric units. However, the term "peptide" does not exclude molecules having more than 50 monomeric units. Long peptides are also called polypeptides, as a rule, they have from 50 to 600 monomer units.

Pharmaceutically effective amount: In the context of the invention, a pharmaceutically effective amount is generally understood to mean an amount sufficient to induce a pharmaceutical effect, such as an immune response, an altered abnormal level of an expressed peptide or protein, or a replacement of a missing gene product, for example, in the case of the presence of a pathological situation.

Protein: A protein typically contains one or more peptides or polypeptides. The protein is typically folded into a 3-dimensional structure, which may be required for the biological function of the protein.

Poly(A) sequence: A poly(A) sequence, also referred to as a poly(A) tail or 3'-poly(A) tail, is typically an adenosine nucleotide sequence containing, for example, up to about 400 adenosine nucleotides, e.g., from about 25 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides . The poly(A) sequence is typically located at the 3' end of the mRNA. In the context of the present invention, the poly(A) sequence may be located within an mRNA or any other nucleic acid molecule, for example within a vector, for example a vector serving as a template for making an RNA, preferably an mRNA, for example, when the vector is transcribed.

Polyadenylation: By polyadenylation is generally meant the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, eg immature mRNA. Polyadenylation can be induced by the so-called polyadenylation signal. Said signal is preferably located in a segment of nucleotides at the 3' end of the nucleic acid molecule, such as the RNA molecule to be polyadenylated. The polyadenylation signal typically contains a hexamer consisting of adenine and/or uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences are also possible, preferably hexameric sequences. Polyadenylation typically occurs during the processing of pre-mRNA (also referred to as immature mRNA). Typically, RNA maturation (conversion of pre-mRNA to mature mRNA) includes a polyadenylation step.

Restriction site: A restriction site, also referred to as a restriction enzyme recognition site, is a nucleotide sequence recognized by a restriction enzyme. The restriction site is typically a short, preferably palindromic, nucleotide sequence, such as a sequence of 4 to 8 nucleotides. The restriction site is preferably specifically recognized by the restriction enzyme. The restriction enzyme typically cleaves a nucleotide sequence containing a restriction site at that site. In a double-stranded nucleotide sequence, such as a double-stranded DNA sequence, the restriction enzyme typically cleaves both strands of the nucleotide sequence.

RNA, mRNA: RNA is the common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer made up of nucleotides. Said nucleotides are, as a rule, monomers of adenosine monophosphate, uridine monophosphate, guanosine monophosphate and cytidine monophosphate, connected to each other along the so-called backbone. The framework is formed with the help of phosphodiester bonds between sugars, i.e. ribose, the first monomer and the phosphate fragment of the second adjacent monomer. A particular sequence of monomers is called an RNA sequence. Typically, RNA can be obtained by transcription of a DNA sequence, for example, within a cell. In eukaryotic cells, transcription usually occurs in the nucleus or mitochondria. In vivo transcription of DNA, as a rule, leads to the formation of the so-called immature RNA, which, as a result of processing, must turn into the so-called messenger (messenger) RNA, which is abbreviated as mRNA. Processing of immature RNA, for example in eukaryotic organisms, involves a number of different post-transcriptional modifications such as splicing, 5' capping, polyadenylation, nuclear or mitochondrial export, and the like. The combination of these processes is also called RNA maturation. Mature messenger RNA is typically a nucleotide sequence that can be translated into the amino acid sequence of a particular peptide or protein. Typically, the mature mRNA contains a 5'cap, a 5'UTR, an open reading frame, optionally a 3'UTR, and a poly(A) sequence. In addition to messenger RNA, there are several non-coding types of RNA that may be involved in the regulation of transcription and/or translation.

Sequence of a Nucleic Acid Molecule: Sequence of a nucleic acid molecule is generally understood to mean a specific and individual arrangement, i. a series of nucleotides in it. By the sequence of a protein or peptide, as a rule, is understood the order of arrangement, i. consecutive series of amino acids included in it.

Sequence Identity: Two or more sequences are identical if they are the same length and nucleotide or amino acid order. Percent identity generally characterizes the degree of identity between two sequences, i.e. it usually characterizes the percentage of nucleotides that correspond in position to identical nucleotides in the reference sequence. When determining the degree of identity, the sequences to be compared are considered to be of the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that the first 8 nucleotide sequence is 80% identical to the second 10 nucleotide sequence that contains the first sequence. In other words, in the context of the present invention, sequence identity preferably refers to the percentage of nucleotides in a sequence that are in the same positions in two or more sequences that are the same length. In a comparative analysis of the primary structure of sequences, gaps are usually considered as non-identical positions, regardless of their actual position.

Stabilized Nucleic Acid Molecule: A stabilized nucleic acid molecule is a nucleic acid molecule, preferably a DNA or RNA molecule, modified to be more resistant to cleavage or degradation, for example by environmental factors, or to enzymatic cleavage, such as cleavage of exo- or endonucleases than the nucleic acid molecule without modification. Preferably, the stabilized nucleic acid molecule in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell. The stabilizing effect can also be exerted outside the cells, for example in a buffer solution, etc., for example during the production of a pharmaceutical composition which contains a stabilized nucleic acid molecule.

Transfection: The term "transfection" refers to the introduction of nucleic acid molecules, such as DNA or RNA (eg mRNA) molecules, into cells, preferably eukaryotic cells. In the context of the present invention, "transfection" refers to any method known to the person skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells, such as mammalian cells. Such methods include, for example, electroporation, lipofection, for example, based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethyleneimine, and etc. Preferably, the introduction is carried out without the use of viruses.

Translation efficiency (or translation efficiency): In the context of the present description, the specified concept, as a rule, refers to a property of a nucleic acid molecule (eg, mRNA) containing an open reading frame (ORF). Translation efficiency is quantifiable. Translation efficiency is generally measured by determining the amount of protein translated from the ORF. When experimentally quantifying translation efficiency, preferably the ORF encodes a reporter protein or any other quantifiable protein. However, without wishing to be bound by any particular theory, it can be assumed that high translation efficiency is usually provided by a specific UTR element (specific 5'-UTR element or specific 3'-UTR element). Thus, in the context of the present invention, the term "translation efficiency" is used primarily in relation to nucleic acid molecules that contain, in addition to ORFs, at least one 5'-UTR element and/or at least one 3'-UTR element , preferably presented in the present description. Although an ORF encoding a reporter protein or any other protein that can be quantified should be used for purposes of experimental quantification of translation efficiency, the present invention is not limited to such purposes; therefore, at least one 5'-UTR element and/or at least one 3'-UTR element of the invention (which provides high translation efficiency) may be contained in a nucleic acid molecule containing an ORF that does not encode reporter protein.

Translation efficiency is a relative concept. Thus, it is possible to determine and compare the efficiency of translation of different, for example, two or more nucleic acid molecules, for example, by experimental quantification of the protein collated by the ORF. This can be done under standardized conditions, for example using the standardized assay provided herein. Thus, the efficiency of translation, determined under standardized conditions, is an objective characteristic. Preferably, the translation efficiency of two nucleic acid molecules is determined and compared. The first of these two nucleic acid molecules may be referred to as "the nucleic acid molecule of interest" or "the construct of interest", and the second of these two nucleic acid molecules may be referred to as the "reference nucleic acid molecule" or "the reference construct". The nucleic acid molecule in question may be an artificial nucleic acid molecule according to the present invention. For the purpose in question, the reference nucleic acid molecule and the nucleic acid molecule in question must have the same ORF (having the same nucleotide sequence); and preferably the nucleotide sequence of the nucleic acid molecule in question is identical to the nucleotide sequence of the reference nucleic acid molecule except for the UTR element to be tested, i. either a 5'-CTK-element or a 3'-UTR element; in other words, the nucleic acid molecule in question and the reference nucleic acid molecule preferably differ from each other only in that either their 5'-UTR elements or their 3'-UTR elements have a different nucleotide sequence from each other; thus, the 5'-UTR element or 3'-UTR element is the only structural feature that distinguishes the nucleic acid molecule in question from the reference nucleic acid molecule.

In the presently described assay, the subject nucleic acid molecule and the reference nucleic acid molecule are transfected into mammalian cells and the efficiency of translation is determined. If the translation efficiency of the nucleic acid molecule in question is higher than the translation efficiency of the reference nucleic acid molecule, then the nucleic acid molecule in question is considered to have a high translation efficiency. In this case, the 5'-UTR element or 3'-UTR element that distinguishes the nucleic acid molecule in question and the reference nucleic acid molecule is considered to provide high translation efficiency (of the nucleic acid molecule in question). In other words, it is believed that high translation efficiency should be "provided" by a specific 5'-UTR element or 3'-UTR element that is present in the nucleic acid molecule in question, but not present in the reference nucleic acid molecule; and it is considered that the subject nucleic acid molecule (or artificial nucleic acid molecule), which contains such a 5'-UTR or 3'-UTR and at least one ORF, "should be characterized" by high translation efficiency.

Vaccine: A vaccine is generally understood to mean a prophylactic or therapeutic product containing at least one antigen, preferably an immunogen. The antigen or immunogen may be derived from any material suitable for vaccination. For example, the antigen or immunogen may be from a pathogen such as bacteria or viral particles, etc., or from a tumor or cancer tissue. The antigen or immunogen stimulates the body's adaptive immune system to produce an adaptive immune response.

Vector: The term "vector" refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule. In the context of the present invention, a vector can be used to insert or store a desired nucleotide sequence, such as a nucleotide sequence containing an open reading frame. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, and the like. A storage vector is a vector that allows convenient storage of a nucleic acid molecule, such as an mRNA molecule. Thus, the vector may contain a sequence corresponding to, for example, the desired mRNA sequence or a portion thereof, such as a sequence corresponding to the open reading frame and the 3'-UTR and/or 5'-UTR of the mRNA. An expression vector can be used to produce expression products such as RNA, eg mRNA, or peptides, polypeptides or proteins. For example, the expression vector may contain sequences necessary for transcription of a segment of the vector's sequence, such as a promoter sequence, such as an RNA polymerase promoter sequence. A cloning vector is typically a vector that contains a cloning site that can be used to insert nucleotide sequences into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage-based vector. The transfer vector may be a vector that is suitable for transferring nucleic acid molecules into cells or organisms, such as viral vectors. In the context of the present invention, the vector may be, for example, an RNA vector or a DNA vector. Preferably the vector is a DNA molecule. In the context of the present description, preferably the vector contains a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for propagating the vector, such as an origin of replication. In the context of the present description, preferably the vector is a plasmid vector.

Excipient: An excipient is generally understood to mean a material suitable for storing, transporting and/or administering a compound, such as a pharmaceutically active compound. It may be, for example, a physiologically acceptable liquid suitable for storing, transporting and/or administering a compound, such as a pharmaceutically active compound.

3'-untranslated region (3'-UTR): In general, the term "3'-UTR" refers to the part of an artificial nucleic acid molecule that is located on the 3'-side (i.e. "downstream") of the open frame reading and which is not translated into protein. Typically, the 3'UTR is the portion of the mRNA that is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the mRNA poly(A) sequence. In the context of the invention, the term "3'-UTR" may also include elements that are not encoded in the template from which the RNA is transcribed, but which are added after transcription during maturation, for example, a poly(A) sequence. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is typically encoded by a gene that is transcribed into the corresponding mRNA during gene expression. The genomic sequence is first transcribed into immature mRNA, which contains optional introns. The immature mRNA is then further processed into mature mRNA during maturation. Said maturation process includes the steps of 5' capping, splicing of the immature mRNA with excision of optional introns and 3' end modifications such as polyadenylation of the 3' end of the immature mRNA, and optional endo-/or exonuclease cleavages, etc. In the context of the present invention, the 3'-UTR corresponds to a mature mRNA sequence that is located between the stop codon of the protein coding region, preferably immediately adjacent 3' to the stop codon of the protein coding region, and the mRNA poly(A) sequence. The term "corresponds" means that the 3'-UTR sequence may be an RNA sequence, such as in the mRNA sequence specified in the definition of the 3'UTR sequence, or a DNA sequence that corresponds to the specified RNA sequence. In the context of the present invention, the term "3'-UTR of a gene" means a sequence that corresponds to the 3'-UTR of a mature mRNA derived from said gene, i.e. mRNA obtained by transcription of a gene and maturation of immature mRNA. The term "3'-UTR of a gene" includes a DNA sequence and an RNA sequence (both sense and antisense strands, and both mature and immature sequences) 3'-UTR. Preferably the 3'-UTRs are greater than 20, 30, 40 or 50 nucleotides in length.

5'-Untranslated Region (5'-UTR): The term "5'-UTR" generally refers to the part of an artificial nucleic acid molecule that is located on the 5' side (i.e., "backward") relative to open reading frame of mRNA and which is not translated into protein. Under the 5'-UTR, as a rule, understand the specific segment of messenger RNA (mRNA), which is located on the 5'-side of the open reading frame of the mRNA. Typically, the 5'-UTR starts at the site of transcription initiation and ends one nucleotide before the start codon of the open reading frame. Preferably the 5'-UTR is greater than 20, 30, 40 or 50 nucleotides in length. The 5'-UTR may contain elements designed to control gene expression, so-called regulatory elements. Said regulatory elements may be, for example, ribosome binding sites. The 5'-UTR may be subject to post-transcriptional modifications, for example by adding a 5'-cap. The 5'-UTR is not translated into an amino acid sequence. The 5'UTR sequence is typically encoded by a gene that is transcribed into the corresponding mRNA during gene expression. The genomic sequence is first transcribed into immature mRNA, which contains optional introns. The immature mRNA is then further processed into mature mRNA during maturation. Said maturation process includes the steps of 5' capping, splicing of the immature mRNA with excision of optional introns and 3' end modifications such as polyadenylation of the 3' end of the immature mRNA, and optional endo-/or exonuclease cleavages, etc. In the context of the present invention, the 5'-UTR corresponds to a mature mRNA sequence located between the start codon and, for example, the 5'-cap. Preferably, the 5'-UTR corresponds to a sequence that extends from the nucleotide located 3'-side of the 5'-cap, more preferably from the nucleotide immediately adjacent 3'-side of the 5'-cap, to the nucleotide located 5'- side of the start codon of the protein coding region, preferably up to the nucleotide immediately adjacent on the 5' side of the start codon of the protein coding region. The nucleotide immediately adjacent on the 3' side to the 5' cap of the mature mRNA usually corresponds to the site of transcription initiation. The term "corresponds" means that the 5'-UTR sequence may be an RNA sequence, such as in the mRNA sequence specified when determining the 5'UTR sequence, or a DNA sequence that corresponds to the specified RNA sequence. In the context of the present invention, the term "5'-UTR of a gene" means a sequence that corresponds to the 5'-UTR of the mature mRNA derived from this gene, i.e. mRNA obtained by transcription of a gene and maturation of immature mRNA. The term "5'UTR of a gene" includes a DNA sequence and an RNA sequence (both sense and antisense strands, and both mature and immature sequences) of the 5'UTR.

5' oligopyrimidine tract (TOR): The 5' oligopyrimidine tract (TOR) generally refers to a segment of pyrimidine nucleotides located at the 5' end of a nucleic acid molecule, such as the 5' end of some mRNA molecules. or in the 5'-terminal region of a functional element, for example, the transcribed region of some genes. The sequence begins with a cytidine, which typically corresponds to the site of transcription initiation, and is followed by a segment typically consisting of about 3-30 pyrimidine nucleotides. For example, TOP can contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidine segment, and hence the 5'-TOP, terminates in one nucleotide located 5'-direction relative to the first purine nucleotide located downstream of the TOP. Messenger RNA that contains the 5' oligopyrimidine tract is often referred to as TOP mRNA. Thus, the genes from which these messenger RNAs are derived are referred to as TOP genes. TOP sequences, for example, are found in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, the TOP motif is a nucleotide sequence that corresponds to the 5'-TOP described above. Thus, the TOP motif in the context of the present invention preferably denotes a 3-30 nucleotide long segment consisting of pyrimidine nucleotides. Preferably the TOP motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, with the pyrimidine nucleotide segment preferably beginning at its 5' end with a cytosine nucleotide. In TOP genes and TOP mRNA, the TOP motif preferably starts at its 5' end from the site of transcription initiation and ends one nucleotide away in the 5' direction from the first purine residue in said gene or mRNA. In the context of the present invention, the TOP motif is preferably located at the 5'end of the sequence which is the 5'UTR or at the 5'end of the sequence which encodes the 5'UTR. Thus, in the context of the present invention, a segment of 3 or more pyrimidine nucleotides is preferably referred to as a "TOP motif" if the segment is located at the 5' end of the corresponding sequence, such as an artificial nucleic acid molecule, the 5'UTR element of the artificial nucleic acid molecule or a nucleotide sequence that is derived from the 5'UTR of the TOP gene provided herein. In other words, a segment of 3 or more pyrimidine nucleotides that is not located at the 5' end of the 5'-UTR or 5'-UTR element, but is located somewhere within the 5'-UTR or 5'-UTR- element is preferably not referred to as a "TOP motif".

TOP gene: TOP genes are generally characterized by the presence of a 5'-terminal oligoprimidine tract. In addition, most TOP genes are characterized by growth-related regulation of translation. However, TOP genes with tissue-specific regulation of translation are also known. As indicated above, the 5'-UTR of the TOP gene corresponds to the sequence of the 5'-UTR of the mature mRNA derived from the TOP gene, which preferably extends from the nucleotide adjacent 3' to the 5' cap to the nucleotide adjacent 5 '-sides to the start codon. The 5'UTR of the TOP gene generally does not contain any start codons, preferably no upstream AUGs (uAUGs) or upstream open reading frames (uOPCs). In this context, reversed AUGs and reversed open reading frames are generally understood to mean AUGs and open reading frames that are 5' from the start codon (AUG) of the open reading frame to be translated. The 5'UTRs of TOP genes tend to be relatively short. The lengths of the 5'UTRs of TOP genes can vary from 20 nucleotides up to 500 nucleotides and are typically less than about 200 nucleotides, preferably less than about 150 nucleotides, more preferably less than about 100 nucleotides. Examples of the 5' UTRs of TOP genes in the context of the present invention are nucleotide sequences extending from the nucleotide at position 5 to the nucleotide immediately adjacent to the 5' end of the start codon (eg, ATG) in the sequences shown in SEQ ID NO: 1 -1363 patent application PCT/EP 2012/002448, the contents of which are incorporated herein by reference. In this context, the most preferred fragment of the 5'UTR of the TOP gene is the 5'UTR of the TOP gene without the 5'TOP motif. The terms "5'-UTR of the TOP gene" or "5'-TOP UTR" preferably refers to the 5'-UTR of the naturally occurring TOP gene. A preferred example is SEQ ID NO: 208 (5'-UTR of the large subunit of human ribosomal protein 32 without a 5'-oligopyrimidine tract); which corresponds to SEQ ID NO: 1368 presented in patent application WO 2013/143700).

Wild type, eg, wild type nucleic acid molecule: The term "wild type" may refer to a naturally occurring sequence. A wild-type nucleic acid molecule is generally understood to mean a nucleic acid molecule, such as DNA or RNA, that occurs naturally. In other words, an artificial nucleic acid molecule can be understood as a naturally occurring nucleic acid molecule. Such a nucleic acid molecule may be considered to be naturally occurring due to its individual sequence (which occurs naturally) and/or due to the presence of other modifications, such as structural modifications of nucleotides, which occur naturally. The wild-type nucleic acid molecule can be a DNA molecule, an RNA molecule, or a hybrid molecule containing DNA and RNA segments. In the context of the present description, the term "wild type" refers to any sequence, if it occurs in nature, it is not required that it be in publicly available collections of sequences, such as GenBank. The National Institutes of Health (NIH) maintains a publicly accessible annotated collection of publicly available nucleotide sequences ("GenBank", accessible through the NCBI Entrez search engine: http://www.ncbi.nlm.nih.gov), (Nucleic Acids Research; 41( D1), 2013, D36-42), which includes publicly available wild-type sequences. Each GenBank record is assigned a unique permanent identifier, called an access code, which appears in the ACCESSION line of the GenBank record; and changes in the sequence data are tracked by the numeric expansion of the access code present in the VERSION line of the GenBank record unit. In addition, the concept of "wild-type nucleic acid molecule" is not limited to the concept of "one single molecule", but, as a rule, it is understood that it includes a set of identical molecules. Therefore, it refers to a plurality of identical molecules contained in an aliquot.

Detailed description

The invention relates to an artificial nucleic acid molecule containing at least one open reading frame and at least one element of the 3'-untranslated region (3'-UTR element), and/or at least one element of the 5'-untranslated region ( 5'-UTR element), where the specified artificial nucleic acid molecule is characterized by high translation efficiency. Translation efficiency is due at least in part to the 5'UTR element or the 3'UTR element, or both the 5'UTR element and the 3'UTR element. The invention also relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. In addition, new 3'-UTR elements and 5'-UTR elements have been proposed.

The first object of the present invention relates to an artificial nucleic acid molecule containing

a. at least one open reading frame (ORF); and

b. at least one element representing a 3'-untranslated region (3'-UTR element) and/or at least one element representing a 5'-untranslated region (5'-UTR element),

wherein said artificial nucleic acid molecule is characterized by high translation efficiency.

For example, an artificial nucleic acid molecule of the invention may differ from other (eg, wild-type or artificial) nucleic acid molecules in that at least one 3'-UTR element (preferably one 3'-UTR element) is replaced at least one non-identical 3'-UTR element (preferably one 3'-UTR element), or that at least one 5'-UTR element (preferably one 5'-UTR element) is replaced by at least one non-identical 5'UTR element (preferably one 5'UTR element).

In general, a substitution of one (or at least one) 3'UTR element, or a substitution of one (or at least one) 5'UTR element (in the parent or reference nucleic acid molecule) is generally , means that a sequence containing at least some of the nucleotides of the specified 3'-UTR element or the specified 5'-UTR element, is replaced with a non-identical nucleic acid sequence. Preferably, when the 5'-UTR element is replaced, the contiguous nucleotide sequence of the 5'-UTR element is replaced with a non-identical contiguous nucleotide sequence (element); and when the 3'-UTR element is replaced, the contiguous nucleotide sequence of the 3'-UTR element is replaced with a non-identical contiguous nucleotide sequence (element). A continuous sequence of nucleotides of the original (to be replaced) element and the replacement element, each independently can be of any length, for example, from 1 to more than 500 nucleotides, from 20 to 500 nucleotides, from 40 to 400 nucleotides, from 60 to 300 nucleotides, from 80 up to 200 nucleotides or from 100 to 150 nucleotides. When a (5'- or 3'-) UTR element is replaced, this does not mean that all nucleotides located 5'-side of the start codon or all nucleotides located 3'-side of the stop codon need to be replaced. In preferred embodiments, a contiguous sequence is replaced that is a subset of all nucleotides located 5' to the start codon or a subset of all nucleotides located 3' to the stop codon, respectively. Such examples are shown in Fig. 1B (5') and 1C (3'). You can replace a well-known 5'-UTR element or a known 3'-UTR element present in the original sequence or reference sequence. For example, if a nucleic acid (for example, a nucleic reference acid) contains a 5'-UTR element corresponding to the sequence shown in SEQ ID NO: 208, then an artificial nucleic acid molecule of the present invention can be created by replacing strictly the entire continuous sequence shown in SEQ ID NO: 208, on a non-identical continuous 5'-UTR element, proposed in the present invention, ie. on the 5'-UTR element, which ensures high translation efficiency. Alternatively, it is possible to replace not only the (strictly) known UTR element, for example, to replace a continuous nucleotide sequence containing additional nucleotides, i. e. that are complementary to a known UTR element, or, for example, replace only part of a known UTR element (usually the main part, i.e. 90% of its length or more). While these possibilities are illustrated herein for 5'UTR elements, the same possibilities exist for 3'UTR elements as well. In other words, the replaced UTR element need not strictly conform to the boundaries of the known UTR element. These possibilities are further illustrated with the example in FIG. 1B and FIG. 1B respectively, in each case in comparison with FIG. 1A. Similarly, a replacement UTR element does not need to strictly conform to the boundaries of a known UTR element.

Preferably, the artificial nucleic acid molecule of the present invention does not contain the 3'-UTR (element) and/or 5'-UTR (element) of the S6 ribosomal protein, RPL36AL, rps16 or the L9 ribosomal protein. More preferably, the artificial nucleic acid molecule of the present invention does not contain the 3'-UTR (element) and/or 5'-UTR (element) of the S6 ribosomal protein, RPL36AL, rps16 or L9 ribosomal protein and the open reading frame of the artificial nucleic acid molecule , proposed in the present invention, does not encode the GFP protein. Even more preferably, the artificial nucleic acid molecule of the present invention does not contain the 3'-UTR (element) and/or 5'-UTR (element) of ribosomal protein S6, RPL36AL, rps16 or ribosomal protein L9 and the open reading frame of the artificial nucleic acid molecule acid of the present invention does not encode a reporter protein, for example, selected from the group consisting of globin proteins (primarily beta-globin), luciferase protein, GFP proteins, glucuronidase proteins (primarily beta-glucuronidase) or their variants, for example, variants whose sequences are at least 70% identical to the sequence of a globin protein, a luciferase protein, a GFP protein, or a glucuronidase protein.

The term "3'-UTR element" refers to a nucleotide sequence that contains or consists of a nucleotide sequence derived from a 3'-UTR or from a 3'-UTR variant or fragment. The term "3'-UTR element" preferably refers to a nucleotide sequence that is contained in a 3'-UTR of an artificial nucleotide sequence, such as an artificial mRNA. Therefore, in the context of the present invention, the 3'UTR element may preferably be the 3'UTR of an mRNA, preferably an artificial mRNA, or may be contained in the 3'UTR of an appropriate transcription template. Preferably, the 3'UTR element is a nucleotide sequence that corresponds to the 3'UTR of an mRNA, preferably the 3'UTR of an artificial mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, the 3'UTR element in the context of the present invention functions as a 3'UTR or encodes a nucleotide sequence that functions as a 3'UTR.

Accordingly, the term "5'-UTR element" refers to a nucleotide sequence that contains or consists of a nucleotide sequence derived from a 5'-UTR or from a 5'-UTR variant or fragment. The term "5'-UTR element" preferably refers to a nucleotide sequence that is contained in a 5'-UTR of an artificial nucleotide sequence, such as an artificial mRNA. Therefore, in the context of the present invention, the 5'UTR element may preferably be the 5'UTR of an mRNA, preferably an artificial mRNA, or may be contained in the 5'UTR of an appropriate transcription template. Preferably the 5'UTR element is a nucleotide sequence that corresponds to the 5'UTR of an mRNA, preferably the 5'UTR of an artificial mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, the 5'UTR element in the context of the present invention functions as a 5'UTR or encodes a nucleotide sequence that functions as a 5'UTR.

The 3'UTR element and/or the 5'UTR element in the artificial nucleic acid molecule of the present invention provides high translation efficiency of said artificial nucleic acid molecule. Thus, an artificial nucleic acid molecule according to the present invention may, in particular, contain:

3'-UTR element, which ensures high efficiency of translation of the specified artificial nucleic acid molecule,

5'-UTR element, which ensures high efficiency of translation of the specified artificial nucleic acid molecule,

a 3'UTR element that provides high translation efficiency of said artificial nucleic acid molecule; and a 5'UTR element that provides high translation efficiency of said artificial nucleic acid molecule.

Preferably, the artificial nucleic acid molecule of the present invention comprises a 3'-UTR element that provides high translation efficiency of said artificial nucleic acid molecule and/or a 5'-UTR element that provides high translation efficiency of said artificial nucleic acid molecule. .

Preferably, the artificial nucleic acid molecule of the present invention contains at least one 3'-UTR element and at least one 5'-UTR element, namely at least one 3'-UTR element, which provides high translation efficiency of said artificial nucleic acid molecule, and at least one 5'-UTR element that provides high translation efficiency of said artificial nucleic acid molecule.

As detailed below, said at least one 3'UTR element that provides high translation efficiency of said artificial nucleic acid molecule, or said at least one 5'UTR element that provides high translation efficiency of said artificial nucleic acid molecule acids, can be selected from naturally occurring (preferably heterologous) 3'-UTR elements and 5'-UTR elements (collectively referred to as naturally occurring UTR elements or wild-type UTR elements), and from artificial 3'UTR elements and artificial 5'UTR elements (collectively referred to as artificial UTR elements). Wild type UTR elements can be selected from the group consisting of wild type UTR elements known from the literature and available in publicly available databases such as GenBank (NCBI) and wild type UTR elements that have not been previously published. The latter can be identified by sequencing mRNAs present in cells, preferably mammalian cells. Using this approach, several previously unpublished wild-type UTR elements were identified in the invention, and UTR elements of this type are proposed in the present invention. The term "artificial UTR element" is not particularly limited and may refer to any nucleotide sequence that is not found naturally, i.e. is not identical to a wild-type UTR element. However, in preferred embodiments of the invention, the artificial UTR element used according to the present invention is a nucleotide sequence that has a certain degree of identity with the sequence of the wild-type UTR element, for example, ranging from 10 to 99.9%, from 20 to 99 %, 30 to 98%, 40 to 97%, 50 to 96%, 60 to 95%, 70 to 90%. In preferred embodiments, the artificial UTR used according to the present invention is identical to the wild-type UTR except that one or two or three or four or five or more than five nucleotides are replaced by the same number of nucleotides (e.g., one nucleotide is replaced by one nucleotide). Preferably, the single nucleotide substitution is a substitution for the corresponding complementary nucleotide. Preferred artificial UTR elements correspond to wild-type UTR elements, except that (i) some or all of the ATG triplets in the wild-type 5' UTR element (if present) are converted to a TAG triplet; and/or (II) the selected restriction enzyme cleavage site(s) in the wild-type 5'-UTR element or in the wild-type 3'-UTR element (if present) are eliminated by replacing one nucleotide in the cleavage site of the specified specific restriction enzyme to a complementary nucleotide, which results in the removal of cleavage sites by said specific restriction enzyme. The latter is generally required when the UTR element (eg, wild-type) contains a cleavage site for said specific restriction enzyme, and when said specific restriction enzyme is (is intended to be) used in subsequent cloning steps.

Since such cleavage within 5'-UTR elements and 3'-UTR elements is undesirable, it is possible to create an artificial UTR element in which the restriction site cleaved by said specific restriction enzyme is eliminated. Such replacement can be carried out using any suitable method known to the person skilled in the art, for example, using PCR using modified primers.

The artificial nucleic acid molecule of the present invention optionally contains at least one 3'UTR element and at least one 5'CTC element, namely at least one 3'UTR element that prolongs and/or increases the production of a protein encoded by said artificial nucleic acid molecule and at least one 5'-UTR element that prolongs and/or increases the production of a protein encoded by said artificial nucleic acid molecule.

Protein production: Assay to determine if there is a prolongation and/or increase in protein production

"Prolonging and/or increasing protein production from said artificial nucleic acid molecule" generally refers to the amount of protein produced from an artificial nucleic acid molecule of the present invention that contains the appropriate 3'-UTR element and/or 5'-UTR element, as compared to the amount of protein produced from the corresponding nucleic acid reference that does not contain a 3'-UTR and/or 5'-UTR or contains a reference 3'-UTR and/or a reference 5'-UTR, such as a 3'UTR and/or a 5'UTR occurring naturally in combination with an ORF.

In particular, at least one 3'-UTR element and/or 5'-UTR element of the artificial nucleic acid molecule of the present invention prolongs the production of protein from the artificial nucleic acid molecule of the present invention, for example, from mRNA , proposed in the present invention, compared with the corresponding nucleic acid, which does not contain 3'-UTR and/or 5'-UTR or contains a reference-3'-UTR and/or reference-5'-UTR, such as 3'- UTR and/or 5'-UTR occurring naturally in combination with ORF.

In particular, at least one 3'-UTR element and/or 5'-UTR element of the artificial nucleic acid molecule of the present invention increases protein production, especially protein expression or overall protein production, from the artificial nucleic acid molecule. , proposed in the present invention, for example, with the mRNA proposed in the present invention, compared with the corresponding nucleic acid that does not contain a 3'-UTR and/or 5'-UTR or contains a reference 3'-UTR and/or a reference 5'-UTR, such as 3'-UTR and/or 5'-UTR, occurring naturally in combination with ORF.

Preferably, at least one 3'-UTR element and/or at least one 5'-UTR element of the artificial nucleic acid molecule of the present invention does not adversely affect the translation efficiency of the nucleic acid compared to the translation efficiency of the corresponding nucleic acid. an acid that does not contain a 3'-UTR and/or a 5'-UTR or contains a reference 3'-UTR and/or a reference 5'-UTR, such as a 3'-UTR and/or a 5'-UTR occurring in natural conditions in combination with ORS. Alternatively, the 3'-UTR and/or 5'-UTR increase the efficiency of translation compared to the efficiency of translation of the protein encoded by the corresponding ORF in its natural environment.

The term "corresponding nucleic acid molecule" or "reference nucleic acid molecule", as used in the present description, means that with the exception of different 3'-UTR and/or 5'-UTR, the reference nucleic acid molecule is comparable, preferably identical, artificial a nucleic acid molecule according to the invention which contains a 3'UTR element and/or a 5'UTR element.

To evaluate the production of a protein from an artificial nucleic acid molecule of the invention in vivo or in vitro as defined herein (i.e., in vitro refers to (“live”) cells and/or tissue, including tissue from a living individual; cells include in particular cell lines, primary cells, cells in tissue or in the body of individuals, mammalian cells such as human cells and mouse cells are preferred, and most preferred are human HeLa, HEPG2 and U-937 cell lines and mouse NIH3T3 cell lines , JAWSII and L929, in addition, the most preferred are primary cells, in the most preferred embodiments of the invention - human dermal fibroblasts (HDF)), determine the expression of the encoded protein after the injection / transfection of the artificial nucleic acid molecule of the invention into cells / tissues -targets and comparisons with protein expression induced by nucleic reference acid. Quantitative methods for determining protein expression are known in the art (eg Western blotting, FACS, ELISA, mass spectrometry). In this context, the most suitable is the determination of the expression of reporter proteins such as luciferase, green fluorescent protein (GFP) or secreted alkaline phosphatase (SEAP). To do this, the artificial nucleic acid according to the invention or the reference nucleic acid is introduced into the target tissue or target cell, for example by transfection or injection, preferably into a mammalian expression system, such as mammalian cells, for example into HDF cells, L929, HepG2 and/or Hela. Hours or days later (eg 6, 12, 24, 48 or 72 hours) after initiation of expression or after introduction of the nucleic acid molecule, a sample of target cells is taken and measured by FACS and/or lysed. The lysates can then be used to detect the expressed protein (and thereby determine the efficiency of protein expression) by several methods, such as Western blotting, FACS, ELISA, mass spectrometry, or by measuring fluorescence or luminescence.

Therefore, if the expression of a protein from an artificial nucleic acid molecule of the invention is compared with the expression of a protein from a reference nucleic acid molecule at certain time points (for example, 6, 12, 24, 48, or 72 hours after the initiation of expression or after the introduction of the molecule nucleic acid), then both nucleic acid molecules are introduced separately into target tissues/cells, a sample of tissue/cells is taken at a certain point in time, protein lysates are prepared according to a specific protocol adjusted for a specific detection method (for example, for Western blotting, ELISA, fluorescence or luminescence measurements, etc., as known in the art) and detect the protein using the selected detection method. As an alternative to measuring the amounts of expressed protein in cell lysates, or in addition to measuring the amounts of protein in cell lysates, FACS analysis can also determine the amount of protein prior to lysis of harvested cells or in parallel using aliquots.

The term "prolongation of protein production" from an artificial nucleic acid molecule, such as an artificial mRNA, preferably means that the production of a protein from an artificial nucleic acid molecule, such as an artificial mRNA, is prolonged compared to the production of a protein from a reference nucleic acid molecule, such as a reference -mRNA, for example, which contains a reference 3'-UTR and/or a reference-5'-UTR or lacks a 3'-UTR and/or a reference-5'-UTR, preferably in a mammalian expression system such as HDF, L929, HEP2G or HeLa cells. Thus, a protein produced from an artificial nucleic acid molecule, such as an artificial mRNA, can be detected for a longer period of time than that during which a protein produced from a reference nucleic acid molecule can be detected. In other words, the amount of protein produced from an artificial nucleic acid molecule, such as an artificial mRNA, measured at a later point in time, for example, 48 hours or 72 hours after transfection, is greater than the amount of protein produced from a reference nucleic acid molecule, such as a reference mRNA, at an appropriate later time point. Such a "later time point" can be, for example, any time point after 24 hours after initiation of expression, such as transfection with a nucleic acid molecule, for example, 36, 48, 60, 72, 96 hours after initiation of expression, i.e. e. after transfection. In addition, for the same nucleic acid, the amount of protein produced at a later time point can be standardized with respect to the amount produced at an earlier (reference) time point, for example, the amount of protein produced at a later time point can be expressed as as a percentage of the amount of protein 24 h after transfection.

Preferably, the effect of prolonging protein production is determined by (i) measuring the amounts of protein, for example, obtained from the expression of an encoded reporter protein such as luciferase, preferably in a mammalian expression system such as HDF, L929, HEP2G or HeLa cell lines, as a function of time , (II) determining the amount of protein detected at the "reference" time t ₁ , for example, t ₁ =24 h after transfection, while the specified amount of protein is taken as 100%, (III) measuring the amount of protein found in one or several later time points t ₂ , t ₃ etc., for example, t ₂ =48 h and t ₃ =72 h after transfection, and calculating the relative amount of protein found at the later time point as a percentage of the amount of protein at time t ₁ . For example, for a protein expressed at time t ₁ in the amount of "80", at time t ₂ in the amount of "20" and at time t ₃ in the amount of "10", the relative amount of protein at time t ₂ will be 25 %, and at time t ₃ 12.5%. These relative amounts at a later time point can then be compared in step (IV) with the relative amounts of protein at the respective time points obtained in the case of a nucleic acid molecule lacking the 3'- and/or 5'-UTR, respectively, or containing reference-3 '-UTR and/or reference-5'-UTR, respectively. By comparing the relative amount of protein produced from the artificial nucleic acid molecule of the present invention with the relative amount of protein produced from the reference nucleic acid molecule, i. molecule that does not contain 3'- and/or 5'-UTR, respectively, or contains a reference-3'-UTR and/or reference-5'-UTR, respectively, it is possible to determine the coefficient characterizing how many times the production of a protein from an artificial nucleic molecule is prolonged acid of the present invention compared to the production of a protein from a reference nucleic acid molecule.

Preferably, at least one 3'- and/or 5'-UTR element in the artificial nucleic acid molecule of the invention prolongs protein production from said artificial nucleic acid molecule by at least 1.2 times, preferably at least 1.5-fold, more preferably at least 2-fold, even more preferably at least 2.5-fold, compared to protein production from a reference nucleic acid molecule that does not contain a 3'- and/or 5'-UTR respectively or contains a reference 3'-UTR and/or a reference 5'-UTR, respectively. In other words, the (relative) amount of protein produced from the artificial nucleic acid molecule according to the invention, at a certain later point in time indicated above, is increased by a factor of at least 1.2, preferably at least 1.5, more preferably at least 2, even more preferably at least 2.5, compared to the (relative) amount of protein produced from a reference nucleic acid molecule that does not contain the 3'- and/or 5'-UTR, respectively, or contains reference-3'-UTR and/or reference-5'-UTR, respectively, at the same later point in time.

Alternatively, the prolongation effect on protein production can also be determined by (I) measuring the amounts of protein, for example, obtained from the expression of an encoded reporter protein such as luciferase, preferably in a mammalian expression system such as HDF, L929, HEP2G or HeLa cell lines, depending on time, (II) determining the point in time at which the amount of protein falls below the amount of protein detected, for example, 1, 2, 3, 4, 5, or 6 h after the initiation of expression, for example, after 1, 2, 3 , 4, 5, or 6 hours after transfection with the artificial nucleic acid molecule, and (III) comparing the time point at which the amount of protein becomes lower than the amount of protein detected, for example, 1, 2, 3, 4, 5, or 6 hours after initiation of expression , with a specified time point defined for a nucleic acid molecule that lacks a 3'-UTR and/or a 5'-UTR, respectively, or that contains a reference-3'-UTR and/or a reference-5'-U TR respectively.

For example, producing a protein from an artificial nucleic acid molecule, such as an artificial mRNA, in an amount that is at least the amount found in the initial phase of expression, for example, 1, 2, 3, 4, 5, or 6 hours after initiation of expression, for example, after transfection with a nucleic acid molecule, prolongs at least about 5 hours, preferably at least about 10 hours, more preferably at least about 24 hours compared to the production of a protein from a reference nucleic acid molecule, such as a reference -mRNA, in a mammalian expression system such as mammalian cells, for example HDF, L929, HEP2G or HeLa cell lines. Thus, the artificial nucleic acid molecule of the present invention preferably allows the prolongation of protein production in an amount that is at least the amount found in the initial phase of expression, for example, 1, 2, 3, 4, 5 or 6 hours after initiation of expression, e.g., after transfection, for at least about 5 hours, preferably at least about 10 hours, more preferably at least about 24 hours, compared to producing a protein from a reference nucleic acid molecule lacking 3 '-UTR and/or 5'-UTR, respectively, or which contains a reference-3'-UTR and/or a reference-5'-UTR, respectively.

In preferred embodiments of the invention, the period of production of a protein from an artificial nucleic acid molecule of the present invention is extended by at least 1.2 times, preferably at least 1.5 times, more preferably at least 2 times, even more preferably at least 2.5 times the period of protein production from a reference nucleic acid molecule that lacks a 3'-UTR and/or a 5'-UTR, respectively, or that contains a reference 3'-UTR and/or a reference -5'-UTR, respectively.

Preferably, said prolongation effect on protein production is achieved when the total amount of protein produced from the artificial nucleic acid molecule of the present invention, for example, over a period of 48 or 72 hours, corresponds to at least the amount of protein produced from the reference a nucleic acid molecule that lacks a 3'-UTR and/or a 5'-UTR, respectively, or that contains a reference-3'-CTK and/or a reference-5'-UTR, respectively, such as a 3'-UTR and/or a 5' -UTR that occurs naturally with the ORF of an artificial nucleic acid molecule. Thus, the present invention provides an artificial nucleic acid molecule that provides prolonged protein production in a mammalian expression system, such as mammalian cells, for example, in HDF, L929, HEP2G or HeLa cells, as described above, while the total amount of protein produced from said artificial nucleic acid molecule, for example, over a period of 48 or 72 hours, is at least the total amount of protein produced, for example, during a specified period of time, from a reference nucleic acid molecule that lacks 3 '-UTR and/or 5'-UTR, respectively, or which contains a reference-3'-UTR and/or a reference-5'-UTR, respectively, such as a 3'-UTR and/or 5'-UTR, naturally occurring with ORF of an artificial nucleic acid molecule.

In addition, the term "prolonged protein expression" also includes the term "stable protein expression", while "stable protein expression" preferably means that there is a more uniform production of protein from the artificial nucleic acid molecule of the present invention for a predetermined period of time. a period of time, e.g., within 24 hours, more preferably within 48 hours, even more preferably within 72 hours, as compared to a reference nucleic acid molecule, e.g. -5'-UTR, respectively, or lacking a 3'-UTR and/or 5'-UTR, respectively.

Therefore, the level of protein production, for example, in a mammalian system, from an artificial nucleic acid molecule containing a 3'-UTR element and/or a 5'-UTR element of the present invention, for example, from an mRNA of the present invention, preferably does not decrease to the level found for a reference nucleic acid molecule, such as the reference mRNA mentioned above. To assess the extent to which the production of protein from a specific nucleic acid molecule is reduced, one can compare, for example, the amount of protein (encoded by the corresponding ORF) found 24 hours after initiation of expression, for example, 24 hours after transfection with an artificial nucleic acid molecule, proposed in the present invention, a cell, such as a mammalian cell, with the amount of protein detected 48 hours after initiation of expression, for example, 48 hours after transfection. Thus, the ratio of the amount of protein encoded by the ORF of the artificial nucleic acid molecule of the present invention, such as the amount of a reporter protein, such as luciferase, detected at a later point in time, such as 48 hours after initiation of expression, such as after transfection, to the amount of protein detected at an earlier time point, e.g. 24 hours after initiation of expression, e.g. after transfection, preferably greater than the corresponding ratio (determined for the same time points) for a reference nucleic acid molecule containing reference-3 '- and/or reference-5'-UTR, respectively, or which lacks a 3'-UTR and/or 5'-UTR, respectively.

Preferably, the ratio of the amount of protein encoded by the ORF of the artificial nucleic acid molecule of the present invention, e.g., the amount of a reporter protein, e.g. protein detected at an earlier time point, e.g., 24 hours after initiation of expression, e.g., after transfection, is preferably at least 0.2, more preferably at least about 0.3, even more preferably at least about 0 .4, even more preferably at least about 0.5, and most preferably at least about 0.7. For the corresponding reference nucleic acid molecule, e.g. mRNA, which contains the reference 3'-UTR and/or the reference 5'-UTR, respectively, or which lacks the 3'-UTR and/or 5'-UTR, respectively, the indicated ratio may be, for example, from about 0.05 to about 0.35.

Thus, the present invention provides an artificial nucleic acid molecule comprising an ORF and a 3'-UTR element and/or a 5'-UTR element as described above, wherein the ratio of the amount of protein, e.g., the amount of luciferase, detected 48 hours after of initiation of expression, to the amount of protein found 24 hours after initiation of expression, preferably in a mammalian expression system such as mammalian cells, for example in HDF cells or HeLa cells, is preferably at least 0.2, more preferably at least at least about 0.3, more preferably at least about 0.4, even more preferably at least about 0.5, even more preferably at least about 0.6, and most preferably at least about 0.7. Thus, preferably, the total amount of protein produced from said artificial nucleic acid molecule, for example, over a period of 48 hours, at least corresponds to the total amount of protein produced, for example, during said period of time, from a reference nucleic acid molecule. an acid which lacks a 3'-UTR and/or a 5'-UTR, respectively, or which contains a reference-3'-UTR and/or a reference-5'-UTR, respectively, such as a 3'-UTR and/or a 5'-UTR , which occurs naturally with the ORF of an artificial nucleic acid molecule.

Preferably, the present invention provides an artificial nucleic acid molecule comprising an ORF and a 3'UTR element and/or a 5'UTR element as described above, wherein the ratio of the amount of protein, e.g. the amount of luciferase, detected 72 hours after initiation of expression , to the amount of protein detected 24 hours after initiation of expression, preferably in a mammalian expression system such as HeLa or HDF cells, preferably greater than about 0.05, more preferably greater than about 0.1, more preferably greater than about 0 ,2, even more preferably greater than about 0.3, preferably the total amount of protein produced from said artificial nucleic acid molecule, for example, over a period of 72 hours, is at least the total amount of protein produced, for example , during the specified period of time from the reference nucleic acid molecule, in which missing 3'-UTR and/or 5'-UTR, respectively, or which contains a reference 3'-UTR and/or a reference-5'-UTR, respectively, such as a 3'-UTR and/or a 5'-UTR occurring naturally conditions c OPC artificial nucleic acid molecule.

"Increased protein expression" or "increased protein expression" in the context of the present invention preferably means increased/increased protein expression at one of the time points after initiation of expression, or increased/increased total protein expressed, compared to expression induced by a reference nucleic acid molecule. . Thus, the level of protein detected at a certain point in time after the initiation of expression, for example, after transfection with an artificial nucleic acid molecule of the present invention, for example, after transfection of an mRNA of the present invention, for example, after 6, 12, 24, 48 or 72 hours after transfection, preferably exceeds the protein level detected at the same time point after initiation of expression, for example, after transfection with a reference nucleic acid molecule, such as a reference mRNA, which contains a reference 3'-UTR and/or a reference 5'-UTR, respectively, or lacking a 3'-UTR and/or 5'-UTR, respectively. In a preferred embodiment of the invention, the maximum amount of protein (determined, for example, by activity or protein weight) expressed from an artificial nucleic acid molecule is increased compared to the amount of protein expressed from a reference nucleic acid that contains a reference 3'-UTR and /or reference-5'-UTR, respectively, or in which there is no 3'-UTR and/or 5'-UTR, respectively. Peak expression levels are preferably reached within 48 hours, more preferably within 24 hours and even more preferably within 12 hours after, for example, transfection.

Preferably, "increased total protein production" or "increased total protein production" from the artificial nucleic acid molecule of the invention refers to increased/increased protein production over the period of time that the protein is produced from the artificial nucleic acid molecule, e.g. , within 48 hours or 72 hours, preferably in a mammalian expression system, such as mammalian cells, for example, in HDF, L929, HEP2G or HeLa cells, compared to a reference nucleic acid molecule that lacks a 3'-UTR and /or 5'-UTR, respectively, or which contains a reference-3'-UTR and/or a reference-5'-UTR, respectively. According to a preferred embodiment of the invention, the cumulative amount of protein expressed over a period of time is increased if an artificial nucleic acid molecule according to the invention is used.

The total amount of protein over a period of time can be determined by (i) collecting tissue or cells at several time points after the introduction of the artificial nucleic acid molecule (e.g., 6, 12, 24, 48, and 72 hours after the initiation of expression or after the introduction of the nucleic acid molecule). acids), and the amount of protein at each time point can be determined as above. To calculate the cumulative amount of protein, a mathematical method for determining the total amount of protein can be used, for example, you can determine the area under the curve (AUC) according to the following formula:

To calculate the area under the curve for total protein, the integral of the equation of the expression curve between each endpoints (a and b) is calculated.

Thus, the term "total protein production" preferably refers to the area under the curve (AUC) describing protein production as a function of time.

Preferably, at least one 3'-UTR element or 5'-UTR element of the present invention increases protein production from said artificial nucleic acid molecule at least 1.5-fold, preferably at least 2-fold, more preferably at least 2.5 times as compared to protein production from a reference nucleic acid molecule lacking the 3'-UTR and/or 5'-UTR, respectively. In other words, the total amount of protein produced from the artificial nucleic acid molecule of the invention at a certain point in time, for example, 48 hours or 72 hours after the initiation of expression, for example, after transfection, is increased by a factor of at least 1, 5, preferably according to. at least 2, more preferably at least 2.5, compared to the (relatively) amount of protein produced from a reference nucleic acid molecule that, for example, lacks a 3'-UTR and/or a 5'-UTR, respectively, or which contains a reference-3'-UTR and/or a reference-5'-CTR, respectively, at a corresponding later time point.

Prolongation of mRNA and/or protein production and/or protein production and/or protein production of 3'-UTR and/or 5'-UTR variants, fragments and/or fragment variants and prolongation of mRNA and/or protein production and/or protein production-enhancing activity and the potency of at least one 3'UTR element and/or at least one 5'UTR element of the artificial nucleic acid molecule of the present invention can be determined using any suitable for this purpose, a method known to the person skilled in the art.

For example, one can create artificial mRNA molecules containing the coding sequence/open reading frame (ORF) of a reporter protein, such as luciferase, and a 3'-UTR element of the present invention, namely one that prolongs and/or increases the production protein from the specified artificial mRNA molecule. In addition, such an inventive mRNA molecule may also contain a 5'-UTR element according to the present invention, namely one that prolongs and/or increases protein production from said artificial mRNA molecule, does not contain a 5'-UTR- element or 5'-UTR element not proposed in the present invention, for example, the reference 5'-UTR. It is therefore possible to create artificial mRNA molecules containing the coding sequence/open reading frame (ORF) of a reporter protein such as luciferase and a 5'-UTR element of the present invention, namely one that prolongs and/or increases protein production from the specified artificial mRNA molecule. In addition, such an inventive mRNA molecule may also contain the 3'-UTR element of the present invention, namely one that prolongs and/or increases protein production from said artificial mRNA molecule, does not contain a 3'-UTR- element or 3'-UTR element not proposed in the present invention, for example, reference-3'-UTR

According to the present invention, mRNAs can be generated, for example, by in vitro transcription of appropriate vectors, such as plasmid vectors, for example, those containing the T7 promoter and a sequence encoding the corresponding mRNA sequences. The generated mRNA molecules can be transfected into cells using any transfection method suitable for mRNA transfection, for example, they can be inserted via lipofection into mammalian cells such as HeLa cells or HDF cells, and samples can be analyzed at specific time points after transfection, for example, via 6 h, 24 h, 48 h and 72 h after transfection. These samples can be analyzed to determine the amounts of mRNA and/or amounts of protein using methods well known to the person skilled in the art. For example, the amount of reporter mRNA present in cells at certain time points can be determined using quantitative PCR methods. The amounts of reporter protein encoded by the respective mRNAs can be determined, for example, by Western blotting, ELISA assays, FACS assays, or reporter assays such as luciferase assays, depending on the reporter protein used. The effect of stabilizing protein expression and/or prolonging protein expression can be analyzed, for example, by determining the ratio of the level of protein detected 48 hours after transfection to the level of protein detected 24 hours after transfection. The closer this value is to 1, the more stable the expression of the protein during this period of time. Such measurements to determine the stability of protein expression can, of course, also be carried out after 72 hours or more, and the ratio of the level of protein detected 72 hours after transfection to the level of protein detected 24 hours after transfection can be determined.

Translation Efficiency: Analysis to Determine Translation Efficiency

As detailed above, translation efficiency is a characteristic of a nucleic acid molecule (e.g., mRNA) that contains an open reading frame (ORF) and is typically conferred by a specific UTR element (specific 5'-UTR element or specific 3 '-UTR element) contained in the nucleic acid molecule. Translation efficiency is generally measured by the amount of protein translated from the ORF. Regarding this, the present invention provides general methods as well as specific assays. The translation efficiency of different, eg two or more, nucleic acid molecules can be compared, for example, by experimental quantification of the protein encoded by the ORF.

In general, cell-based methods are used to evaluate translation efficiency. Translation efficiency of an artificial nucleic acid molecule of the invention is experimentally tested in vivo or in vitro as described herein (i.e., in vitro refers to (“live”) cells and/or tissue, including tissue from a living individual; cells include, in particular cell lines, primary cells, cells in tissue or in the body of individuals, mammalian cells such as human cells and mouse cells are preferred, and most preferred are human HeLa, HEPG2 and U-937 cell lines and mouse NIH3T3 cell lines, JAWSII and L929, in addition, the most preferred are primary cells, in the most preferred embodiments of the invention - human dermal fibroblasts (HDF)), the expression of the encoded protein is determined after injection / transfection of the artificial nucleic acid molecule proposed in the invention into cells - / tissues - target and compared with protein expression induced by ucleic reference acid. Quantitative methods for determining protein expression are known in the art (eg Western blotting, FACS, ELISA, mass spectrometry). In this context, the most suitable is the determination of the expression of reporter proteins such as luciferase, green fluorescent protein (GFP) or secreted alkaline phosphatase (SEAP). Thus, an artificial nucleic acid according to the invention or a reference nucleic acid is introduced into the target tissue or target cell, for example by transfection or injection, preferably into a mammalian expression system, such as mammalian cells, for example, into the HDF cell line, L929, HepG2 and/or Hela. Hours or days (eg, 6, 12, 24, 48, or 72 hours) after initiation of expression or after introduction of the nucleic acid molecule, a sample of target cells is taken and measured by FACS and/or lysed. The lysates can then be used to detect the expressed protein (and thereby determine the efficiency of protein expression) by several methods, such as Western blot, FACS, ELISA, mass spectrometry, or by measuring fluorescence or luminescence.

Thus, if the expression of a protein from an artificial nucleic acid molecule of the invention is compared with the expression of a protein from a reference nucleic acid molecule at a specific time point (for example, 6, 12, 24, 48 or 72 hours after the initiation of expression or after the introduction of the molecule nucleic acid), then both nucleic acid molecules are injected independently into the target tissue/target cells, a sample of the target tissue/target cells is taken after a certain point in time, protein lysates are prepared according to a specific protocol adapted to a specific detection method (such as, for example, such as Western blot, ELISA, fluorescence or luminescence measurement, etc. known in the art) and perform protein detection using the selected detection method. As an alternative to measuring the amounts of expressed protein in cell lysates, or in addition to measuring the amounts of expressed protein in cell lysates, protein amounts can also be determined by FACS before harvested cells are lysed or in parallel using aliquots.

Other general entities correspond to those presented above in the description of protein production, unless the context dictates otherwise.

Preferably, the high translation efficiency of the artificial nucleic acid molecule of the present invention is provided by at least one 3'-untranslated region element (3'-UTR element) and/or at least one 5'-untranslated region element (5 '-UTR-element). The expression "provided" means that in the absence of at least one element of the 3'-untranslated region (3'-UTR element) and/or at least one element of the 5'-untranslated region (5'-UTR element), the translation efficiency is not high, as indicated in the present description.

The term "high translation efficiency" can be used to compare the translation efficiency of an artificial nucleic acid molecule of the present invention with a reference nucleic acid molecule. The term "reference nucleic acid molecule" in the context of the present description means that, with the exception of different 3'-UTR and/or 5'-UTR, the reference nucleic acid molecule is comparable, preferably identical, to an artificial nucleic acid molecule of the invention, which contains 3'UTR element and/or 5'UTR element. Thus, one can compare the translation efficiency of an artificial nucleic acid molecule with the translation efficiency of a reference nucleic acid molecule. An appropriate method for comparing translation efficiency is provided herein.

All of the matters presented above in describing the definition of protein production also apply to the translation efficiency comparison method, unless the context dictates otherwise. Since the method for comparing translation efficiency uses a comparison of two nucleic acid molecules, two parallel experiments are carried out, the only difference between these two experiments is the nature of the nucleic acid molecule. In other words, all other conditions, such as growth medium, temperature, pH, are identical in the experiments. A person skilled in the art, using conventional approaches, can select suitable conditions based on general competence and the guidance provided in this description, provided that identical conditions should be selected for both parallel experiments. Typically, one of the parallel experiments is carried out using the reference nucleic acid molecule (which may be a wild-type molecule or an artificial molecule), and the other is carried out with an artificial nucleic acid molecule (which is also referred to as the nucleic acid molecule in question). This method makes it possible to compare whether a given nucleic acid molecule has a high translation efficiency; ie, whether it is an artificial nucleic acid molecule according to the present invention. Specific aspects of the method for comparing translation efficiency are as follows:

In the present method, the reference nucleic acid molecule comprises at least one open reading frame (ORF) that is identical to at least one open reading frame (ORF) of the artificial nucleic acid molecule; and the reference nucleic acid molecule does not contain at least one element of the 3'-untranslated region (3'-UTR element) of the artificial nucleic acid molecule and/or at least one element of the 5'-untranslated region (5'-UTR element ) an artificial nucleic acid molecule. The translation efficiency of an artificial nucleic acid molecule and the translation efficiency of a reference nucleic acid molecule are compared by a method comprising the steps of:

(i) transfecting mammalian cells with the artificial nucleic acid molecule and measuring the amount of expressed protein encoded by the ORF of the artificial nucleic acid molecule at a certain point in time after transfection (for example, 24 or 48 hours),

(ii) transfecting mammalian cells with the reference nucleic acid molecule and measuring the amounts of expressed protein encoded by the ORF of the reference nucleic acid molecule at the same time point after transfection,

(III) calculate the ratio of the amount of protein expressed from the artificial nucleic acid molecule to the amount of protein expressed from the reference nucleic acid molecule,

where the ratio calculated in step (III) is ≥1, preferably >1.

Thus, by comparing the relative amount of protein produced from the subject nucleic acid molecule of the present invention with the relative amount of protein produced from the reference nucleic acid molecule, it is possible to determine the ratio by which the translation efficiency of the artificial nucleic acid molecule of the present invention is improved. of the present invention, compared with the efficiency of translation of the reference nucleic acid molecule.

The term "after transfection" is used to refer to the point in time at which cells were transfected with the nucleic acid molecule in question and the reference nucleic acid molecule.

Preferably, at least one 3'- and/or 5'-UTR element in the artificial nucleic acid molecule of the invention increases the efficiency of translation of said artificial nucleic acid molecule by at least 1.2 times, preferably at least 1. .5 times, more preferably at least 2 times, even more preferably at least 2.5 times, compared to the production of a protein from a reference nucleic acid molecule containing a reference-3'-UTR and/or reference-5 '-UTR, respectively (i.e. the ratio calculated in step (III) is at least 1.2, preferably at least 1.5, more preferably at least 2, even more preferably at least 2, 5). In other words, the translation efficiency is increased by a factor of at least 1.2, preferably at least 1.5, more preferably at least 2, even more preferably at least 2.5, compared to the translation efficiency of the reference molecule. nucleic acid at the same time.

High translation efficiency is provided by the 5'-UTR or 3'-UTR, in which the nucleic acid molecule in question differs from the reference nucleic acid molecule.

In a preferred embodiment of the translation efficiency comparison method of the present invention, (i) the reference nucleic acid molecule contains a 3'-UTR element that is not present in the artificial nucleic acid molecule, or (ii) the reference nucleic acid molecule contains a 5' -UTR element that is not present in an artificial nucleic acid molecule. In more preferred embodiments of the invention, (I) the reference nucleic acid molecule contains a 3'-UTR element that is derived from the 3'-UTR of the albumin gene, preferably the 3'-UTR, which corresponds to the sequence shown in SEQ ID NO: 207 , or (ii) the reference nucleic acid molecule contains a 5'-UTR element that is derived from the 5'-UTR of the TOP gene, preferably the 5'-UTR of the human ribosomal large subunit protein 32 without the 5'-oligopyrimidine tract, preferably 5 '-UTR which corresponds to the sequence shown in SEQ ID NO: 208.

In an alternative preferred embodiment of the invention (I) the reference nucleic acid molecule does not contain any element of the 3'-untranslated region (3'-UTR element), or (II) the reference nucleic acid molecule does not contain any element of the 5'-untranslated region (5'-UTR element).

In a preferred embodiment, the cells used in the transfection step with the artificial nucleic acid molecule and the reference nucleic acid molecule are mammalian cells, preferably selected from the group of HDF, L929, HEP2G and HeLa cell lines. In addition, preferably the method is carried out in parallel on more than one type of mammalian cells, for example, in parallel on a combination of any two, preferably any three, more preferably all four of the above cell lines. When the method is carried out in parallel on more than one type of mammalian cell, it can be considered that the nucleic acid molecule in question should be characterized by high translation efficiency if the ratio calculated in step (III) is ≥ 1, preferably > 1, for at least one of mammalian cell types used. However, it is preferable to consider that the nucleic acid molecule in question has a high translation efficiency if the ratio calculated in step (III) is ≥ 1, preferably > 1, for at least two types, for example, for at least three types, for example, for four types of mammalian cells used.

The amounts of expressed protein encoded by the ORF of the artificial nucleic acid molecule and the reference nucleic acid molecule are measured at a specific time point after transfection (eg, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours). Preferably, the amount of expressed protein encoded by the ORF is measured 24 hours after transfection.

The present invention also provides a specific preferred reference construct that can be used in the method of the present invention. Most preferably, the reference nucleic acid molecule is an mRNA having SEQ ID NO: 205 (FIG. 1A). In this case, it is also preferred that the reference nucleic acid molecule differs from the artificial nucleic acid molecule only in that at least one 3'-UTR element is replaced by at least one other 3'-UTR element (preferably one 3 '-UTR element), or by replacing at least one 5'-UTR element with at least one other 5'-UTR element (preferably one 5'-UTR element). In any case, this allows direct testing of whether said other 3'UTR element or another 5'UTR element provides correspondingly high translation efficiency.

Most preferably, the method for determining translation efficiency is further characterized in that:

(a) in the transfection step, the cells are mammalian cells selected from a group of HDF, L929, HEP2G and HeLa cell lines; and

(b) the time point for measurement is the time point 24 hours after transfection; and

(c) the reference nucleic acid molecule is an mRNA having SEQ ID NO: 205 (FIG. 1A); and

preferably (d) the reference nucleic acid molecule differs from the artificial nucleic acid molecule only in that (i) at least one 3'-UTR element (preferably one 3'-UTR element) is replaced by at least one other 3 '-UTR element (preferably one 3'-UTR element), or (II) at least one 5'-UTR element (preferably one 5'-UTR element)) replaced by at least one other 5' -UTR element (preferably one 5'-UTR element); in other words, the nucleic acid molecule in question is identical to the reference nucleic acid molecule, except that at least one 5'-UTR element differs from the 5'-UTR shown in FIG. 1A, or in that at least one 3'UTR element differs from the 3'UTR shown in FIG. 1A. An example of a 5'UTR element replacement is shown in FIG. 1B; an example of a 3'UTR element replacement is shown in FIG. 1B.

If all conditions (a) to (d) are met, then the method is also referred to as "standardized translation efficiency assay" or simply "translation efficiency assay". Most preferably, the artificial nucleic acid molecule according to the invention has a high translational efficiency, which can be determined using said standard translational efficiency assay. This standard assay allows direct identification of the most advantageous artificial nucleic acid molecules.

Standard analysis was used in example 3 of the present invention. In this way, the present invention has found that a group of novel artificial nucleic acids have the advantageous property of high translation efficiency, namely, higher translation efficiency than the reference nucleic acid molecule illustrated in FIG. 1A (see example 3.5). This is a very unexpected and advantageous discovery, primarily because said reference nucleic acid molecule contains a 3'-UTR element that is derived from the 3'-UTR of the albumin gene (the 3'-UTR corresponds to the DNA sequence shown in SEQ ID NO: 207), and a 5'-UTR element that is derived from the 5'-UTR of the TOP gene, primarily the 5'-UTR of the human ribosome 32 large subunit protein without the 5'-terminal oligopyrimidine tract (5'-UTR corresponds to the DNA sequence shown in SEQ ID NO: 208). Such 3'-UTR elements and 5'-UTR elements are known to be advantageous in terms of protein translation in the cell (data for the 3'-UTR derived from the albumin gene are presented in WO 2013/143700; for the 5'-TOP -UTRs are presented in WO 2013/143700). The present invention proposes a further improvement.

In some embodiments, the ORF of the artificial nucleic acid molecule encodes a protein that can be quantified, preferably a reporter protein. These embodiments of the invention are preferred for use in the translation efficiency comparison method of the present invention. In other embodiments, the ORF of the artificial nucleic acid molecule does not encode a quantifiable protein, preferably a reporter protein. For example, if using the method of the invention, using the ORF of an artificial nucleic acid molecule encoding a protein that can be quantified, a specific 5'-UTR or 3'-UTR is determined that provides high translation efficiency, then the specified 5'-UTR or the 3'-UTR can be recombined with any ORF. For example, when an ORF encodes a reporter protein in a standardized assay for determining translation efficiency, the artificial nucleic acids of the present invention are not limited to the ORF used in the standardized assay.

These methods can be applied iteratively; namely, an artificial nucleic acid molecule found to have high translation efficiency by the method of the present specification can itself be used as a "reference construct" for the next round of the present method. This makes it possible to test whether another artificial nucleic acid molecule has even higher translation efficiency (or whether the UTR contained in another artificial nucleic acid molecule provides even higher translation efficiency). This allows the identification of even more advanced artificial nucleic acid molecules and UTRs.

stable mRNA

In some embodiments, at least one 3'UTR element and/or at least one 5'UTR element in the artificial nucleic acid molecule of the present invention is derived from stable mRNA. In this case, "derived (derived from)" from a stable mRNA means that the sequence of at least one 3'-UTR element and/or at least one 5'-UTR element is at least 50% identical, preferably at least 60%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90% , even more preferably at least 95%, and most preferably at least 98% of the sequence of the 3'-UTR element and/or 5'-UTR element of the stable mRNA. Preferably the stable mRNA is a naturally occurring mRNA and thus the 3'UTR element and/or the 5'UTR element of the stable mRNA refers to the 3'UTR and/or 5'UTR of the naturally occurring mRNA , or their fragments or variants. In addition, a 3'UTR element and/or a 5'UTR element derived from a stable mRNA preferably also refers to a 3'UTR element and/or a 5'UTR element that is modified from that found in natural conditions 3'-UTR element and/or 5'-UTR element, for example, in order to further improve the stability of the RNA and/or to prolong and/or increase protein production. It goes without saying that modifications are preferred which do not impair the stability of the RNA, eg compared to a naturally occurring (unmodified) 3'UTR element and/or a 5'UTR element. In particular, the term "mRNA" in the context of the present description refers to the mRNA molecule, however, it can also refer to the types of mRNA presented in the present description.

Preferably the stability of the mRNA, ie. the cleavage time and/or half-life of the mRNA is evaluated under standard conditions, eg under standard conditions (standard medium, incubation, etc.) for the specific cell line used.

The term "stable mRNA" in the context of the present description refers generally to mRNA, characterized by slow cleavage of mRNA. Thus, "stable mRNA" typically has a long half-life. The half-life of an mRNA is the time required to cleave 50% of the mRNA molecules present under in vivo or in vitro conditions. Therefore, mRNA stability is generally assessed in vivo or in vitro. As used herein, in vitro refers specifically to (“live”) cells and/or tissues, including tissue from a living individual. Cells include, in particular, cell lines, primary cells, cells in a tissue or an individual's body. In specific embodiments of the invention suitable for use according to the present invention may be cell types that allow the cultivation of cells. Most preferred are mammalian cells, eg human cells and mouse cells. In the most preferred embodiments of the invention, human HeLa, HEPG2 and U-937 cell lines and mouse NIH3T3, JAWSII and L929 cell lines are used. In addition, primary cells are most suitable, in the most preferred embodiments of the invention, human dermal fibroblasts (HDF) can be used. Alternatively, tissue from the individual may also be used.

Preferably, the "stable mRNA" half-life is at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, and/or at least 15 hours. The half-life of an mRNA of interest can be determined by various methods known to one of skill in the art. Generally, the half-life of the mRNA of interest is determined by determining the cleavage constant, generally assuming that there is an ideal situation in vivo (or in vitro as above) in which transcription of the mRNA of interest can be completely "turned off". ” (or reduced to at least an undetectable level). In this ideal situation, it is generally assumed that such mRNA cleavage occurs according to first order kinetics. Therefore, mRNA cleavage can generally be described by the following equation:

where A ₀ is the amount (or concentration) of mRNA of interest at time 0, i. before the start of cleavage, A(t) is the amount (or concentration) of mRNA of interest at time t in the cleavage process, and λ is the cleavage constant. Thus, if the amount (or concentration) of mRNA of interest at time 0 (A ₀ ) and the amount (or concentration) of mRNA of interest at time t in the cleavage process (A(t) and t) are known, then the constant can be calculated splitting λ. Based on the splitting constant λ, the half-life t _1/2 can be calculated according to the following equation:

because by definition A(t)/A0=1/2 at t _1/2 . Thus, in order to estimate the half-life of an mRNA of interest, the amount or concentration of the mRNA is usually determined during in vivo (or in vitro, as above) cleavage of the RNA.

To determine the amount or concentration of mRNA in the process of cleavage of RNA in vivo (or in vitro, as indicated above), you can use various methods known to the person skilled in the art. Examples of such methods include, but are not limited to, general transcription inhibition, e.g., with a transcription inhibitor such as actinomycin D, the use of inducible promoters to specifically enhance transient transcription, e.g., the serum inducible c-fos promoter system and the regulatory promoter system. Tet-off, and kinetic labeling methods, eg pulse labeling, eg using 4-thiouridine (4sU), 5-ethynyluridine (EU), or 5'-bromouridine (BrU). Other details and preferred options regarding how to determine the amount or concentration of mRNA during RNA cleavage are described below in the context of the method for identifying a 3'-UTR element and/or at least one 5'-UTR element of the present invention. . The corresponding description and preferred options for how to determine the amount or concentration of mRNA during RNA cleavage apply here as well.

Preferably "stable mRNA" in the context of the present invention is characterized by slower cleavage of mRNA compared to average mRNA, which is preferably measured in vivo (or in vitro as above). For example, "average mRNA cleavage" can be assessed by examining mRNA cleavage for multiple mRNA species, preferably 100, at least 300, at least 500, at least 1000, at least 2000, at least 3000, at least 4000 at least 5000 at least 6000 at least 7000 at least 8000 at least 9000 at least 10000 at least 11000 at least 12000 at least 13000 at least 14000 at least 15000 at least 16000 at least 17000 at least 18000 at least 19000 at least 20000 at least 21000 at least 22000 at least 23000 at least 24,000, at least 25,000, at least 26,000, at least 27,000, at least 28,000, at least 29,000, at least 30,000 mRNA species. It is most preferable to evaluate the entire transcriptome or the maximum possible number of mRNA species in the transcriptome. This can be done, for example, using a microarray that covers all transcripts.

The concept of "types of mRNA" in the context of the present description corresponds to a genomic transcription unit, namely, as a rule, to a gene. Thus, in one "type of mRNA" there may be different transcripts, for example, due to the processing of mRNA. mRNA species can be introduced as spots on the microarray. Therefore, the microarray is the preferred quantification "tool" for multiple mRNA species, for example, at a specific point in time during mRNA cleavage. However, other methods known to the person skilled in the art, such as RNA sequencing, quantitative PCR, etc., can also be used.

According to the present invention, it is most preferred that the stable mRNA is characterized by mRNA cleavage such that the ratio of the amount of said mRNA at the second time point to the amount of said mRNA at the first time point is at least 0.5 (50%), at least 0.6 (60%), at least 0.7 (70%), at least 0.75 (75%), at least 0.8 (80%), at least 0.85 (85%), by at least 0.9 (90%) or at least 0.95 (95%). This implies that the second time occurs later than the first time in the splitting process.

Preferably, the first time point is chosen such that only the mRNA that is undergoing the cleavage process is considered, i.e. so as not to consider mRNA that occurs, for example, during transcription. For example, if kinetic labeling methods are used, such as pulsed labeling, the first time point is preferably chosen such that incorporation of the label into the mRNA is completed, i.e. there was no continued incorporation of the label into the mRNA. Thus, if kinetic labeling is used, the first time point can be at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, or at least 90 minutes after completion of the experimental labeling procedure, for example, after completion of incubation of the labeled cells.

For example, the first time point may preferably occur between 0 and 6 hours after transcription has ceased (eg, with a transcription inhibitor), promoter induction has ceased for inducible promoters, or after the pulse or label has ceased, such as after labeling has ended. More preferably, the first time point may be between 30 minutes and 5 hours, even more preferably between 1 hour and 4 hours, and most preferably about 3 hours after transcription has ceased (e.g., with a transcription inhibitor), promoter induction has ceased in the case of inducible promoters or after the termination of the pulse or label, for example, after the end of labeling.

Preferably, the second time point is chosen as late as possible in the mRNA cleavage process. However, if multiple mRNA species are considered, the second time point is preferably chosen such that a significant amount of the plurality of mRNA species, preferably at least 10% of the mRNA species, is still present in a detectable amount, i. in an amount greater than 0. Preferably, the second time occurs after at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, or at least 15 hours after the end of transcription or after the end of the experimental labeling procedure.

Thus, the time interval between the first time and the second time is preferably as long as possible within the above limits. Therefore, the time interval between the first time point and the second time point is preferably at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours.

In addition, at least one 3'UTR element and/or at least one 5'UTR element in the artificial nucleic acid molecule of the present invention can be identified using the 3'UTR element identification method, and /or 5'-UTR-element proposed in the present invention, which is presented in the present description. Most preferably, at least one 3'-UTR element and/or at least one 5'-UTR element in the artificial nucleic acid molecule of the present invention is identified using the 3'-UTR-identification method provided herein. element and/or 5'-UTR element, which provides high translation efficiency of the artificial nucleic acid molecule.

Preferred embodiments of the artificial nucleic acid molecule of the present invention

Preferably, at least one 3'-UTR element and/or at least one 5'-UTR element in the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is derived from a 3'-UTR and /or the 5'-UTR of a gene encoding a eukaryotic protein, preferably from the 3'-UTR and/or 5'-UTR of a gene encoding a vertebrate protein, more preferably from the 3'-UTR and/or 5'-UTR of a gene encoding a mammalian protein , for example, from genes encoding mouse and human protein, even more preferably from the 3'-UTR and/or 5'-UTR of a gene encoding a primate or rodent protein, especially the 3'-UTR and/or 5'-UTR of the gene, encoding a human or mouse protein.

In general, it should be understood that at least one 3'-UTR element in the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is preferably derived from naturally occurring 3 '-UTR, while at least one 5'-UTR element in the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is preferably derived from naturally occurring 5' -UTR.

Preferably, at least one open reading frame is heterologous to at least one 3'UTR element and/or at least one 5'UTR element. The term "heterologous" in this context means that two sequence elements contained in an artificial nucleic acid molecule, such as an open reading frame and a 3'-UTR element and/or an open reading frame and a 5'-UTR element, do not occur in natural conditions (in nature) in the considered combination. As a rule, they are recombinant. Preferably, the 3'UTR element and/or the 5'UTR element is/is derived from a different gene from that from which the open reading frame is derived. For example, the ORF may be derived from a different gene from which the 3'UTR element and/or at least one 5'UTR element is derived, such as encoding a different protein or the same protein from a different species. etc. Those. the open reading frame is derived from a gene that is different from the gene from which the 3'UTR element and/or at least one 5'UTR element is derived. In a preferred embodiment, the ORF does not encode human ribosomal protein or plant ribosomal protein (e.g. from Arabidopsis), preferably does not encode human ribosomal protein S6 (RPS6), human ribosomal protein L36a-like protein (RPL36AL) or Arabidopsis ribosomal protein S16 (RPS16). ). In another preferred embodiment of the invention, the open reading frame (ORF) does not encode ribosomal S6 protein (RPS6), ribosomal protein L36a like protein (RPL36AL) or ribosomal protein S16 (RPS16).

In specific embodiments of the invention, preferably the open reading frame does not encode a reporter protein, for example, selected from the group consisting of globin proteins (primarily beta-globin), luciferase protein, GFP proteins, or variants thereof, for example, variants whose sequences are identical in at least 70% of the globin protein, luciferase protein or GFP protein sequence. Most preferably, the open reading frame (ORF) does not encode the GFP protein. It is also most preferred that the open reading frame (ORF) does not encode a reporter gene or is not derived from a reporter gene, where the reporter gene is preferably not selected from the group consisting of (genes) globin proteins (primarily beta-globin), luciferase protein, beta -glucuronidase (GUS) and GFP proteins or variants thereof, preferably not selected from EGFP or from variants of any of the above genes, the sequences of which are generally at least 70% identical to the sequence of any of the above reporter genes, preferably globin protein genes, luciferase protein or GFP protein.

Even more preferably, the 3'UTR element and/or the 5'UTR element is heterologous with respect to any other element contained in the artificial nucleic acid provided herein. For example, if an artificial nucleic acid according to the invention contains a 3'-UTR element from the gene in question, then it preferably does not contain any other nucleotide sequence, in particular any functional nucleotide sequence (for example, a coding or regulatory sequence element) from the same gene, including its regulatory sequences at the 5'- and 3'-end of the ORF gene. Therefore, for example, if an artificial nucleic acid according to the invention contains a 5'-UTR element from the gene in question, then it preferably does not contain any other nucleotide sequence, in particular any functional nucleotide sequence (for example, a coding or regulatory sequence element) from of the same gene, including its regulatory sequences at the 5' and 3' ends of the ORF gene.

It is further preferred that the artificial nucleic acid of the present invention comprises at least one open reading frame, at least one 3'-UTR (element) and at least one 5'-UTR (element), wherein or at least one 3'-UTR (element) is a 3'-UTR element of the present invention, and/or at least one 5'-UTR (element) is a 5'-UTR element of the present invention in the present invention. In such a preferred artificial nucleic acid of the present invention, which contains at least one open reading frame, at least one 3'-UTR (element) and at least one 5'-UTR (element), most preferably when each of at least one open reading frame, at least one 3'-UTR (element) and at least one 5'-UTR (element) are heterologous, i. neither at least one 3'-UTR (element) and at least one 5'-UTR (element), nor an open reading frame and a 3'-UTR (element) or 5'-UTR (element), respectively, occur in natural conditions (in nature) in the considered combination. This means that an artificial nucleic acid molecule contains an ORF, a 3'-UTR (element) and a 5'-UTR (element), which are all heterologous to each other, for example, they are recombinant, since each of these elements comes from different genes ( and their 5'- and 3'-UTRs). In another preferred embodiment of the invention, the 3'-UTR (element) is not derived from the 3'-UTR (element) of a viral gene or is not derived from a virus.

Preferably, at least one 3'UTR element and/or at least one 5'UTR element is operably linked to the ORF. This means that preferably the 3'UTR element and/or at least one 5'UTR element is associated with the ORF in such a way that it can perform a function, such as the function of increasing or stabilizing the expression of the encoded peptide or protein, or the function stabilization of an artificial nucleic acid molecule. Preferably, the ORF and the 3'UTR element are associated in the 5'→3' direction and/or the 5'UTR element and the ORF are associated in the 5'→3' direction. Thus, preferably, the artificial nucleic acid molecule contains, as a rule, the structure nucleic acid may contain only a 5'-UTR element and no 3'-UTR element, only a 3'-UTR element and no 5'-UTR element, or both and a 3'-UTR element, and 5'-UTR element. In addition, the linker may or may not be present. For example, the linker may consist of one or more nucleotides, for example, be a segment consisting of 1-50 or 1-20 nucleotides, for example, containing or consisting of one or more sites recognized by restriction enzymes (restriction sites).

Preferably, at least one 3'-UTR element and/or at least one 5'-UTR element contains or consists of a nucleotide sequence that is derived from the 3'-UTR and/or 5'-UTR of a gene transcript selected from group which consists of ZNF460, TGM2, IL7R, BGN, TK1, RAB3B, CBX6, FZD2, COL8A1, NDUFS7, PHGDH, PLK2, TSPO, PTGS1, FBXO32, NID2, ATP5D, EXOSC4, NOL9, UBB4B, VPS18, ORMDL2, FSCN1 , TMEM33, TUBA4A, EMP3, TMEM201, CRIP2, BRAT1, SERPINH1, CD9, DPYSL2, CDK9, TFRC, PSMB3 5'-UTR, FASN, PSMB6, PRSS56, KPNA6, SFT2D2, PARD6B, LPP, SPARC, SCAND1, VASN, SLC26A1 , LCLAT1, FBXL18, SLC35F6, RAB3D, MAP1B, VMA21, CYBA, SEZ6L2, PCOLCE, VTN, ALDH16A1, RAVER1, KPNA6, SERINC5, JUP, CPN2, CRIP2, EPT1, PNPO, SSSCA1, POLR2L, LIN7C, UQCR10, PYCRL, AMN , MAP1S, NDUFS7, PHGDH, TSPO, ATP5D, EXOSC4, TUBB4B, TUBA4A, EMP3, CRIP2, BRAT1, CD9, CDK9, PSMB3, PSMB6, PRSS56, SCAND1, AMN, CYBA, PCOLCE, MAP1S, VTN, ALDH16A1 (preferably all human ) and Dpysl2, Ccnd1, Acox2, Cbx6, Ubc, Ldlr, Nudt22 , Pcyoxl1, Ankrd1, Tmem37, Tspyl4, Slc7a3, Cst6, Aacs, Nosip, Itga7, Ccnd2, Ebp, Sf3b5, Fasn, Hmgcs1, Osr1, Lmnb1, Vma21, Kif20a, Cdca8, Slc7a1, Ubqln2, Prps2, Shmt2, Aurkb, Fignl1 , Cad, Anln, Slfn9, Ncaph, Pole, Uhrf1, Gja1, Fam64a, Kif2c, Tspan10, Scand1, Gpr84, Fads3, Cers6, Cxcr4, Gprc5c, Fen1, Cspg4, Mrpl34, Comtd1, Armc6, Emr4, Atp5d, 1110001J03Rik, Csf2ra , Aarsd1, Kif22, Cth, Tpgs1, Ccl17, Alkbh7, Ms4a8a, Acox2, Ubc, Slpi, Pcyox1l, Igf2bp1, Tmem37, Slc7a3, Cst6, Ebp, Sf3b5, Plk1, Cdca8, Kif22, Cad, Cth, Pole, Kif2c, Scand1 , Gpr84, Tpgs1, Ccl17, Alkbh7, Ms4a8a, Mrpl34, Comtd1, Armc6, Atp5d, 1110001J03Rik, Nudt22, Aarsd1 (preferably all mouse).

Preferably, at least one 3'-UTR element and/or at least one 5'-UTR element of the artificial nucleic acid molecule of the present invention contains or consists of a "functional fragment", "functional variant" or "functional fragment of the variant" 3'-UTR and/or 5'-UTR of the gene transcript.

Preferably, at least one 5'-UTR element contains a nucleotide sequence that is derived from the 5'-UTR of a gene transcript selected from the group consisting of ZNF460-5'-UTR, TGM2-5'-UTR, IL7R-5' -UTR, BGN-5'-UTR, TK1-5'-UTR, RAB3B-5'-UTR, CBX6-5'-UTR, FZD2-5'-UTR, COL8A1-5'-UTR, NDUFS7-5'- UTR, PHGDH-5'-UTR, PLK2-5'-UTR, TSPO-5'-UTR, PTGS1-5'-UTR, FBXO32-5'-UTR, NID2-5'-UTR, ATP5D-5'-UTR , EXOSC4-5'-UTR, NOL9-5'-UTR, UBB4B-5'-UTR, VPS18-5'-UTR, ORMDL2-5'-UTR, FSCN1-5'-UTR, TMEM33-5'-UTR, TUBA4A-5'-UTR, EMP3-5'-UTR, TMEM201-5'-UTR, CRIP2-5'-UTR, BRAT1-5'-UTR, SERPINH1-5'-UTR, CD9-5'-UTR, DPYSL2 -5'-UTR, CDK9-5'-UTR, TFRC-5'-UTR, PSMB3 5'-UTR, FASN-5'-UTR, PSMB6-5'-UTR, PRSS56-5'-UTR, KPNA6-5 '-UTR, SFT2D2-5'-UTR, PARD6B-5'-UTR, LPP-5'-UTR, SPARC-5'-UTR, SCAND1-5'-UTR, VASN-5'-UTR, SLC26A1-5' -UTR, LCLAT1-5'-UTR, FBXL18-5'-UTR, SLC35F6-5'-UTR, RAB3D-5'-UTR, MAP1B-5'-UTR, VMA21-5'-UTR, CYBA-5'- UTR, SEZ6L2-5'-UTR, PCOLCE-5'-UTR, VTN-5'-UTR, ALDH16A1-5'-UTR, RAVER1-5'-UTR, KP NA6-5'-UTR, SERINC5-5'-UTR, JUP-5'-UTR, CPN2-5'-UTR, CRIP2-5'-UTR, EPT1-5'-UTR, PNPO-5'-UTR, SSSCA1 -5'-UTR, POLR2L-5'-UTR, LIN7C-5'-UTR, UQCR10-5'-UTR, PYCRL-5'-UTR, AMN-5'-UTR, MAP1S-5'-UTR, (preferred all human) and Dpysl2-5'-UTR, Ccnd1-5'-UTR, Acox2-5'-UTR, Cbx6-5'-UTR, Ubc-5'-UTR, Ldlr-5'-UTR, Nudt22-5' -UTR, Pcyox1l-5'-UTR, Ankrd1-5'-UTR, Tmem37-5'-UTR, Tspyl4-5'-UTR, Slc7a3-5'-UTR, Cst6-5'-UTR, Aacs-5'- UTR, Nosip-5'-UTR, Itga7-5'-UTR, Ccnd2-5'-UTR, Ebp-5'-UTR, Sf3b5-5'-UTR, Fasn-5'-UTR, Hmgcs1-5'-UTR , Osr1-5'-UTR, Lmnb1-5'-UTR, Vma21-5'-UTR, Kif20a-5'-UTR, Cdca8-5'-UTR, Slc7a1-5'-UTR, Ubqln2-5'-UTR, Prps2-5'-UTR, Shmt2-5'-UTR, Aurkb-5'-UTR, Fignl1-5'-UTR, Cad-5'-UTR, Anln-5'-UTR, Slfn9-5'-UTR, Ncaph -5'-UTR, Pole-5'-UTR, Uhrf1-5'-UTR, Gja1-5'-UTR, Fam64a-5'-UTR, Kif2c-5'-UTR, Tspan10-5'-UTR, Scand1- 5'-UTR, Gpr84-5'-UTR, Fads3-5'-UTR, Cers6-5'-UTR, Cxcr4-5'-UTR, Gprc5c-5'-UTR, Fen1-5'-UTR, Cspg4-5 '-UTR, Mrpl34-5'-UTR, Comtd1-5'-UTR, Armc6-5'-UTR, Emr4-5'-UTR, Atp5d-5'-UTR , 1110001J03Rik-5'-UTR, Csf2ra-5'-UTR, Aarsd1-5'-UTR, Kif22-5'-UTR, Cth-5'-UTR, Tpgs1-5'-UTR, Ccl17-5'-UTR, Alkbh7-5'-UTR, Ms4a8a-5'-UTR (preferably all mouse).

Preferably, at least one 3'-UTR element contains or consists of a nucleotide sequence that is derived from the 3'-UTR of a gene transcript selected from the group consisting of NDUFS7-3'-UTR, PHGDH-3'-UTR, TSPO -3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'-UTR, BRAT1- 3'-UTR, CD9-3'-UTR, CDK9-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, PRSS56-3'-UTR, SCAND1-3'-UTR, AMN-3 '-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1-3'-UTR (preferably all human) and Acox2-3'-UTR , Ubc-3'-UTR, Slpi-3'-UTR, Pcyox1l-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'-UTR, Slc7a3-3'-UTR, Cst6-3'-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Plk1-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR, Cad-3'-UTR, Cth-3'-UTR, Pole -3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3'-UTR, Ms4a8a- 3'-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik-3'-UTR, Nudt22-3'-UTR, Aarsd1-3 '-UTR (preferably all mouse).

It is also preferred that the artificial nucleic acid of the invention contains both (I) at least one preferred 5'-UTR and (I) at least one preferred 3'-UTR.

In the most preferred embodiment of the invention, at least one 5'-UTR element contains a nucleotide sequence that is derived from the 5'-UTR of a gene transcript selected from the group consisting of ZNF460-5'-UTR, TGM2-5'-UTR , IL7R-5'-UTR, COL8A1-5'-UTR, NDUFS7-5'-UTR, PLK2-5'-UTR, FBXO32-5'-UTR, ATP5D-5'-UTR, TUBB4B-5'-UTR, ORMDL2-5'-UTR, FSCN1-5'-UTR, CD9-5'-UTR, PYSL2-5'-UTR, PSMB3-5'-UTR, PSMB6-5'-UTR, KPNA6-5'-UTR, SFT2D2 -5'-UTR, LCLAT1-5'-UTR, FBXL18-5'-UTR, SLC35F6-5'-UTR, VMA21-5'-UTR, SEZ6L2-5'-UTR, PCOLCE-5'-UTR, VTN- 5'-UTR, ALDH16A1-5'-UTR, KPNA6-5'-UTR, JUP-5'-UTR, CPN2-5'-UTR, PNPO-5'-UTR, SSSCA1-5'-UTR, POLR2L-5 '-UTR, LIN7C-5'-UTR, UQCR10-5'-UTR, PYCRL-5'-UTR, AMN-5'-UTR, MAP1S-5'-UTR (all human), Dpysl2-5'-UTR, Acox2-5'-UTR, Ubc-5'-UTR, Nudt22-5'-UTR, Pcyox1l-5'-UTR, Ankrd1-5'-UTR, Tspyl4-5'-UTR, Slc7a3-5'-UTR, Aacs -5'-UTR, Nosip-5'-UTR, Itga7-5'-UTR, Ccnd2-5'-UTR, Ebp-5'-UTR, Sf3b5-5'-UTR, Fasn-5'-UTR, Hmgcs1- 5'-UTR, Osr1-5'-UTR, Lmnb1 -5'-UTR, Vma21-5'-UTR, Kif20a-5'-UTR, Cdca8-5'-UTR, Slc7a1-5'-UTR, Ubqln2-5'-UTR, Prps2-5'-UTR, Shmt2- 5'-UTR, Fignl1-5'-UTR, Cad-5'-UTR, Anln-5'-UTR, Slfn9-5'-UTR, Ncaph-5'-UTR, Pole-5'-UTR, Uhrf1-5 '-UTR, Gja1-5'-UTR, Fam64a-5'-UTR, Tspan10-5'-UTR, Scand1-5'-UTR, Gpr84-5'-UTR, Cers6-5'-UTR, Cxcr4-5' -UTR, Gprc5c-5'-UTR, Fen1-5'-UTR, Cspg4-5'-UTR, Mrpl34-5'-UTR, Comtd1-5'-UTR, Armc6-5'-UTR, Emr4-5'- UTR, Atp5d-5'-UTR, Csf2ra-5'-UTR, Aarsd1-5'-UTR, Cth-5'-UTR, Tpgs1-5'-UTR, Ccl17-5'-UTR, Alkbh7-5'-UTR , Ms4a8a-5'-UTR (all murine).

It has been found that such UTR elements contribute to high translation efficiency.

In the most preferred embodiment of the invention, at least one 3'-UTR element contains a nucleotide sequence that is derived from the 3'-UTR of a gene transcript selected from the group consisting of NDUFS7-3'-UTR, PHGDH-3'-UTR , TSPO-3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'-UTR, BRAT1-3'-UTR, CD9-3'-UTR, CDK9-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, PRSS56-3'-UTR, SCAND1-3'-UTR, AMN -3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1-3'-UTR (preferably all human) and Acox2-3' -UTR, Ubc-3'-UTR, Slpi-3'-UTR, Pcyox1l-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'-UTR, Slc7a3-3'-UTR, Cst6-3'- UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Plk1-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR, Cad-3'-UTR, Cth-3'-UTR , Pole-3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3'-UTR, Ms4a8a-3'-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik-3'-UTR, Nudt22-3'-UTR, Aarsd1 -3'-UTR, (preferably all mice). It has been found that such UTR elements contribute to high translation efficiency.

These most preferred embodiments of the invention can be combined such that the artificial nucleic acid of the invention contains both (I) at least one most preferred 5'-UTR and (I) at least one most preferred 3'-UTR .

The phrase "a nucleotide sequence which is derived from the 3'-UTR and/or 5'-UTR of a gene transcript" preferably refers to a nucleotide sequence which is based on the 3'-UTR sequence of a gene and/or the 5'-UTR sequence of a gene transcript or a fragment thereof. or a region, preferably a naturally occurring gene or fragment or region thereof. In this context, "naturally occurring" is synonymous with "wild type". Said phrase includes sequences corresponding to the full 3'-UTR sequence and/or the full 5'-UTR sequence, i.e. a full-length 3'-UTR and/or 5'-UTR sequence of the gene transcript, and sequences corresponding to a fragment of the 3'-UTR sequence and/or 5'-UTR sequence of the gene transcript. Preferably, the 3'-UTR and/or 5'-UTR fragment of the gene transcript consists of a contiguous nucleotide segment corresponding to the nucleotide contiguous segment of the full-length 3'-UTR and/or 5'-UTR of the gene transcript, which includes at least 5%, 10% , 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, still more preferably at least 70%, even more preferably at least at least 80% and most preferably at least 90% of the full length 3'UTR and/or 5'UTR of the gene transcript. In the context of the present invention, such a fragment is preferably a functional fragment presented in the present description. Preferably, the fragment retains the function of regulating translation of the ORF linked to the 3'-UTR and/or 5'-UTR, or a fragment thereof.

The terms "3'-UTR variant and/or 5'-UTR variant of the gene transcript and" its variant" in the context of the 3'-UTR and/or 5'-UTR of the gene transcript refer to the 3'-UTR and/or 5'-UTR variant. UTR of a naturally occurring gene transcript, preferably a 3'-UTR and/or 5'-UTR variant of a vertebrate gene transcript, more preferably a 3'-UTR and/or 5'-UTR variant of a mammalian gene transcript, even more preferably a variant The 3'-UTR and/or 5'-UTR of a primate gene transcript, especially the human gene, as described above. Such a variant may be a modified 3'-UTR and/or 5'-UTR of the gene transcript. For example, a 3'-UTR variant and/or a 5'-UTR variant may have one or more deletions, insertions, additions, and/or substitutions of nucleotides compared to a naturally occurring 3'-UTR and/or 5'-UTR, of which the variant is received. Preferably the 3'-UTR variant and/or the 5'-UTR variant of the gene transcript by at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the naturally occurring 3'-UTR and/or 5'-UTR from which the variant is derived. Preferably, the variant is the functional variant described herein from which the variant is derived.

The phrase "a nucleotide sequence that is derived from a 3'-UTR variant and/or a 5'-UTR variant of a gene transcript" preferably refers to a nucleotide sequence based on a 3'-UTR and/or 5'-UTR sequence variant of a gene transcript or its fragment or section presented above. This phrase includes sequences corresponding to the complete sequence of the 3'-UTR variant and/or 5'-UTR of the gene transcript, i.e. the full-length sequence of the 3'-UTR variant and/or the full-length sequence of the 5'-UTR variant of the gene transcript, and sequences corresponding to a fragment of the 3'-UTR sequence variant and/or a fragment of the 5'-UTR sequence variant of the gene transcript. Preferably, the 3'-UTR and/or 5'-UTR variant fragment of the gene transcript consists of a contiguous nucleotide segment corresponding to the nucleotide contiguous segment of the full-length 3'-UTR and/or 5'-UTR variant of the gene transcript, which includes at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full length variant of the 3'-UTR and/or 5'-UTR of the gene transcript. In the context of the present invention, such a variant fragment is preferably a functional fragment of the variant presented in the present description.

In the context of the present description, the terms "functional variant" "functional fragment" and "functional fragment of the variant" (which is also referred to as "functional variant fragment") mean that the fragment 3'-UTR and/or 5'-UTR, variant 3'- UTR and/or 5'-UTR or a fragment of a 3'-UTR and/or 5'-UTR variant of a gene transcript performs at least one, preferably more than one, function of a naturally occurring 3'-UTR and/or 5'-UTR the gene transcript from which the variant, fragment, or fragment of the variant is derived. Such a function may be, for example, to stabilize the mRNA and/or increase, stabilize and/or prolong protein production from mRNA and/or increase protein expression or total protein production from mRNA, preferably in a mammalian cell such as a human cell. Preferably, the function of the 3'-UTR and/or 5'-UTR is to translate the protein encoded by the ORF. More preferably, the function includes increasing the efficiency of translation of the ORF associated with the 3'-UTR and/or 5'-UTR, or a fragment or variant thereof. In the context of the present invention, it is most preferred that the variant, fragment and fragment variant have the function of stabilizing the mRNA, preferably in a mammalian cell such as a human cell, compared to an mRNA that contains a reference-3'-UTR and/or a reference-5'- UTR, or lacking a 3'-UTR and/or 5'-UTR, and/or the function of increasing, stabilizing and/or prolonging protein production with an mRNA, preferably in a mammalian cell such as a human cell, compared to an mRNA that contains a reference 3'-UTR and/or a reference-5'-UTR, or which lacks a 3'-UTR and/or a 5'-UTR, and/or has the function of increasing protein production from mRNA, preferably in a mammalian cell, such as a human cell, compared to an mRNA that contains a reference 3'-UTR and/or a reference 5'-UTR, or lacks a 3'-UTR and/or a 5'-UTR. The reference 3'-UTR and/or the reference-5'-UTR may be, for example, a 3'-UTR and/or a 5'-UTR occurring naturally in combination with an ORF. In addition, the functional variant, functional fragment or functional variant of the 3'-UTR fragment and/or 5'-UTR of the gene transcript preferably does not significantly reduce the efficiency of translation of the mRNA that contains such a variant, fragment or variant of the 3'-UTR fragment and /or 5'-UTR, compared with the wild-type 3'-UTR and/or wild-type 5'-UTR from which the variant, fragment or fragment variant is derived. In the context of the present invention, the most preferred function of a "functional fragment", "functional variant" or "functional fragment variant" of the 3'-UTR and/or 5'-UTR of a gene transcript is to increase, stabilize and/or prolong the production of a protein through the expression of mRNA, carrying the functional variant, the functional fragment or the functional variant of the fragment presented above.

Preferably, the efficiency of one or more functions performed by the functional variant, functional fragment, or functional fragment variant, such as providing translation efficiency, is increased by at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least at least 80%, most preferably at least 90% of the translation efficiency provided by the naturally occurring 3'-UTR and/or 5'-UTR of the gene transcript from which the variant, fragment or fragment variant is derived.

In the context of the present invention, a fragment of the 3'-UTR and/or 5'-UTR of a gene transcript or a variant of the 3'-UTR and/or 5'-UTR of a gene transcript is preferably at least about 3 nucleotides in length, preferably at least about 5 nucleotides , more preferably at least about 10, 15, 20, 25 or 30 nucleotides, even more preferably at least about 50 nucleotides, most preferably at least about 70 nucleotides. Preferably, such a 3'-UTR and/or 5'-UTR fragment of a gene transcript, or a 3'-UTR and/or 5'-UTR variant of a gene transcript, is a functional fragment as described above. In a preferred embodiment, the 3'-UTR and/or 5'-UTR of the gene transcript, or fragment or variant thereof, is 3 to about 500 nucleotides in length, preferably 5 to about 150 nucleotides, more preferably 10 to 100 nucleotides, even more preferably 15 to 90, most preferably 20 to 70 nucleotides. Typically, a 3'UTR element and/or a 5'UTR element is characterized by being less than 500, 400, 300, 200, 150, or less than 100 nucleotides.

5'-UTR elements

Preferably, at least one 5'-UTR element contains or consists of a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 10, 15, 20, 30, or 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-151, or the corresponding RNA sequence, respectively, or at least one 5'-UTR element contains or consists of a fragment of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of the nucleotide sequence, selected from the group consisting of SEQ ID NO: 1-151, or the corresponding RNA sequence, respectively.

Among the sequences detailed below, SEQ ID NOs: 1-136 can be considered as wild-type 5'-UTR sequences, and SEQ ID NOs: 136-151 can be considered as artificial 5'-UTR sequences.

SEQ ID NO: 1 - 5'-UTR ZNF460 Homo sapiens

NM_006635.3

SEQ ID NO: 2 - 5'-UTR TGM2 Homo sapiens

NM_004613.2

SEQ ID NO: 3-5'-UTR IL7R Homo sapiens

NM_002185.3

SEQ ID NO: 4-5'-UTR BGN Homo sapiens

NM_001711.4

SEQ ID NO: 5 - 5'-UTR TK1 Homo sapiens

NM_003258.4

SEQ ID NO: 6-5'-UTR RAB3B Homo sapiens

NM_002867.3

SEQ ID NO: 7-5'-UTR CBX6 Homo sapiens

NM_014292.3

SEQ ID NO: 8-5'-UTR FZD2 Homo sapiens

NM_001466.3

SEQ ID NO: 9-5'-UTR COL8A1 Homo sapiens

NM_001850.4

SEQ ID NO: 10-5'-UTR NDUFS7 Homo sapiens

NM_024407.4

SEQ ID NO: 11 - 5'-UTR PHGDH Homo sapiens

NM_006623.3

SEQ ID NO: 12 - 5'-UTR PLK2 Homo sapiens

NM_006622.3

SEQ ID NO: 13 - 5'-UTR TSPO Homo sapiens

NM_000714.5

SEQ ID NO: 14 - 5'-UTR PTGS1 Homo sapiens

NM_000962.3

SEQ ID NO: 15-5'-UTR FBXO32 Homo sapiens

NM_058229.3

SEQ ID NO: 16-5'-UTR NID2 Homo sapiens

NM_007361.3

SEQ ID NO: 17 - 5'-UTR ATP5D Homo sapiens

NM_001687.4

SEQ ID NO: 18-5'-UTR EXOSC4 Homo sapiens

NM_019037.2

SEQ ID NO: 19-5'-UTR NOL9 Homo sapiens

NM_024654.4

SEQ ID NO: 20 - 5'-UTR TUBB4B Homo sapiens

NM_006088.5

SEQ ID NO: 21 - 5'-UTR VPS18 Homo sapiens

NM_020857.2

SEQ ID NO: 22 - 5'-UTR ORMDL2 Homo sapiens

NM_014182

SEQ ID NO: 23 - 5'-UTR FSCN1 Homo sapiens

NM_003088.3

SEQ ID NO: 24-5'-UTR TMEM33 Homo sapiens

NM_018126.2

SEQ ID NO: 25 - 5'-UTR TUBA4A Homo sapiens

NM_006000.2

SEQ ID NO: 26 - 5'-UTR EMP3 Homo sapiens

NM_001425.2

SEQ ID NO: 27 - 5'-UTR TMEM201 Homo sapiens

NM_001010866.3

SEQ ID NO: 28 - 5'-UTR CRIP2 Homo sapiens

NM_001312.3

SEQ ID NO: 29 - 5'-UTR BRAT1 Homo sapiens

NM_152743.3

SEQ ID NO: 30 - 5'-UTR SERPINH1 Homo sapiens

NM_001235.3

SEQ ID NO: 31 - 5'-UTR CD9 Homo sapiens

NM_001769.3

SEQ ID NO: 32 - 5'-UTR DPYSL2 Homo sapiens

NM_001386.5

SEQ ID NO: 33-5'-UTR CDK9 Homo sapiens

NM_001261.3

SEQ ID NO: 34 - 5'-UTR SSSCA1 Homo sapiens

NM_006396.1

SEQ ID NO: 35-5'-UTR POLR2L Homo sapiens

NM_021128.4

SEQ ID NO: 36 - 5'-UTR LIN7C Homo sapiens

NM_018362

SEQ ID NO: 37 - 5'-UTR UQCR10 Homo sapiens

NM_001003684.1

SEQ ID NO: 38 - 5'-UTR TFRC Homo sapiens

NM_001128148.1

SEQ ID NO: 39 - 5'-UTR PSMB3 Homo sapiens

NM_002795.2

SEQ ID NO: 40 - 5'-UTR FASN Homo sapiens

NM_004104.4

SEQ ID NO: 41 - 5'-UTR PSMB6 Homo sapiens

NM_002798.2

SEQ ID NO: 42 - 5'-UTR PYCRL Homo sapiens

NM_023078.3

SEQ ID NO: 43 - 5'-UTR PRSS56 Homo sapiens

NM_001195129.1

SEQ ID NO: 44 - 5'-UTR KPNA6 Homo sapiens

NM_012316.4

SEQ ID NO: 45 - 5'-UTR SFT2D2 Homo sapiens

NM_199344.2

SEQ ID NO: 46 - 5'-UTR PARD6B Homo sapiens

NM_032521.2

SEQ ID NO: 47 - 5'-UTR LPP Homo sapiens

SEQ ID NO: 48 - 5'-UTR SPARC Homo sapiens

NM_003118.3

SEQ ID NO: 49 - 5'-UTR SCAND1 Homo sapiens

SEQ ID NO: 50 - 5'-UTR VASN Homo sapiens

NM_138440.2

SEQ ID NO: 51 - 5'-UTR SLC26A1 Homo sapiens

NM_022042.3

SEQ ID NO: 52 - 5'-UTR LCLAT1 Homo sapiens

NM_182551.3

SEQ ID NO: 53 - 5'-UTR FBXL18 Homo sapiens

NM_024963.4

SEQ ID NO: 54 - 5'-UTR SLC35F6 Homo sapiens

NM_017877.3

SEQ ID NO: 55 - 5'-UTR RAB3D Homo sapiens

NM_004283.3

SEQ ID NO: 56 - 5'-UTR MAP1B Homo sapiens

NM_005909.3

SEQ ID NO: 57 - 5'-UTR VMA21 Homo sapiens

NM_001017980.3

SEQ ID NO: 58 - 5'-UTR AMN Homo sapiens

NM_030943.3

SEQ ID NO: 59 - 5'-UTR CYBA Homo sapiens

NM_000101.3

SEQ ID NO: 60 - 5'-UTR SEZ6L2 Homo sapiens

NM_012410.3

SEQ ID NO: 61 - 5'-UTR PCOLCE Homo sapiens

NM_002593.3

SEQ ID NO: 62 - 5'-UTR MAP1S Homo sapiens

NM_018174.4

SEQ ID NO: 63 - 5'-UTR VTN Homo sapiens

NM_000638.3

SEQ ID NO: 64 - 5'-UTR ALDH16A1 Homo sapiens

NM_001145396.1

SEQ ID NO: 65 - 5'-UTR RAVER1 Homo sapiens

NM_133452.2

SEQ ID NO: 66 - 5'-UTR KPNA6 Homo sapiens (identical to SEQ ID NO: 44)

NM_012316.4

SEQ ID NO: 67 - 5'-UTR SERINC5 Homo sapiens

NM_178276.5

SEQ ID NO: 68 - 5'-UTR JUP Homo sapiens

NM_002230.2

SEQ ID NO: 69 - 5'-UTR CPN2 Homo sapiens

NM_001080513.2

SEQ ID NO: 70 - 5'-UTR CRIP2 Homo sapiens

NM_001312.3

SEQ ID NO: 71 - 5'-UTR EPT1 Homo sapiens

NM_033505.2

SEQ ID NO: 72 - 5'-UTR PNPO Homo sapiens

NM_018129.3

SEQ ID NO: 73 - 5'-UTR Dpysl2 Mus musculus

NM_009955.3

SEQ ID NO: 74 - 5'-UTR Ccnd1 Mus musculus

NM_007631.2

SEQ ID NO: 75 - 5'-UTR Acox2 Mus musculus

NM_001161667.1

SEQ ID NO: 76 - 5'-UTR Cbx6 (Npcd) Mus musculus

NM_001013360.2

SEQ ID NO: 77 - 5'-UTR Ubc Mus musculus

SEQ ID NO: 78 - 5'-UTR Ldlr Mus musculus

NM_001252659.1

SEQ ID NO: 79 - 5'UTR Nudt22 Mus musculus

SEQ ID NO: 80 - 5'-UTR Pcyox11 Mus musculus

NM_172832.4

SEQ ID NO: 81 - 5'-UTR Ankrdl Mus musculus

NM_013468.3

SEQ ID NO: 82 - 5'-UTR Tmem37 Mus musculus

NM_019432.2

SEQ ID NO: 83 - 5'-UTR Tspyl4 Mus musculus

NM_030203.2

SEQ ID NO: 84 - 5'-UTR Slc7a3 Mus musculus

SEQ ID NO: 85 - 5'-UTR Cst6 Mus musculus

SEQ ID NO: 86 - 5'-UTR Aacs Mus musculus

NM_030210.1

SEQ ID NO: 87 - 5'-UTR Nosip Mus musculus

NM_001163684.1

SEQ ID NO: 88 - 5'UTR Itga7 Mus musculus

NM_008398.2

SEQ ID NO: 89 - 5'-UTR Ccnd2 Mus musculus

NM_009829.3

SEQ ID NO: 90 - 5'-UTR Ebp Mus musculus

SEQ ID NO: 91 - 5'-UTR Sf3b5 Mus musculus

NM_009829.3

SEQ ID NO: 92 - 5'-UTR Fasn Mus musculus

SEQ ID NO: 93 - 5'-UTR Hmgcs1 Mus musculus

SEQ ID NO: 94 - 5'-UTR Osr1 Mus musculus

NM_011859.3

SEQ ID NO: 95 - 5'-UTR Lmnb1 Mus musculus

NM_011121.3

SEQ ID NO: 96 - 5'-UTR Aarsd1 Mus musculus

NM_144829.1

SEQ ID NO: 97 - 5'-UTR Vma21 Mus musculus

NM_001081356.2

SEQ ID NO: 98 - 5'-UTR Kif20a Mus musculus

SEQ ID NO: 99 - 5'-UTR Cdca8 Mus musculus

NM_026560.4

SEQ ID NO: 100 - 5'-UTR Slc7a1 Mus musculus

NM_007513.4

SEQ ID NO: 101 - 5'-UTR Ubqln2 Mus musculus

NM_018798.2

SEQ ID NO: 102 - 5'-UTR Prps2 Mus musculus

NM_026662.4

SEQ ID NO: 103 - 5'-UTR Shmt2 Mus musculus

NM_026662.4

SEQ ID NO: 104 - 5'-UTR Kif22 Mus musculus

NM_145588.1

SEQ ID NO: 105 - 5'-UTR Aurkb Mus musculus

NM_011496.1

SEQ ID NO: 106 - 5'-UTR Fign11 Mus musculus

SEQ ID NO: 107 - 5'-UTR Cad Mus musculus

NM_023525.2

SEQ ID NO: 108 - 5'-UTR Anln Mus musculus

NM_028390.3

SEQ ID NO: 109 - 5'-UTR Slfn9 Mus musculus

SEQ ID NO: 110 - 5'-UTR Ncaph Mus musculus

NM_144818.3

SEQ ID NO: 111 - 5'-UTR Cth Mus musculus

NM_145953.2

SEQ ID NO: 112 - 5'-UTR Pole Mus musculus

NM_011132.2

SEQ ID NO: 113 - 5'-UTR Uhrf1 Mus musculus

NM_001111079.1

SEQ ID NO: 114 - 5'-UTR Gja1 Mus musculus

NM_010288.3

SEQ ID NO: 115 - 5'-UTR Fam64a Mus musculus

SEQ ID NO: 116 - 5'-UTR Kif2c Mus musculus

NM_134471.4

SEQ ID NO: 117 - 5'-UTR Tspan10 Mus musculus

NM_145363.2

SEQ ID NO: 118 - 5'-UTR Scand1 Mus musculus

SEQ ID NO: 119 - 5'-UTR Gpr84 Mus musculus

NM_030720.1

SEQ ID NO: 120 - 5'-UTR Tpgs1 Mus musculus

BC138516.1

SEQ ID NO: 121 - 5'-UTR Ccl17 Mus musculus

NM_011332.3

SEQ ID NO: 122 - 5'-UTR Fads3 Mus musculus

NM_021890.3

SEQ ID NO: 123 - 5'-UTR Cers6 Mus musculus

NM_172856.3

SEQ ID NO: 124 - 5'-UTR Alkbh7 Mus musculus

NM_027372.1

SEQ ID NO: 125 - 5'-UTR Ms4a8a Mus musculus

SEQ ID NO: 126 - 5'-UTR Cxcr4 Mus musculus

NM_009911.3

SEQ ID NO: 127 - 5'-UTR Gprc5c Mus musculus

SEQ ID NO: 128 - 5'-UTR Fen1 Mus musculus

NM_007999.4

SEQ ID NO: 129 - 5'-UTR Cspg4 Mus musculus

NM_139001.2

SEQ ID NO: 130 - 5'-UTR Mrpl34 Mus musculus

NM_053162.2

SEQ ID NO: 131 - 5'-UTR Comtd1 Mus musculus

SEQ ID NO: 132 - 5'-UTR Armc6 Mus musculus

NM_133972.2

SEQ ID NO: 133 - 5'-UTR Emr4 Mus musculus

NM_139138.3

SEQ ID NO: 134 - 5'-UTR Atp5d Mus musculus

NM_025313.2

SEQ ID NO: 135-5'-UTR 1110001J03 Rik Mus musculus

SEQ ID NO: 136 - 5'-UTR Csf2ra Mus musculus

Some of these wild-type 5'-UTR elements have sequences that differ from the publicly available 5'-UTR sequences presented in GenBank NCBI, see Table 1 (Example 1). This table reflects the sequencing results obtained in the creation of the present invention.

Thus, 5'UTR elements selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135 are elements of the present invention and may be used in any aspect of the present invention.

Examples of artificial 5'-UTR elements that can be used according to the present invention

In some embodiments, the 5' UTR element of the present invention is different from the wild-type 5' UTR element. Such 5'-UTR elements are referred to as "artificial 5'-UTR elements".

Preferably, the artificial 5'-UTR element has a degree of sequence identity with the wild-type 5'-UTR element of (a) less than 100%, and that, at the same time, it is (b) more than 10% , more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, such 5'- The wild-type UTR element is selected from the group consisting of SEQ ID NOs: 1-136. Generally, an artificial 5'-UTR element differs from a wild-type 5'-UTR element in that at least one nucleotide(s) has been replaced, e.g., two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, or more than ten nucleotides. For example, such a nucleotide substitution may be recommended when the wild-type 5' UTR element contains a nucleotide element that is considered "unfavorable". For example, in some embodiments, the nucleotide element that is considered "unfavorable" is selected from (I) an internal ATG triplet (i.e., an ATG triplet other than the start codon of the nucleic acid open reading frame of the invention) or (II ) a restriction enzyme recognition site (cleavage site), especially a restriction enzyme recognition site (cleavage site) that is recognized (can be cleaved) by a restriction enzyme used in the process of creating (cloning) an artificial nucleic acid of the present invention. Thus, it is possible to deliberately introduce certain base(s) (to exchange, preferably replace, the corresponding wild-type base(s)), so that the artificial 5'-UTR element does not contain a nucleotide element that is considered as "disadvantageous". In particular embodiments, the artificial 5'UTR elements are selected from the group consisting of SEQ ID NOs: 137-151:

SEQ ID NO: 137 - Artificial sequence (based on SEQ ID NO: 3)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 138 - Artificial sequence (based on SEQ ID NO: 9)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 139 - Artificial sequence (based on SEQ ID NO: 14)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 140 - Artificial sequence (based on SEQ ID NO: 17)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 141 - Artificial sequence (based on SEQ ID NO: 18)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 142 - Artificial sequence (based on SEQ ID NO: 29)

заменены на два

оба подчеркнуты выше для большей выразительности][This sequence corresponds to its underlying wild-type sequence, except that two

replaced by two

both underlined above for emphasis]

SEQ ID NO: 143 - Artificial sequence (based on SEQ ID NO: 31)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 144 - Artificial sequence (based on SEQ ID NO: 42)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 145 - Artificial sequence (based on SEQ ID NO: 43)

заменен на

и что (II)

заменен на

каждый подчеркнут выше для большей выразительности][The indicated sequence corresponds to its underlying wild-type sequence, except that (I)

replaced by

and what (II)

replaced by

each underlined above for emphasis]

SEQ ID NO: 146 - Artificial sequence (based on SEQ ID NO: 52)

заменен на

каждый подчеркнут выше для большей выразительности][This sequence corresponds to its underlying wild-type sequence, except that each of the three

replaced by

each underlined above for emphasis]

SEQ ID NO: 147 - Artificial sequence (based on SEQ ID NO: 109)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 148 - Artificial sequence (based on SEQ ID NO: 110)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 149 - Artificial sequence (based on SEQ ID NO: 119)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 150 - Artificial sequence (based on SEQ ID NO: 120)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

SEQ ID NO: 151 - Artificial sequence (based on SEQ ID NO: 136)

заменен на

подчеркнут выше для большей выразительности][The specified sequence corresponds to its underlying wild-type sequence, except that

replaced by

underlined above for emphasis]

3'-UTR elements

Preferably, at least one 3'-UTR element contains or consists of a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 10, 15, 20, 30, or 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 152-204, or the corresponding DNA or RNA sequence, respectively, or at least one 3'-UTR- the element contains or consists of a nucleotide sequence fragment that is at least about 40% identical, preferably at least about 50% identical, preferably at least about 60% identical, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of the nucleotide a sequence selected from the group consisting of SEQ ID NO: 152-204, or the corresponding DNA or RNA sequence, respectively.

Among the sequences detailed below, SEQ ID NO: 152-203 can be considered wild-type 3'-UTR sequences, and SEQ ID NO: 204 can be considered an artificial 3'-UTR sequence.

SEQ ID NO: 152 - 3'-UTR NDUFS7 Homo sapiens

NM_024407.4

SEQ ID NO: 153 - 3'-UTR PHGDH Homo sapiens

NM_006623.3

SEQ ID NO: 154 - 3'-UTR TSPO Homo sapiens

NM_000714.5

SEQ ID NO: 155 - 3'-UTR ATP5D Homo sapiens

NM_001687.4

SEQ ID NO: 156 - 3'-UTR EXOSC4 Homo sapiens

NM_019037.2

SEQ ID NO: 157 - 3'-UTR TUBB4B Homo sapiens

NM_006088.5

SEQ ID NO: 158-3'-UTR TUBA4A Homo sapiens

NM_006000.1

SEQ ID NO: 159 - 3'-UTR EMP3 Homo sapiens

SEQ ID NO: 160 - 3'-UTR CRIP2 Homo sapiens

NM_001312.3

SEQ ID NO: 161 - 3'-UTR BRAT1 Homo sapiens

NM_152743.3

SEQ ID NO: 162 - 3'-UTR CD9 Homo sapiens

NM_001769.3

SEQ ID NO: 163 - 3'-UTR CDK9 Homo sapiens

NM_001261.3

SEQ ID NO: 164 - 3'-UTR PSMB3 Homo sapiens

NM_002795.2

SEQ ID NO: 165 - 3'-UTR PSMB6 Homo sapiens

NM_002798.2

SEQ ID NO: 166 - 3'-UTR PRSS56 Homo sapiens

NM_001195129.1

SEQ ID NO: 167 - 3'-UTR SCAND1 Homo sapiens

NM_016558

SEQ ID NO: 168 - 3'-UTR AMN Homo sapiens

NM_030943.3

SEQ ID NO: 169 - 3'-UTR CYBA Homo sapiens

NM_000101.3

SEQ ID NO: 170 - 3'-UTR PCOLCE Homo sapiens

NM_002593.3

SEQ ID NO: 171 - 3'-UTR MAP1S Homo sapiens

NM_018174.4

SEQ ID NO: 172 - 3'-UTR VTN Homo sapiens

NM_000638.3

SEQ ID NO: 173 - 3'-UTR ALDH16A1 Homo sapiens

NM_001145396.1

SEQ ID NO: 174 - 3'-UTR Acox2 Mus musculus

NM_001161667.1

SEQ ID NO: 175 - 3'-UTR Ubc Mus musculus

BC006680.1

SEQ ID NO: 176 - 3'-UTR Slpi Mus musculus

NM_011414.3

SEQ ID NO: 177 - 3'-UTR Nudt22 Mus musculus

NM_026675.2

SEQ ID NO: 178 - 3'-UTR Pcyox11 Mus musculus

NM_172832.4

SEQ ID NO: 179 - 3'-UTR Igf2bp1 Mus musculus

SEQ ID NO: 180 - 3'-UTR Tmem37 Mus musculus

SEQ ID NO: 181 - 3'-UTR (240) Slc7a3 Mus musculus

NM_007515

SEQ ID NO: 182 - 3'-UTR Cst6 Mus musculus

SEQ ID NO: 183 - 3'-UTR Ebp Mus musculus

SEQ ID NO: 184 - 3'-UTR Sf3b5 Mus musculus

NM_009829.3

SEQ ID NO: 185 - 3'-UTR Plk1 Mus musculus

NM_011121.3

SEQ ID NO: 186 - 3'-UTR Aarsd1 Mus musculus

NM_144829.1

SEQ ID NO: 187 - 3'-UTR Cdca8 Mus musculus

NM_026560.4

SEQ ID NO: 188 - 3'-UTR Kif22 Mus musculus

NM_145588.1

SEQ ID NO: 189 - 3'-UTR Cad Mus musculus

NM_023525.2

SEQ ID NO: 190 - 3'-UTR Cth Mus musculus

NM_145953.2

SEQ ID NO: 191 - 3'-UTR Pole Mus musculus

NM_011132.2

SEQ ID NO: 192 - 3'-UTR Kif2c

Mus musculus NM_134471.4

SEQ ID NO: 193 - 3'-UTR Scand1 Mus musculus

NM_020255.3

SEQ ID NO: 194 - 3'-UTR Gpr84 Mus musculus

NM_030720.1

SEQ ID NO: 195 - 3'-UTR Tpgs1 Mus musculus

NM_148934.2

SEQ ID NO: 196 - 3'-UTR Cel17 Mus musculus

NM_011332.3

SEQ ID NO: 197 - 3'-UTR Alkbh7 Mus musculus

SEQ ID NO: 198 - 3'-UTR Ms4a8a Mus musculus

NM_022430.2

SEQ ID NO: 199 - 3'-UTR Mrp134 Mus musculus

NM_053162.2

SEQ ID NO: 200 - 3'-UTR Comtd1 Mus musculus

SEQ ID NO: 201 - 3'-UTR Armc6 Mus musculus

NM_133972.2

SEQ ID NO: 202 - 3'-UTR Atp5d Mus musculus

NM_025313.2

SEQ ID NO: 203 - 3'-UTR 1110001J03 Rik Mus musculus

NM_025363.3

Some of these wild-type 3'-UTR elements differ from the publicly available 3'-UTR sequences presented in GenBank NCBI, see Table 2 (Example 1). This table reflects the results of sequencing obtained during the creation of the present invention.

Thus, 3'UTR elements selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 197, SEQ ID NO: 200 are the elements proposed in the present invention, and they can be used in any of the objects of the present invention.

An example of artificial 3'-UTR elements that can be used according to the present invention

In some embodiments, the 3'UTR element of the present invention is different from the wild-type 3'UTR element. Such 3'UTR elements are referred to as "artificial 3'UTR elements".

Preferably, the artificial 3'-UTR element has a degree of identity with the wild-type 3'-UTR element of (a) less than 100%, and that, at the same time, it is (B) more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, such a 3'-UTR the wild-type element is selected from the group consisting of SEQ ID NOs: 152-203. Generally, an artificial 3'-UTR element differs from a wild-type 3'-UTR element in that at least one nucleotide(s) has been replaced, e.g., two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, or more than ten nucleotides. For example, such a nucleotide substitution may be recommended when the wild-type 3' UTR element contains a nucleotide element that is considered disadvantageous. For example, in some embodiments of the invention, the nucleotide element that is considered disadvantageous is a site recognized by a restriction enzyme (cleavage site), primarily a site recognized by a restriction enzyme (cleavage site), which is recognized (may be cleaved) by a restriction enzyme used in the creation process ( cloning) of the artificial nucleic acid of the present invention. Thus, it is possible to deliberately introduce certain base(s) (to exchange, preferably replace, the corresponding wild-type base(s)), so that the artificial 3'-UTR element does not contain a nucleotide element that is considered as disadvantageous. In specific embodiments, the artificial 3'-UTR elements have the sequence of SEQ ID NO: 204:

SEQ ID NO: 204 - Artificial sequence (based on SEQ ID NO: 192)

[The indicated sequence corresponds to its underlying wild-type sequence, except that AGATCT is replaced by TGATCT , underlined above for emphasis]

New 5'-UTR elements and new 3'-UTR elements

The present invention also provides new 5'UTR elements and new 3'UTR elements, i.e. 5'-UTR elements and 3'-UTR elements, which were found during the creation of the present invention in human cells and mouse cells, respectively, but which are not presented in public databases (see example 1). Any 5'-UTR selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 may be preferred in some embodiments of the present invention. Any 5'UTR selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO : 131, SEQ ID NO: 135, may be preferred in some embodiments of the present invention. Any 3'-UTR selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 197, SEQ ID NO: 200 may be preferred in some embodiments of the present invention.

Description of 5'UTR Elements and Preferred 3'UTR Elements

Preferably, at least one 3'-UTR element of the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the sequence of the 3'-UTR of a gene transcript selected from the group consisting of NDUFS7-3'-UTR, PHGDH-3'-UTR, TSPO-3'-UTR, ATP5D- 3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'-UTR, BRAT1-3'-UTR, PSMB3-3 '-UTR, PSMB6-3'-UTR, SCAND1-3'-UTR, AMN-3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3' -UTR, ALDH16A1-3'-UT R (all human), Acox2-3'-UTR, Ubc-3'-UTR, Slpi-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'-UTR, Slc7a3-3'-UTR, Cst6- 3'-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR, Cad-3'-UTR, Pole-3'-UTR, Kif2c-3 '-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3'-UTR, Ms4a8a-3'-UTR, Mrpl34-3' -UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001 J03Rik-3'-UTR, Nudt22-3'-UTR (all murine). Most preferably, at least one 3'-UTR element of the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of a sequence selected from the group consisting of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO : 172, SEQ ID NO: 173; SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 204 (alternatively SEQ ID NO: 192), SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 177, or corresponding RNA sequences, respectively.

Preferably, at least one 5'-UTR element of the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the 5'-UTR sequence of the transcript ZNF460-5'-UTR, TGM2-5'-UTR, IL7R-5'-UTR, COL8A1-5'-UTR, NDUFS7-5' -UTR, PLK2-5'-UTR, FBXO32-5'-UTR, ATP5D-5'-UTR, TUBB4B-5'-UTR, ORMDL2-5'-UTR, FSCN1-5'-UTR, CD9-5'- UTR, PYSL2-5'-UTR, PSMB3-5'-UTR, PSMB6-5'-UTR, KPNA6-5'-UTR, SFT2D2-5'-UTR, LCLAT1-5'-UTR, FBXL18-5'-UTR , SLC35F6-5'-UTR, VMA21-5'-UTR, SEZ6L2-5'-UT R, PCOLCE-5'-UTR, VTN-5'-UTR, ALDH16A1-5'-UTR, KPNA6-5'-UTR, JUP-5'-UTR, CPN2-5'-UTR, PNPO-5'-UTR , SSSCA1-5'-UTR, POLR2L-5'-UTR, LIN7C-5'-UTR, UQCR10-5'-UTR, PYCRL-5'-UTR, AMN-5'-UTR, MAP1S-5'-UTR ( all human), Dpysl2-5'-UTR, Acox2-5'-UTR, Ubc-5'-UTR, Nudt22-5'-UTR, Pcyox1l-5'-UTR, Ankrd1-5'-UTR, Tspyl4-5' -UTR, Slc7a3-5'-UTR, Aacs-5'-UTR, Nosip-5'-UTR, Itga7-5'-UTR, Ccnd2-5'-UTR, Ebp-5'-UTR, Sf3b5-5'- UTR, Fasn-5'-UTR, Hmgcs1-5'-UTR, Osr1-5'-UTR, Lmnb1-5'-UTR, Vma21-5'-UTR, Kif20a-5'-UTR, Cdca8-5'-UTR , Slc7a1-5'-UTR, Ubqln2-5'-UTR, Prps2-5'-UTR, Shmt2-5'-UTR, Fignl1-5'-UTR, Cad-5'-UTR, Anln-5'-UTR, Slfn9-5'-UTR, Ncaph-5'-UTR, Pole-5'-UTR, Uhrf1-5'-UTR, Gja1-5'-UTR, Fam64a-5'-UTR, Tspan10-5'-UTR, Scand1 -5'-UTR, Gpr84-5'-UTR, Cers6-5'-UTR, Cxcr4-5'-UTR, Gprc5c-5'-UTR, Fen1-5'-UTR, Cspg4-5'-UTR, Mrpl34- 5'-UTR, Comtd1-5'-UTR, Armc6-5'-UTR, Emr4-5'-UTR, Atp5d-5'-UTR, Csf2ra-5'-UTR, Aarsd1-5'-UTR, Cth-5 '-UTR, Tpgs1-5'-UTR, Ccl17-5'-UTR, Alkbh7-5'-UTR, Ms4a8a-5'-UTR (all murine). Most preferably, at least one 5'-UTR element of the artificial nucleic acid molecule of the present invention contains or consists of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (alternatively, SEQ ID NO: 3), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (alternatively SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO : 144 (alternatively SEQ ID NO: 42), SEQ ID NO: 58, SEQ ID NO: 62; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119 ), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125 or the corresponding RNA sequence, respectively.

At least one 3'-UTR element of the artificial nucleic acid molecule of the present invention may also contain or consist of a nucleotide sequence fragment that is at least about 40% identical, preferably at least about 50% identical, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the nucleotide sequence of the 3'UTR of the gene transcript, such as the 3'UTR sequence shown in SEQ ID NOs: 152-204, wherein the fragment is preferably a functional fragment or a functional variant of the fragment described above. Such a fragment is preferably at least about 3 nucleotides in length, preferably at least about 5 nucleotides, more preferably at least about 10, 15, 20, 25 or 30 nucleotides, even more preferably at least about 50 nucleotides, most preferably at least about 70 nucleotides. In a preferred embodiment, the fragment or variant thereof is 3 to about 500 nucleotides in length, preferably 5 to about 150 nucleotides, more preferably 10 to 100 nucleotides, even more preferably 15 to 90, most preferably 20 to 70 nucleotides. Preferably, said variants, fragments, or fragment variants are functional variants, functional fragments, or functional variants of 3'-UTR fragments that prolong protein production from the artificial nucleic acid molecule of the invention with an efficiency of at least 30%, preferably with an efficiency of at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% on the efficiency of protein production prolongation provided by an artificial nucleic acid molecule that contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID N O: 161, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173; SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 204 (alternatively SEQ ID NO: 192), SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 177.

At least one 5'-UTR element of the artificial nucleic acid molecule of the present invention may also contain or consist of a nucleotide sequence fragment that is at least about 40% identical, preferably at least about 50% identical, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the nucleotide sequence of the 5'UTR of the gene transcript, such as the 5'UTR sequence shown in SEQ ID NOs: 1-151, wherein the fragment is preferably a functional fragment or a functional variant of the fragment described above. Such a fragment is preferably at least about 3 nucleotides in length, preferably at least about 5 nucleotides, more preferably at least about 10, 15, 20, 25 or 30 nucleotides, even more preferably at least about 50 nucleotides, most preferably at least about 70 nucleotides. In a preferred embodiment, the fragment or variant thereof is 3 to about 500 nucleotides in length, preferably 5 to about 150 nucleotides, more preferably 10 to 100 nucleotides, even more preferably 15 to 90, most preferably 20 to 70 nucleotides. Preferably, said variants, fragments, or fragment variants are functional variants, functional fragments, or functional variants of 3'-UTR fragments that prolong protein production from the artificial nucleic acid molecule of the invention with an efficiency of at least 30%, preferably with an efficiency of at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% from the efficiency of protein production prolongation provided by an artificial nucleic acid molecule that contains a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (alternatively, SEQ ID NO: 3 ), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 ( alternatively SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 144 (alternatively SEQ ID NO: 42), SEQ ID N0:58, SEQ ID NO: 62; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119 ), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125.

Other preferred embodiments of the invention

Preferably, at least one 3'-UTR element and/or at least one 5'-UTR element of the artificial nucleic acid molecule of the present invention is at least about 3 nucleotides in length, preferably at least about 5 nucleotides. , more preferably at least about 10, 15, 20, 25 or 30 nucleotides, even more preferably at least about 50 nucleotides, most preferably at least about 70 nucleotides. The upper limit of the length of at least one 3'UTR element and/or at least one 5'UTR element may be 500 nucleotides or less, such as 400, 300, 200, 150 or 100 nucleotides. In other embodiments of the invention, the upper limit can be selected in the range from 50 to 100 nucleotides. For example, a fragment or variant thereof may be from 3 to about 500 nucleotides in length, preferably from 5 to about 150 nucleotides, more preferably from 10 to 100 nucleotides, even more preferably from 15 to 90, most preferably from 20 to 70 nucleotides.

It may also be preferred that the UTR element contained in the artificial nucleic acid molecule contains (or consists of) a contiguous sequence that is shorter than any sequence selected from SEQ ID NOs: 1-204 provided herein, provided that it is still 70% or more, 80% or more, 90% or more, or 95% or more identical to the corresponding sequence of any sequence selected from SEQ ID NO: 1-204. In such a case, preferably, the replacement UTR element contains a contiguous sequence that is identical throughout its length to any sequence selected from SEQ ID NOs: 1-204; however, the replacement UTR element is shorter, for example, has a length of 70% or more, 80% or more, 90% or more, or 95% or more of the entire length of the sequence, and is otherwise identical to the specified sequence, which is selected from SEQ ID NO: 1-204.

In addition, the artificial nucleic acid molecule of the present invention may contain more than one 3'UTR element and/or more than one 5'UTR element as described above. For example, an artificial nucleic acid molecule of the present invention may contain one, two, three, four or more 3'-UTR elements, and/or one, two, three, four or more 5'-UTR elements. , while the individual 5'-UTR elements may be the same or they may be different, and similarly, the individual 5'-UTR elements may be the same or they may be different. For example, an artificial nucleic acid molecule of the present invention may contain two substantially identical 3'-UTR elements as described above, for example, two 3'-UTR elements containing or consisting of a nucleotide sequence that is derived from a 3'- The UTR of the gene transcript, for example, from the sequence shown in SEQ ID NO: 152-204, or from a fragment or variant of the 3'-UTR of the gene transcript, its functional variants, its functional fragments or its functional variants of the fragments described above. Therefore, for example, an artificial nucleic acid molecule of the present invention may contain two substantially identical 5'-UTR elements as described above, for example, two 5'-UTR elements containing or consisting of a nucleotide sequence that is derived from 5 The '-UTR of a gene transcript, such as, for example, from the sequence shown in SEQ ID NO: 1-151, or from a fragment or variant of the 5'-UTR of a gene transcript, its functional variants, its functional fragments or its functional variants of the fragments described above.

In the course of the invention, it has surprisingly been found that an artificial nucleic acid molecule containing a 3'UTR element as described above and/or a 5'UTR element as described above may be, or may allow the creation of, an mRNA molecule that provides a high translation efficiency. Thus, a 3'-UTR element as provided herein and/or a 5'-UTR element as provided herein can increase the efficiency of translation of an mRNA molecule.

In particular, an artificial nucleic acid molecule according to the invention may contain (i) at least one 3'-UTR element and at least one 5'-UTR element, which provide high translation efficiency; (II) at least one 3'UTR element that provides high translation efficiency, but does not contain a 5'UTR element that provides high translation efficiency; or (iii) at least one 5'UTR element that provides high translation efficiency, but does not contain a 3'UTR element that provides high translation efficiency.

However, primarily in cases (II) and (III), but possibly also in case (I), the artificial nucleic acid molecule of the present invention may additionally contain one or more "additional 3'-UTRs -elements and/or 5'-UTR-elements", i. e. 3'UTR elements and/or 5'UTR elements that do not meet the requirements described above. For example, an artificial nucleic acid molecule of the invention that contains a 3'-UTR element of the invention, i.e. The 3'-UTR element that provides high translation efficiency of said artificial nucleic acid molecule may further comprise any additional 3'-UTR and/or any additional 5'-UTR, in particular an additional 5'-UTR, for example, 5' -TOP UTR, or any other 5'-UTR or other 5'-UTR element. Similarly, for example, an artificial nucleic acid molecule according to the invention, which contains a 5'-UTR element according to the present invention, ie. The 5'-UTR element that provides high translation efficiency of said artificial nucleic acid molecule may further comprise any additional 3'-UTR and/or any additional 5'-UTR, in particular an additional 3'-UTR, for example, 3' -UTR derived from the 3'-UTR of the albumin gene, most preferably a 3'-UTR containing the sequence shown in SEQ ID NO: 206 or 207, especially SEQ ID NO: 207, or any other 3'-UTR or other 3 '-UTR element.

If, in addition to the inventive at least one 5'-UTR element and/or the inventive at least one 3'-UTR element that provides high translation efficiency, an additional 3'-UTR (element) and/or additional 5'-UTR (element), then additional 5'-UTR (element) and/or additional 3'-UTR (element) can interact with the inventive 3'-UTR- element and/or inventive 5'-UTR element and thus maintain an impact on translation efficiency of the inventive 3'-UTR element and/or inventive 5'-UTR element, respectively. Such additional 3'-UTR and/or 5'-UTR (elements) may further support translational stability and efficiency. In addition, if both the inventive 3'-UTR element and the inventive 5'-UTR element are present in the artificial nucleic acid molecule of the invention, there is preferably a synergistic effect on the effect of the inventive 3'UTR element and/or proposed in the invention 5'UTR element in terms of ensuring translation efficiency.

Preferably, the additional 3'-UTR contains or consists of a nucleotide sequence that is derived from the 3'-UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen gene. alpha, such as the alpha 1(I) collagen gene, or from a 3'-UTR variant of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene such as the alpha 1(I) collagen gene shown in SEQ ID NO: 1369-1390 of patent application WO 2013/143700, the contents of which are incorporated herein by reference. In a most preferred embodiment, the other 3'UTR comprises or consists of a nucleotide sequence that is derived from the 3'UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably a human albumin gene, having SEQ ID NO: 206:

SEQ ID NO: 206:

(3'-UTR of human albumin; corresponding to SEQ ID NO: 1369 presented in patent application WO 2013/143700).

In another most preferred embodiment of the invention, the other 3'UTR comprises or consists of a nucleotide sequence that is derived from the 3'UTR of an α-globin gene, preferably a vertebrate α- or β-globin gene, more preferably an α- or β-globin gene. a mammalian, most preferably a human α- or β-globin gene having SEQ ID NO: 1370 of patent application WO 2013/143700 (Hemoglobin alpha 1 (HBA1) 3'UTR of Homo sapiens), or SEQ ID NO: 1371 , presented in patent application WO 2013/143700 (3'-UTR of hemoglobin alpha 2 (HBA2) Homo sapiens), and/or SEQ ID NO: 1372, presented in patent application WO 2013/143700 (3'-UTR of hemoglobin beta (NVV) Homo sapiens).

For example, the additional 3'-UTR may contain or consist of the central, α-complex-binding fragment of the 3'-UTR of the α-globin gene having SEQ ID NO: 1393, presented in patent application WO 2013/143700.

In this context, it is most preferred that the nucleic acid molecule according to the invention contains an additional 3'-UTR element derived from nucleic acids having SEQ ID NO: 1369-1390, which are presented in patent application WO 2013/143700, or fragments thereof. , homologues or variants.

Most preferably, the additional 3'-UTR contains a nucleotide sequence derived from the human albumin gene fragment having SEQ ID NO: 207:

SEQ ID NO: 207:

(3'-UTR. albumin7; corresponding to SEQ ID NO: 1376 of patent application WO 2013/143700)

In this context, it is most preferred that the additional 3'-UTR of the artificial nucleic acid molecule of the invention comprises or consists of a nucleotide sequence having SEQ ID NO: 207 or a corresponding RNA sequence.

In some embodiments, the additional 3'-UTR comprises or consists of a nucleotide sequence derived from a gene encoding a ribosomal protein, preferably described in International Patent Applications WO 2015/101414 or WO 2015/101415, the content of which is incorporated herein as links.

The additional 3'-UTR may also comprise or consist of a nucleotide sequence derived from a gene encoding a ribosomal protein, wherein the genes encoding the ribosomal protein from which the additional 3'-UTR can be derived include, but are not limited to, ribosomal protein genes. protein L9 (RPL9), ribosomal protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosomal protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomal protein L7a (RPL7A), ribosomal protein L11 (RPL11), ribosomal protein L12 (RPL12), ribosomal protein L13 (RPL13), ribosomal protein L23 (RPL23), ribosomal protein L18 (RPL18), ribosomal protein L18a (RPL18A), ribosomal protein L19 (RPL19), ribosomal protein L21 (RPL21), ribosomal protein L22 (RPL22), ribosomal protein L23a (RPL23A), ribosomal protein L17 (RPL17), ribosomal protein L24 (RPL24), ribosomal protein L26 (RPL26), ribosomal protein L27 (RPL27), ribosomal ribosomal protein L30 (RPL30), ribosomal protein L27a (RPL27A), ribosomal protein L28 (RPL28), ribosomal protein L29 (RPL29), ribosomal protein L31 (RPL31), ribosomal protein L32 (RPL32), ribosomal protein L35a (RPL35A), ribosomal protein L37 (RPL37), ribosomal protein L37a (RPL37A), ribosomal protein L38 (RPL38), ribosomal protein L39 (RPL39), ribosomal protein, large subunit, P0 (RPLP0), ribosomal protein, large subunit, P1 (RPLP1), ribosomal protein, large subunit, P2 (RPLP2), ribosomal protein S3 (RPS3), ribosomal protein S3A (RPS3A), ribosomal protein S4, X-linked (RPS4X), ribosomal protein S4, Y-linked 1 (RPS4Y1), ribosomal protein S5 (RPS5), ribosomal protein S6 (RPS6), ribosomal protein S7 (RPS7), ribosomal protein S8 (RPS8), ribosomal protein S9 (RPS9), ribosomal protein S10 (RPS10), ribosomal protein S11 (RPS11), ribosomal protein S12 ( RPS12), ribosomal protein S13 (RPS13), rib osomal protein S15 (RPS15), ribosomal protein S15a (RPS15A), ribosomal protein S16 (RPS16), ribosomal protein S19 (RPS19), ribosomal protein S20 (RPS20), ribosomal protein S21 (RPS21), ribosomal protein S23 (RPS23), ribosomal ribosomal protein S25 (RPS25), ribosomal protein S26 (RPS26), ribosomal protein S27 (RPS27), ribosomal protein S27a (RPS27a), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), ribosomal protein L15 (RPL15), ribosomal protein S2 (RPS2), ribosomal protein L14 (RPL14), ribosomal protein S14 (RPS14), ribosomal protein L10 (RPL10), ribosomal protein L10a (RPL10A), ribosomal protein L35 (RPL35), ribosomal protein L13a (RPL13A), ribosomal protein L36 (RPL36), ribosomal protein L36a (RPL36A), ribosomal protein L41 (RPL41), ribosomal protein S18 (RPS18), ribosomal protein S24 (RPS24), ribosomal protein L8 (RPL8), ribosomal protein L34 (RPL34), ribosomal protein S17 ( RPS17), ribosomal th protein SA (RPSA), fusion product 1: ubiquitin residue A-52-ribosomal protein (UBA52), ubiquitously expressed (FAU) mouse Finkel-Biskeys-Reilly sarcoma virus, protein 1, like ribosomal protein L22 (RPL22L1), protein, ribosomal L39-like protein (RPL39L), ribosomal protein L10-like protein (RPL10L), ribosomal protein L36a-like protein (RPL36AL), ribosomal protein L3-like protein (RPL3L), ribosomal protein S27-like protein (RPS27L), protein 1 ribosomal protein L26-like 1 (RPL26L1), ribosomal protein L7-like protein 1 (RPL7L1), ribosomal protein L13a pseudogene (RPL13AP), ribosomal protein L37a pseudogene 8 (RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomal pseudogene 11 protein S26 (RPS26P11), pseudogene 5 ribosomal protein L39 (RPL39P5), pseudogene 6 ribosomal protein, large subunit, P0 (RPLP0P6) and pseudogene 14 ribosomal protein L36 (RPL36P14).

Preferably, the additional 5'UTR contains or consists of a nucleotide sequence that is derived from the 5'UTR of the TOP gene, or that is derived from a fragment, homologue or variant of the 5'UTR of the TOP gene.

Most preferably, the 5'UTR element does not contain the TOP motif or 5'TOP as defined above. In particular, it is preferred that the 5'UTR of the TOP gene is the 5'UTR of the TOP gene without a TOP motif.

The nucleotide sequence which is derived from the 5'UTR of the TOP gene is derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as the human TOR gene.

For example, the additional 5'UTR element is preferably selected from 5'UTR elements containing or consisting of a nucleotide sequence that is derived from a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-1363, SEQ ID NO: 1395 , SEQ ID NO: 1421 and SEQ ID NO: 1422, presented in patent application WO 2013/143700, the description of which is incorporated herein by reference, from the homologues of SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422, presented in patent application WO 2013/143700, from its variant or preferably from the corresponding RNA sequence. The term "homologues of SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 presented in patent application WO 2013/143700" refers to sequences from species other than Homo sapiens that are homologous the sequences SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 presented in patent application WO 2013/143700.

In a preferred embodiment of the invention, the additional 5'UTR contains or consists of a nucleotide sequence that is derived from a nucleotide sequence extending from nucleotide position 5 (i.e., the nucleotide located in position 5 in the sequence) to the nucleotide position immediately adjacent to 5' - side to the start codon (located at the 3' end of the sequences), for example, to the nucleotide position immediately adjacent on the 5' side to the ATG sequence of the nucleotide sequence selected from SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of patent application WO 2013/143700 from homologues of SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422, presented in patent application WO 2013/143700, from its variant, or the corresponding RNA sequence. Most preferably, the additional 5' UTR is derived from a nucleotide sequence extending from the nucleotide position immediately 3'-side of the 5'-TOP to the nucleotide position immediately 5'-side of the start codon (located at the 3'-end sequences), for example, to the nucleotide position immediately adjacent on the 5' side to the ATG sequence of the nucleotide sequence selected from SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422, presented in patent application WO 2013/143700, from the homologues of SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 presented in patent application WO 2013/143700, from its variant , or the corresponding RNA sequence.

In the most preferred embodiment of the invention, the additional 5'-UTR contains or consists of a nucleotide sequence derived from the 5'-UTR of the TOP gene encoding the ribosomal protein, or from the variant 5'-UTR of the TOP gene encoding the ribosomal protein. For example, the 5'-UTR element contains or consists of a nucleotide sequence derived from the 5'-UTR nucleotide sequence shown in any of SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1313, 1313. 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1338, 1338, 1338. 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360 patent application20 143700 corresponding to the RNA sequence, its homologue or its variant presented in the present description, preferably without the 5'-TOP motif. As described above, the sequence extending from position 5 to the nucleotide immediately adjacent 5' to the ATG (which is at the 3' end of the sequences) corresponds to the 5' UTR of the indicated sequences.

Preferably, the additional 5'UTR contains or consists of a nucleotide sequence that is derived from the 5'UTR of the TOP gene encoding the ribosomal large subunit protein (RPL) or from a homologue or variant of the 5'UTR of the TOP gene encoding the ribosomal large subunit protein (RPL). For example, the 5'-UTR element contains or consists of a nucleotide sequence derived from the 5'-UTR nucleotide sequence shown in any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422 described in patent application WO 2013/143700 corresponding to the RNA sequence, its homologue or its variant presented in the present description, preferably without a 5'-TOP motif.

In a most preferred embodiment, the 5'-UTR element comprises or consists of a nucleotide sequence derived from the 5'-UTR of the large subunit 32 ribosomal protein gene, preferably from the vertebrate large subunit 32 ribosomal protein (L32) gene, more preferably the ribosomal protein gene mammalian 32 large subunit (L32) ribosomal protein gene, most preferably from the human 32 large subunit ribosomal protein (L32) protein gene, or from the 5'-UTR variant of the 32 large subunit ribosomal protein gene, preferably from the vertebrate 32 large subunit ribosomal protein (L32) protein gene, more preferably from a mammalian 32 large subunit ribosomal protein (L32) gene, most preferably from a human 32 large subunit ribosomal protein (L32) gene, where preferably the additional 5'-UTR does not contain the 5'-TOP of said gene.

Therefore, in the most preferred embodiment of the invention, the additional 5'-UTR contains or consists of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of the nucleotide sequence shown in SEQ ID NO: 208 (5'-UTR of human large subunit 32 ribosomal protein without 5'-oligopyrimidine tract:

(SEQ ID NO: 208); соответствующая SEQ ID NO: 1368, указанной в заявке на патент WO 2013/143700) или предпочтительно соответствующей последовательности РНК, или где по меньшей мере одна дополнительная 5'UTR содержит или состоит из фрагмента нуклеотидной последовательности, идентичного по меньшей мере примерно на 40%, предпочтительно по меньшей мере примерно на 50%, предпочтительно по меньшей мере примерно на 60%, предпочтительно по меньшей мере примерно на 70%, более предпочтительно по меньшей мере примерно на 80%, более предпочтительно по меньшей мере примерно на 90%, еще более предпочтительно по меньшей мере примерно на 95%, еще более предпочтительно по меньшей мере примерно на 99% нуклеотидной последовательности, представленной в SEQ ID NO: 208, или более предпочтительно соответствующей последовательности РНК, где предпочтительно фрагмент представляет собой описанный выше фрагмент, непрерывный сегмент нуклеотидов, представляющий собой по меньшей мере 20% и т.д. полноразмерной 5'-UTR. Предпочтительно фрагмент имеет длину по меньшей мере примерно 20 нуклеотидов или более, предпочтительно по меньшей мере примерно 30 нуклеотидов или более, более предпочтительно по меньшей мере примерно 40 нуклеотидов или более. Предпочтительно фрагмент представляет собой функциональный фрагмент, представленный в настоящем описании.

(SEQ ID NO: 208); corresponding to SEQ ID NO: 1368 specified in patent application WO 2013/143700) or preferably the corresponding RNA sequence, or where at least one additional 5'UTR contains or consists of a fragment of a nucleotide sequence that is at least about 40% identical, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of the nucleotide sequence shown in SEQ ID NO: 208, or more preferably the corresponding RNA sequence, where preferably the fragment is the fragment described above, a continuous segment of nucleotides representing at least 20%, etc. full-length 5'-UTR. Preferably, the fragment is at least about 20 nucleotides or more in length, preferably at least about 30 nucleotides or more, more preferably at least about 40 nucleotides or more. Preferably the fragment is a functional fragment, presented in the present description.

In some embodiments, the artificial nucleic acid molecule comprises an additional 5'-UTR that contains or consists of a nucleotide sequence derived from the 5'-UTR of a vertebrate, e.g., mammalian TOP gene, e.g., a human TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL41, RPL40 RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1 , UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or from a homologue or variant thereof, where preferably the additional 5'-UTR does not contain the TOP motif or 5'-TOP of these genes, and where optionally the 5'-UTR element begins with at its 5' end from a nucleotide at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 downstream of the 5' oligopyrimidine tract (TOP), and where optionally also an additional 5 '-UTR, derived from the 5'-UTR of the TOP gene, ends at its 3'-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 in the opposite direction from the start codon (A(U/T)G) of the gene from which it originates.

In specific embodiments, the additional 5'-UTR comprises or consists of a nucleotide sequence that is derived from the 5'-UTR described in International Patent Application WO 2016/107877, the contents of which are incorporated herein by reference.

The artificial nucleic acid molecule of the present invention may be RNA such as mRNA or viral RNA or replicon, DNA such as plasmid DNA or viral DNA, or may be a modified RNA or DNA molecule. It may be a double-stranded molecule having a sense strand and an antisense strand, such as a DNA molecule having a sense strand and an antisense strand.

The artificial nucleic acid molecule of the present invention may optionally also contain a 5' cap. The optional 5' cap is preferably located 5' to the ORF, more preferably 5' to at least one 5'UTR or any additional 5'UTR in the artificial nucleic acid molecule of the present invention.

Preferably, the artificial nucleic acid molecule of the present invention also contains a poly(A) sequence and/or a polyadenylation signal. Preferably the optional poly(A) sequence is located 3' to at least one 3' UTR element or any additional 3' UTR, more preferably the optional poly(A) sequence is fused to the 3' end of the 3' -UTR element. The connection can be direct or indirect, for example, using a segment consisting of 2, 4, 6, 8, 10, 20, etc. nucleotides, for example, using a linker consisting of 1-50, preferably 1-20 nucleotides, for example, containing or consisting of one or more restriction sites. However, even if the artificial nucleic acid molecule of the present invention does not contain a 3'-UTR, for example, if it contains only at least one 5'-UTR element, it preferably still contains the poly(A) sequence and/or a polyadenylation signal.

In one embodiment of the invention, the optional polyadenylation signal is located downstream of the transcription from the 3' side of the 3' UTR element. Preferably, the polyadenylation signal contains the NN(U/T)ANA consensus sequence in which N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA. Such a consensus sequence can be recognized by most animal cell systems and bacterial cell systems, for example, polyadenylation factors such as cleavage/polyadenylation specificity factor (CPSF) in combination with CstF, PAP, PAB2, CFI and/or CFII. Preferably, the polyadenylation signal, preferably the NNUANA consensus sequence, is less than about 50 nucleotides away, more preferably less than about 30 bases, most preferably less than about 25 bases, e.g., 21 bases downstream of transcription from 3'- the end of the 3'UTR element, or ORF if no 3'UTR element is present.

Transcription of an artificial nucleic acid molecule of the present invention, for example, an artificial DNA molecule containing a polyadenylation signal downstream of the 3'UTR element (or ORF), should result in an immature RNA containing a polyadenylation signal downstream. transcription relative to its 3'-UTR element (or ORF).

The addition of the poly(A) sequence to the immature RNA must then occur using an appropriate transcription system. For example, an artificial nucleic acid molecule according to the invention may be a DNA molecule containing a 3'-UTR element as described above and a polyadenylation signal that can lead to RNA polyadenylation after transcription of said DNA molecule. Therefore, the resulting RNA may contain a combination of the 3'UTR element of the invention followed by the poly(A) sequence.

Possible transcription systems are in vitro transcription systems or cellular transcription systems, etc. Thus, transcription of an artificial nucleic acid molecule of the invention, for example, transcription of an artificial nucleic acid molecule containing an open reading frame, a 3'-UTR element and/or a 5'-UTR element, and optionally a polyadenylation signal, can result in an mRNA molecule containing an open reading frame, a 3'-UTR element, and optionally a poly(A) sequence.

Thus, the invention also provides an artificial nucleic acid molecule, which is an mRNA molecule containing an open reading frame, a 3'-UTR element as described above, and/or a 5'-UTR element as described above, and optionally a poly( A) is a sequence.

In another embodiment, the 3'UTR of the artificial nucleic acid molecule of the invention does not contain a polyadenylation signal or a poly(A) sequence. In addition, preferably the artificial nucleic acid molecule of the invention does not contain a polyadenylation signal or a poly(A) sequence. More preferably, the 3'-UTR of the artificial nucleic acid molecule, or the artificial nucleic acid molecule itself according to the invention, does not contain a polyadenylation signal, in particular does not contain an AAU/TAAA polyadenylation signal.

In a preferred embodiment, the invention provides an artificial nucleic acid molecule, which is an artificial RNA molecule containing an open reading frame and an RNA sequence corresponding to a DNA sequence selected from the group consisting of the sequences shown in SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO: 137 (alternatively SEQ ID NO: 3), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (alternatively SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO : 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO : 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 144 (alternatively SEQ ID NO: 42), SEQ ID NO: 58, SEQ ID NO: 62; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119 ), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125 or a fragment thereof as described above. In addition, a corresponding artificial DNA molecule has been proposed.

In another preferred embodiment, the invention provides an artificial nucleic acid molecule, which is an artificial DNA molecule containing an open reading frame and a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (alternatively SEQ ID NO: 3), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (alternatively SEQ ID NO: 52), SEQ ID NO: 53 , SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, S EQ ID NO: 37, SEQ ID NO: 144 (alternatively SEQ ID NO: 42), SEQ ID NO: 58, SEQ ID NO: 62; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119 ), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125.

Thus, the invention provides an artificial nucleic acid molecule that can serve as a template for an RNA molecule, preferably an mRNA molecule, which has a high translation efficiency. In other words, the artificial nucleic acid molecule may be DNA, which can be used as a template to obtain mRNA. mRNA that can be obtained in this way can, in turn, be translated to obtain the desired peptide or protein encoded by the open reading frame. If the artificial nucleic acid molecule is DNA, then it can, for example, be used as a double-stranded storage form to continue and repeat the production of mRNA in vitro or in vivo. As used herein, in vitro refers specifically to (“live”) cells and/or tissues, including tissue from a living individual. Cells include, in particular, cell lines, primary cells, cells in a tissue or an individual's body. In specific embodiments of the invention suitable for use according to the present invention may be cell types that allow the cultivation of cells. Most preferred are mammalian cells, eg human cells and mouse cells. In the most preferred embodiments of the invention, human HeLa, HEPG2 and U-937 cell lines and mouse NIH3T3, JAWSII and L929 cell lines are used. In addition, most preferred are primary cells, in the most preferred embodiments of the invention, human dermal fibroblasts (HDF) can be used. Alternatively, tissue from the individual may also be used.

In one embodiment, the artificial nucleic acid molecule of the present invention also contains a poly(A) sequence. For example, a DNA molecule containing an ORF, optionally followed by a 3'UTR, may contain a thymidine nucleotide segment that can be transcribed into a poly(A) sequence in the resulting mRNA. The length of the poly(A) sequence may vary. For example, the poly(A) sequence may be from about 20 adenine nucleotides up to 300 adenine nucleotides in length, preferably from about 40 to about 200 adenine nucleotides, more preferably from about 50 to about 100 adenine nucleotides, for example, about 60, 70, 80, 90 or 100 adenine nucleotides. Most preferably, the inventive nucleic acid comprises a poly(A) sequence of about 60 to about 70 nucleotides, most preferably 64 adenine nucleotides.

Artificial RNA molecules can also be produced in vitro using conventional chemical synthesis without the need for transcription from a precursor DNA.

In the most preferred embodiment of the invention, the artificial nucleic acid molecule of the present invention is an RNA molecule, preferably an RNA molecule containing in the 5'→3' direction an open reading frame, a 3'-UTR element as described above, and a poly (A)-sequence, or containing in the 5'→3'-direction 5'-UTR element described above, an open reading frame and poly(A)-sequence.

In a preferred embodiment of the invention, the open reading frame is derived from a gene other than the gene from which the 3'UTR element and/or the 5'UTR element of the artificial nucleic acid of the invention is derived. In some other preferred embodiments, the open reading frame does not encode a gene selected from the group that consists of ZNF460, TGM2, IL7R, BGN, TK1, RAB3B, CBX6, FZD2, COL8A1, NDUFS7, PHGDH, PLK2, TSPO, PTGS1, FBXO32 , NID2, ATP5D, EXOSC4, NOL9, UBB4B, VPS18, ORMDL2, FSCN1, TMEM33, TUBA4A, EMP3, TMEM201, CRIP2, BRAT1, SERPINH1, CD9, DPYSL2, CDK9, TFRC, PSMB3, FASN, PSMB6, PRSS56, KPNA6, SFT2D2 , PARD6B, LPP, SPARC, SCAND1, VASN, SLC26A1, LCLAT1, FBXL18, SLC35F6, RAB3D, MAP1B, VMA21, CYBA, SEZ6L2, PCOLCE, VTN, ALDH16A1, RAVER1, KPNA6, SERINC5, JUP, CPN2, CRIP2, EPT1, PNPO , SSSCA1, POLR2L, LIN7C, UQCR10, PYCRL, AMN, MAP1S, NDUFS7, PHGDH, TSPO, ATP5D, EXOSC4, TUBB4B, TUBA4A, EMP3, CRIP2, BRAT1, CD9, CDK9, PSMB3, PSMB6, PRSS56, SCAND1, AMN, CYBA , PCOLCE, MAP1S, VTN, ALDH16A1 (preferably all human) and Dpysl2, Ccnd1, Acox2, Cbx6, Ubc, Ldlr, Nudt22, Pcyox1l, Ankrd1, Tmem37, Tspyl4, Slc7a3, Cst6, Aacs, Nosip, Itga7, Ccnd2, Ebp, Sf3b5, Fasn, Hmgcs1, Osr1, Lmnb1, Vma21, Kif20a, Cdca8, Slc7a1, Ubqln2, Prps2, Shmt2, Aurkb, Fignl1, Cad, Anln, Slfn9, Ncaph, Pole, Uhrf1, Gja1, Fam64a, Kif2c, Tspan10, Scand1, Gpr84, Fads3, Cers6, Cxcr4, Gprc5c, Fen1, Cspg4, Mrpl34, Comtd1, Armc6, Emr4, Atp5d, 1110001J03Rik, Csf2ra, Aarsd1, Kif22, Cth, Tpgs1, Cel17, Alkbh7, Ms4a8a, Acox2, Ubc, Slpi, Pcyox1l, Igf2bp1, Tmem3, Cst6, Ebp, Sf3b5, Plk1, Cdca8, Kif22, Cad, Cth, Pole, Kif2c, Scand1, Gpr84, Tpgs1, Ccl17, Alkbh7, Ms4a8a, Mrpl34, Comtd1, Armc6, Atp5d, 1110001J03Rik, Nudt22, Aarsd1 (preferably all mice) , or variants thereof, provided that the 3'-UTR element and/or 5'-UTR element has a sequence selected from the group which consists of the sequences shown in SEQ ID NO: 1-204.

In a preferred embodiment of the invention, the ORF does not encode human or plant ribosomal proteins, especially Arabidopsis, primarily does not encode human ribosomal protein S6 (RPS6), human ribosomal protein L36a like protein (RPL36AL), or Arabidopsis ribosomal protein S16 (RPS16). In another preferred embodiment, the open reading frame (ORF) does not encode ribosomal protein S6 (RPS6), ribosomal protein L36a (RPL36AL) or ribosomal protein S16 (RPS16), regardless of its origin.

In one embodiment, the invention provides an artificial DNA molecule comprising an open reading frame, preferably an open reading frame, derived from a gene other than the gene from which the 3'-UTR element and/or 5'-UTR element is derived; wherein the 3'-UTR element contains or consists of a sequence identical to at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90 %, even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of a DNA sequence selected from the group consisting of the sequences shown in SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173; SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 204 (alternatively SEQ ID NO: 192), SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 177, and/or the 5'-UTR element contains or consists of a sequence identity of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of a DNA sequence selected from the group consisting of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (alternatively SEQ ID NO: 3), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (alternatively SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 144 (alternatively SEQ ID NO: 42), SEQ ID NO: 58, SEQ ID NO: 62; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119 ), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125; and a polyadenylation signal and/or a poly(A) sequence.

In addition, the invention provides an artificial RNA molecule, preferably an artificial mRNA molecule or an artificial viral RNA molecule, containing an open reading frame, preferably an open reading frame, derived from a gene other than the gene from which the 3'-UTR element is derived and/or 5'-UTR element; wherein the 3'-UTR element contains or consists of a sequence identical to at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90 %, even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of an RNA sequence that corresponds to a DNA sequence selected from the group consisting of the sequences shown in SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO : 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173; SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 204 (alternatively SEQ ID NO: 192), SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 177, and/or the 5'-UTR element contains or consists of a sequence identity of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of an RNA sequence that corresponds to a DNA sequence selected from the group consisting of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (alternatively, SEQ ID NO: 3), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (alternatively SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57 , SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 144 (alternatively SEQ ID NO: 42), SEQ ID NO: 58, SEQ ID NO: 62 ; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125; and a polyadenylation signal and/or a poly(A) sequence.

The invention provides an artificial nucleic acid molecule, preferably an artificial mRNA, which is characterized by high translation efficiency. Without wishing to be bound by any theory, it is believed that the high efficiency of translation may be due to reduced cleavage of an artificial nucleic acid molecule, such as an artificial mRNA molecule of the present invention. Therefore, the inventive 3'UTR element and/or the inventive 5'UTR element can prevent cleavage and degradation of the artificial nucleic acid.

Preferably, the artificial nucleic acid molecule may further comprise a histone stem-loop structure. Thus, an artificial nucleic acid molecule of the present invention may contain, for example, ORFs in the 5'→3' direction, a 3'UTR element, an optional stem-loop histone sequence, an optional poly(A )-sequence or polyadenylation signal and an optional poly(C)-sequence, or located in the 5'→3'-direction of the 5'-UTR element, ORF, an optional stem-loop histone sequence, an optional poly(A) - a polyadenylation sequence or signal and an optional poly(C) sequence, or located in the 5'→3' direction 5'-UTR element, ORF, 3'-UTR element, optional stem-loop histone structure sequence , an optional poly(A) sequence or polyadenylation signal, and an optional poly(C) sequence. It may also contain, in the 5'→3' direction, an ORF, a 3'UTR element, an optional poly(A) sequence, an optional poly(C) sequence, and an optional stem-loop histone sequence, or located in the 5'→3' direction of the 5'UTR element, ORF, an optional poly(A) sequence, an optional poly(C) sequence, and an optional stem-loop histone sequence, or located in the 5' →3'-direction 5'-UTR element, ORF, 3'-UTR element, optional poly(A) sequence, optional poly(C) sequence and optional stem-loop histone structure sequence.

In a preferred embodiment of the invention, the artificial nucleic acid molecule of the invention further comprises at least one stem-loop histone sequence.

Such histone stem-loop sequences are preferably selected from the stem-loop histone sequences provided in WO 2012/019780, the contents of which are incorporated herein by reference.

The stem-loop histone sequence that can be used in the present invention is preferably selected from at least one of the following formulas (I) or (II):

formula (I) (stem-loop structure sequence without stem boundary elements):

formula (II) (stem-loop structure sequence with stem boundary elements):

in which:

where

stem1 and stem2 elements can base-pair with each other to form a reverse complementary sequence, where base-pairing can take place between stem1 and stem2, for example, through Watson-Crick base-pairing of nucleotides A and U/T or G and C, or by non-Watson-Crick base-pairing, e.g., by "swinging" base-pairing ("swinging" base-pairing), the reverse of Watson-Crick base-pairing, by Hoogsteen base-pairing, by the reverse of Hoogsteen base-pairing, or they may be pairing bases with each other to form a partially reverse complementary sequence, while incomplete base pairing can occur between stem1 and stem2, due to the fact that one or more bases in one stem do not have a complementary base in the reverse complementary sequence of the other stem.

According to another preferred embodiment of the invention, the sequence of the histone stem-loop structure can be selected from at least one of the following specific formulas (Ia) or (IIa):

formula (Ia) (stem-loop structure sequence without stem boundary elements):

formula (IIa) (stem-loop structure sequence with stem boundary elements):

wherein:

N, C, G, T and U are as defined above.

According to another preferred embodiment of the invention, the mRNA of the present invention may contain or encode at least one stem-loop histone sequence corresponding to at least one of the following specific formulas (Ib) or (IIb):

formula (Ib) (stem-loop structure sequence without stem boundary elements):

formula (IIb) (stem-loop structure sequence with stem boundary elements):

wherein:

N, C, G, T and U are as defined above.

или более предпочтительно последовательность РНК, соответствующая нуклеотидной последовательности, представленной в SEQ ID NO: 209.The most preferred sequence of the histone stem-loop structure is the one shown in

or more preferably an RNA sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 209.

For example, individual elements may be present in an artificial nucleic acid molecule in the following order:

5'-cap - 5'-UTR (element) - ORF - 3'-UTR (element) - histone stem-loop structure - poly(A)/(C)-sequence;

5'-cap - 5'-UTR (element) - ORF - 3'-UTR (element) - poly(A)/(C)-sequence - histone stem-loop structure;

5'-cap - 5'-UTR (element) - ORF - IRES - ORF - 3'-UTR (element) - histone stem-loop structure - poly(A)/(C)-sequence;

5'-cap - 5'-UTR (element) - ORF - IRES - ORF - 3'-UTR (element) - histone stem-loop structure - poly(A)/(C)-sequence - poly(A )/(C)-sequence;

5'-cap - 5'-UTR (element) - ORF - IRES - ORF - 3'-UTR (element) - poly(A)/(C)-sequence - histone stem-loop structure;

5'-cap - 5'-UTR (element) - ORF - IRES - ORF - 3'-UTR (element) - poly(A)/(C)-sequence - poly(A)/(C)-sequence - histone stem-loop structure;

5'-cap - 5'-UTR (element) - ORF - 3'-UTR (element) - poly(A)/(C)-sequence - poly(A)/(C)-sequence;

5'-cap - 5'-UTR (element) - ORF - 3'-UTR (element) - poly(A)/(C)-sequence - poly(A)/(C)-sequence - histone stem type structure -the loop"; etc.

In some embodiments, the artificial nucleic acid molecule contains additional elements such as a 5' cap, a poly(C) sequence, and/or an IRES motif. The 5' cap can be added during or after transcription to the 5' end of the RNA. In addition, the artificial nucleic acid molecule according to the invention, especially when the nucleic acid is in the form of mRNA or encodes mRNA, can be modified with a sequence of at least 10 cytidines, preferably at least 20 cytidines, more preferably at least 30 cytidines (the so-called "poly(C)-sequence"). In particular, the artificial nucleic acid molecule according to the invention may contain, especially when the nucleic acid is in the form of an mRNA or encodes an mRNA, a poly(C)-sequence which, as a rule, contains from about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytidine nucleotides, more preferably about 10 to 70 cytidine nucleotides, or even more preferably about 20 to 50 or even 20 to 30 cytidine nucleotides. Most preferably, the nucleic acid according to the invention contains a poly(C) sequence consisting of 30 cytidine residues. Thus, preferably, the artificial nucleic acid molecule of the present invention comprises, preferably located in the 5'→3' direction, at least one 5'-UTR element as described above, an ORF, at least one 3'-UTR- element as described above, poly(A) sequence or polyadenylation signal and poly(C) sequence, or located in the 5'→3' direction optionally additional 5'-UTR, ORF, at least one 3'-UTR element , as described above, a poly(A) sequence or polyadenylation signal and a poly(C) sequence, or located in the 5'→3' direction, at least one 5'-UTR element as described above, an ORF, optionally an additional 3' -UTR, poly(A) sequence or polyadenylation signal and poly(C) sequence.

An internal ribosome binding site (IRES) sequence or IRES motif may share multiple open reading frames, such as when an artificial nucleic acid molecule encodes two or more peptides or proteins. The IRES sequence may be most useful when the artificial nucleic acid molecule is a bi- or polycistronic nucleic acid molecule.

In addition, the artificial nucleic acid molecule may contain additional 5'-elements, preferably a promoter or a sequence containing a promoter. A promoter can control or regulate transcription of an artificial nucleic acid molecule of the present invention, such as an artificial DNA molecule of the present invention.

Preferably, at least the G/C content is modified in the artificial nucleic acid molecule of the present invention, preferably in the open reading frame. Thus, the artificial nucleic acid molecule according to the invention can be stabilized in terms of its thermodynamic characteristics by modifying the content of G (guanosine)/C (cytidine) in the molecule. The G/C content in the open reading frame of the artificial nucleic acid molecule of the present invention can be increased compared to the G/C content in the open reading frame of the corresponding wild-type sequence, preferably based on the degeneracy of the genetic code. Thus, the amino acid sequence encoded by the artificial nucleic acid molecule is preferably not modified by modifying the G/C content as compared to the particular wild type encoded amino acid sequence. Thus, it is possible to vary the codons of a coding sequence or of an entire artificial nucleic acid molecule, e.g. mRNA, compared to the wild-type coding sequence so that they contain more G/C nucleotides while maintaining the translated amino acid sequence. Due to the fact that several codons code for the same amino acid (so-called degeneracy of the genetic code), it is possible to change codons without changing the encoded sequence of the peptide/protein (use of so-called alternative frequent codons). Therefore, it is possible to specifically introduce certain codons (by exchanging the corresponding wild-type codons encoding the same amino acid) that are more preferable in terms of RNA stability and/or in terms of frequently occurring codons in the individual's body (so-called codon optimization).

Depending on the amino acid to be encoded by the coding region of the artificial nucleic acid molecule of the invention provided herein, there are different possibilities for modifying the nucleotide sequence, such as the open reading frame, compared to its wild-type coding region. In the case of amino acids that are encoded by codons containing only G or C nucleotides, codon modification is not required. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG), and Gly (GGC or GGG) do not require modification because they lack A or U/T.

In contrast, codons that contain A and/or U nucleotides can be modified by substitution with other codons that code for the same amino acids but do not contain A and/or U. For example,

encoding Pro codons CC(U/T) or CCA can be modified to CCC or CCG;

Arg-coding codons CG(U/T) or CGA or AGA or AGG can be modified to CGC or CGG;

Ala encoding GC(U/T) or GCA codons can be modified to GCC or GCG;

codons encoding Gly GG(U/T) or GGA can be modified to GGC or GGG.

In other cases where A or U nucleotides cannot be eliminated from codons, A and U content can be reduced by using codons that contain A and/or U nucleotides in a lower amount. Examples of these codons are:

Phe encoding codons (U/T)(U/T)(U/T) can be modified to (U/T)(U/T)C;

codons encoding Leu (U/T) (U/T)A, (U/T) (U/T)G, C(U/T) (U/T) or C(U/T)A can be modified to C (U/T)C or C(U/T)G;

Ser encoding codons (U/T)C(U/T) or (U/T)CA or AG(U/T) can be modified to (U/T)CC, (U/T)CG or AGC;

Tyr encoding codon (U/T)A(U/T) can be modified to (U/T)AC;

the Cys coding codon (U/T)G(U/T) can be modified to (U/T)GC;

the His coding codon CA(U/T) can be modified to CAC;

the Gin coding codon CAA can be modified to CAG;

codons encoding Ile A(U/T)(U/T) or A(U/T)A can be modified to A(U/T)C

coding for Thr codons AC(U/T) or ACA can be modified to ACC or ACG

the Asn encoding codon AA(U/T) can be modified to AAC;

Lys encoding AAA codons can be modified to AAG;

Val-coding codons G(U/T)(U/T) or G(U/T)A can be modified to G(U/T)C or G(U/T)G;

the Asp encoding codon GA(U/T) can be modified to GAC;

the Glu coding codon GAA can be modified to GAG;

the stop codon (U/T)AA can be modified to either (U/T)AG or (U/T)GA.

On the other hand, in the case of Met (AUG) and Trp (UGG) codons, there is no possibility of modifying the sequence without changing the encoded amino acid sequence.

The substitutions listed above can be used either alone or in any possible combination to increase the G/C content in the open reading frame of the inventive artificial nucleic acid molecule provided herein, compared to a specific wild-type open reading frame (i.e., e. the original sequence). Thus, for example, all Thr codons occurring in the wild-type sequence can be modified to ACC (or ACG).

Preferably, the open reading frame G/C content of the inventive artificial nucleic acid molecule described herein is increased by at least 7%, more preferably by at least 15%, most preferably by at least 20%, compared to the content of G/C in the coding region of the wild-type mRNA without changing the encoded amino acid sequence, i.e. using the degeneracy of the genetic code. In a particular embodiment of the invention, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least at least 90%, 95%, or even 100% of codon substitutions in the open reading frame of the inventive artificial nucleic acid molecule or fragment, variant or derivative thereof, thereby increasing the G/C content in said open reading frame.

In this context, it is most preferable to increase the content of G/C in the open reading frame of the inventive artificial nucleic acid molecule presented herein to a maximum (i.e. up to 100% usable codon substitution) compared to the open reading frame of the wild type without changing the encoded amino acid sequence.

In addition, the open reading frame preferably performs at least partially codon optimization. Codon optimization is based on the discovery that the efficiency of translation can also be determined by the different frequencies of transfer RNAs (tRNAs) in cells. Thus, if so-called "rare codons" are present in the coding region of the artificial nucleic acid molecule of the present invention, the level of translation of the corresponding modified nucleotide sequence is lower than when codons are present. encoding a relatively "frequent" tRNA.

Thus, the open reading frame of the artificial nucleic acid molecule according to the invention is preferably modified in comparison with the corresponding wild-type coding region so that at least one codon in the wild-type sequence that codes for a tRNA that is relatively rare in the cell is changed to a codon that codes for a tRNA that is relatively common in the cell and "carries" the same amino acid as a relatively rare tRNA. By such modification, the open reading frame of the artificial nucleic acid molecule of the invention described herein is modified so that codons for which frequently occurring tRNAs are available are replaced with codons that correspond to rare tRNAs. In other words, according to the invention, with this modification, all codons in the wild-type open reading frame that encode a tRNA that is rare in the cell can be replaced with a codon that encodes a tRNA that is more common in the cell, and which in each case "carries" the same amino acid as the rare tRNA. tRNAs that are relatively common in a cell, and vice versa, that are relatively rare, are known to those skilled in the art, see, for example, Akashi, Curr. Opin. Genet. Dev., 11(6), 2001, pp. 660-666. Thus, preferably the open reading frame has optimized codons, preferably in relation to the system in which the artificial nucleic acid molecule of the present invention is to be expressed, preferably in relation to the system in which the artificial nucleic acid molecule of the present invention is to be translated. Preferably, codon optimization for frequently occurring codons in the open reading frame is performed according to frequently occurring mammalian codons, more preferably according to frequently occurring human codons. Preferably, both codon optimization and G/C content modification are performed in the open reading frame.

To further increase resistance to cleavage, for example resistance to in vivo (or in vitro as above) cleavage by an exo- or endonuclease, and/or to further increase the stability of protein expression from the artificial nucleic acid molecule of the present invention, one can perform additional modifications to the artificial nucleic acid molecule, such as backbone modifications, sugar modifications, and/or base modifications, eg, lipid modifications, or the like. Preferably, such modifications do not significantly impair transcription and/or translation of the artificial nucleic acid molecule of the present invention.

Typically, the artificial nucleic acid molecule of the present invention may contain any native (naturally occurring) nucleotide, such as guanosine, uracil, adenosine and/or cytosine, or their analogues. In this context, nucleotide analogs are classified as naturally occurring and non-naturally occurring variants of the naturally occurring nucleotides of adenosine, cytosine, thymidine, guanosine, and uridine. Thus, analogs are, for example, chemically derivatized nucleotides bearing non-naturally occurring functional groups, which are preferably added or removed by deletion from a naturally occurring nucleotide, or by which a naturally occurring functional group of a nucleotide is substituted. Therefore, it is possible to modify each component of a naturally occurring nucleotide, namely the base component, the sugar component (ribose), and/or the phosphate component forming the backbone (see above) of the RNA sequence. Guanosine, uridine, adenosine, thymidine, and cytosine analogs include, but are not limited to, any naturally occurring or non-naturally occurring guanosine, uridine, adenosine, thymidine, or cytosine that has been altered chemically, e.g., by acetylation, methylation , hydroxylation, etc., including, for example, 1-methyladenosine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino -2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurine riboside, 2-aminopurine riboside, 2'-araadenosine, 2'-aracitidine, 2' -arauridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloradenosine, 2'- fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-fluorothymidine, 2-methyladenosine, 2-methylguanosine, 2-methylthio-N6-isopenenyladenosine, 2'-O-methyl-2-aminoaden osine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine, 2'-O-methyl-2'-deoxyguanosine, 2'-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O-methylinosine, 2'-O-methylpseudouridine, 2-thiocytidine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 4-thiouridine, 5-(carboxyhydroxymethyl) uracil, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyldeoxyuridine, 5-bromuridine, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 5-chloro-aracytosine, 5-fluorouridine, 5-ioduridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 5-methyl-2-thiouridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deazaguanosine, 6-chloropurinoribozide, 6-mercaptoguanosine, 6-methylmercaptopurine riboside, 7-deaza-2'-deoxyguanosine, 7 -deazaadenosine, 7-methylguanosine, 8-azaadenosine, 8-broadenosine, 8-bromoguanosine, 8-mercaptorguanosine, 8-oxoguanosine, benzimidazole riboside, beta-D-mannosylqueosine, dihydrouracil, inosine, N1-methyladenosine, N6-([6-aminohexyl ]carbamoylmethyl)adenosine, N6-isopentenyladenosine, N6-methylade nosine, N7-methylxanthosine, N-uracil-5-hydroxyacetic acid methyl ester, puromycin, queosine, uracil-5-hydroxyacetic acid, uracil-5-hydroxyacetic acid methyl ester, vibutoxosine, xanthosine, and xyladenosine. The preparation of such analogues is known to the person skilled in the art, for example, described in US 4373071, US 4401796, US 4415732, US 4458066, US 4500707, US 4668777, US 4973679, US 5047524, US 5132418, US 5153319, US 5262530 and 27 described above, most preferred analogs in some embodiments of the invention may be those analogs that increase the protein expression of the encoded peptide or protein, or increase the immunogenicity of the artificial nucleic acid molecule of the invention, and/or do not interact with another introduced modification of the artificial nucleic acid molecule .

According to a specific embodiment of the invention, the artificial nucleic acid molecule of the present invention may contain a lipid modification.

In a preferred embodiment of the invention, the artificial nucleic acid molecule contains, preferably in the 5'→3' direction, the following elements:

5'-UTR element, providing high translation efficiency of the specified artificial nucleic acid molecule, preferably the nucleotide sequence presented in one of SEQ ID NO: 1-151; or an additional 5'-UTR, preferably a 5'-TOP UTR;

at least one open reading frame (ORF), where the ORF preferably contains at least one modification compared to the wild-type sequence;

A 3'-UTR element providing high translation efficiency of said artificial nucleic acid molecule, preferably the nucleotide sequence shown in one of SEQ ID NOs: 152-204; or an additional 3'-UTR, preferably the 3'-UTR of albumin7;

poly(A)-sequence, preferably containing 64 adenylate;

poly(C)-sequence, preferably containing 30 cytidylates;

histone stem-loop sequence.

In the most preferred embodiment of the invention, the artificial nucleic acid molecule of the invention may also contain one or more of the following modifications:

Chemical modifications:

In the context of the present description, the term "modification" in relation to an artificial nucleic acid molecule can refer to chemical modifications, including modifications of the backbone, as well as modifications of the sugar or modification of the base.

In this context, an artificial nucleic acid molecule, preferably an RNA molecule, as described herein, may contain nucleotide analogs/modifications, for example backbone modifications, sugar modifications, or base modifications. The backbone modification according to the present invention is a modification in which backbone phosphates of the nucleotides contained in the nucleic acid molecule described herein are chemically modified. The sugar modification of the present invention is the chemical modification of the sugar of the nucleotides in the nucleic acid molecule described herein. In addition, the modification of the base according to the present invention is a chemical modification of the main fragment of the nucleotides in the nucleic acid molecule. In this context, nucleotide analogs or modifications are preferably selected from nucleotide analogs suitable for transcription and/or translation.

Sugar modifications:

Modified nucleosides and nucleotides that can be included in an artificial nucleic acid molecule, preferably RNA, as described herein, can be modified in the sugar moiety. For example, the 2'-hydroxy group (OH) in an RNA molecule can be modified or replaced by numerous "oxy" or "deoxy" substituents. Examples of modifications that include "oxy"-2'-hydroxy groups are (but not limited to) alkoxy or aryloxy groups (-OR, for example, R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycols (PEG), -O(CH ₂ CH ₂ O) _n CH ₂ CH ₂ OR; "closed" nucleic acids (LNA), in which the 2'-hydroxyl is attached, for example, via a methylene bridge to the 4'-carbon in the same ribose sugar moiety; and amino groups (-O-amino groups, in which the amino group, for example, NRR, may be an alkylamino group, a dialkylamino group, a heterocyclyl, an arylamino group, a diarylamino group, a heteroarylamino group or a diheteroarylamino group, an ethylenediamine, a polyamino group) or an aminoalkoxy group.

The "deoxy" modification includes hydrogen, an amino group (eg, NH ₂ ; an alkylamino group, a dialkylamino group, a heterocyclyl, an arylamino group, a diarylamino group, a heteroarylamino group, a diheteroarylamino group, or an amino acid); or an amino group can be attached to the sugar through a linker, where the linker contains one or more C, N and O atoms.

The sugar group may also contain one or more carbon atoms, which may be in a stereochemical configuration different from that of the corresponding carbon atom in the ribose. Thus, the modified nucleic acid molecule may include, for example, arabinose as a sugar.

Frame mods:

It is also possible to modify the phosphate backbone in modified nucleosides and nucleotides, which can be inserted into an artificial nucleic acid molecule, preferably RNA, as described herein. Phosphate groups in the framework can be modified by replacing one or more oxygen atoms with various substituents. In addition, in modified nucleosides and nucleotides, the unmodified phosphate moiety can be completely replaced by a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, primary phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In phosphorodithioates, both non-bonding oxygen atoms are replaced by sulfur. The phosphate linker can also be modified by replacing the bonding oxygen atom with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates), and carbon (bridged methylenephosphonates).

Base modifications:

Modified nucleosides and nucleotides that can be inserted into an artificial nucleic acid molecule, preferably an RNA molecule as described herein, can also be modified in a nucleobase moiety. Examples of nucleobases included in RNA are (but not limited to) adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides provided herein can be chemically modified on the surface of the major groove. In some embodiments, the chemical modifications in the major groove include an amino group, a thiol group, an alkyl group, or a halo group.

In the most preferred embodiments of the present invention, the nucleotide analogs/modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate , 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine- 5'-triphosphate, 5-iodcytidine-5'-triphosphate, 5-ioduridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5 '-triphosphate, benzimidazole riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5 '-triphosphate or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. The most preferred nucleotides for base modifications are selected from the group of base-modified nucleotides including 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.

In some embodiments, the modified nucleosides include pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine , 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyluridine, 1-methylpseudouridine, 4-thio-1 -methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy -4-thiouridine, 4-methoxypseudouridine and 4-methoxy-2-thiopseudouridine.

In some embodiments, the modified nucleosides include 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thiocytidine, 2-thio- 5-methylcytidine, 4-thiopseudisocytidine, 4-thio-1-methylpseudoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularine, 5-azazebularine, 5-methylzebularine, 5-aza- 2-thiozebularine, 2-thiosebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine and 4-methoxy-1-methylpseudoisocytidine.

In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deazaadenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6 -(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine and 2-methoxyadenine.

In other embodiments, the modified nucleosides include inosine, 1-methylinosine, viosin, vibutosin, 7-deazaguanosine, 7-deaza-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza- 8-azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine and N2,N2-dimethyl-6-thioguanosine.

In some embodiments, the nucleotide may be modified at the surface of the major groove, and the modification may include replacing the hydrogen atom on the C-5 uracil with a methyl group or a halo group. In specific embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5' -O-(1-thiophosphate)-uridine or 5'-O-(1-thiophosphate)-pseudouridine.

In other specific embodiments, the artificial nucleic acid molecule, preferably an RNA molecule, may contain nucleoside modifications selected from 6-azacytidine, 2-thiocytidine, alpha-thiocytidine, pseudoisocytidine, 5-aminoallyluridine, 5-yoduridine, N1-methylpseudouridine, 5, 6-dihydrouridine, alpha-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, alpha-thioguanosine, 6-methylguanosine, 5-methylcytidine, 8-oxoguanosine, 7-deazaguanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudoisocytidine, 6-chloropurine, N6-methyladenosine, alpha-thioadenosine, 8-azidoadenosine, 7-deazaadenosine.

Lipid modification:

According to a further embodiment of the invention, the artificial nucleic acid molecule, preferably RNA, provided herein may contain a lipid modification. Such lipid-modified RNA typically contains the RNA described herein. Such a lipid-modified RNA molecule as described herein typically further comprises at least one linker covalently linked to said RNA molecule and at least one lipid covalently linked to the corresponding linker. Alternatively, a lipid-modified RNA molecule comprises (at least one) an RNA molecule as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) to said RNA molecule. In a third alternative, the lipid-modified RNA molecule comprises an artificial nucleic acid molecule, preferably an RNA molecule, as defined herein, at least one linker covalently linked to said RNA molecule, and at least one lipid covalently linked to a corresponding linker, and also at least one (bifunctional) lipid covalently linked (without a linker) to said mRNA. In this context, most preferably the lipid modification is carried out at the ends of the linear RNA sequence.

Modification of the 5'-end of the modified RNA:

According to another preferred embodiment of the invention, the artificial nucleic acid molecule, preferably the RNA molecule as described herein, can be modified by adding a so-called "5' cap" structure.

A 5' cap is a structure, typically a modified nucleotide structure, that typically caps the 5' end of a mature mRNA. The 5' cap can generally be formed by a modified nucleotide, primarily a derivative of a guanine nucleotide.

Preferably, the 5' cap is linked to the 5' end via a 5'-5' triphosphate bond. The 5' cap may be methylated, for example with m7GpppN, where N is the 5' terminal nucleotide of the nucleic acid bearing the 5' cap, typically the 5' end of the RNA. m7GpppN is a 5'cap structure that occurs naturally in mRNA transcribed by polymerase II and is therefore not considered a modification contained in the modified RNA of the invention. This means that an artificial nucleic acid molecule, preferably an RNA molecule, according to the present invention may contain m7GpppN as a 5'-cap, but additionally, an artificial nucleic acid molecule, preferably an RNA molecule, contains at least one additional modification represented by in this description.

Other examples of 5'-cap structures are glyceryl, inverted deoxy group (fragment) devoid of a nitrogenous base, 4',5'-methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, base-modified nucleotide, treopentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'- 3'-inverted nucleotide fragment, 3'-3'-inverted baseless fragment, 3'-2'-inverted nucleotide fragment, 3'-2'-inverted baseless fragment, 1,4-butanediolphosphate, 3'- a phosphoramidate, hexylphosphate, aminohexylphosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate or bridged or unbridged methylphosphonate moiety. These modified 5' cap structures are considered to be at least one modification contained in an artificial nucleic acid molecule, preferably an RNA molecule, according to the present invention.

The most preferred modified 5'-cap structures are CAP1 (ribose methylation of nucleotide adjacent to m7GpppN), CAP2 (methylation of ribose 2nd nucleotide downstream of m7GpppN), CAP3 (methylation of ribose 3rd nucleotide downstream of m7GpppN). relative m7GpppN), CAP4 (ribose methylation of the 4th nucleotide located downstream of m7GpppN), ARCA (correctly oriented cap analog modified by ARCA (for example, modified by ARCA phosphothioate), inosine, N1-methylguanosine, 2'- fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine and 2-azidoguanosine.

In a preferred embodiment of the invention, at least one open reading frame encodes a therapeutic protein or peptide. In another embodiment, the at least one open reading frame encodes an antigen, such as a pathogenic antigen, a tumor antigen, an allergenic antigen, or an autoimmune antigen. Thus, the introduction of an artificial nucleic acid molecule encoding an antigen is used in the approach of genetic vaccination against a disease in which said antigen is involved.

In an alternative embodiment of the invention, at least one open reading frame of the artificial nucleic acid molecule of the invention encodes an antibody or an antigen-specific T-cell receptor, or a fragment thereof.

Antigens:

Pathogenic antigens:

The artificial nucleic acid molecule of the present invention may encode a protein or peptide that contains a pathogenic antigen or fragment, variant or derivative thereof. Such pathogenic antigens are derived from pathogenic organisms, primarily bacteria, viruses or protozoan (multicellular) pathogenic organisms, which elicit an immunological response in an individual, primarily a mammal, more preferably a human. More specifically, pathogenic antigens are preferably surface antigens, eg proteins (or fragments of proteins, eg the exposed portion of a surface antigen) located on the surface of a virus or bacterium or protozoan.

Pathogenic antigens are peptide or protein antigens, preferably derived from pathogens associated with an infectious disease, which are preferably selected from antigens derived from pathogens Acinetobacter baumannii, genus Anaplasma, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, genus Aspergillus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and others Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostr idium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Coryneb acterium diphtheriae, Coxiella burnetii, Crimean Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN- 1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebola virus (EBOV), genus Echinococcus, Ehrlichia chaffeensis, Ehrlichia ewingii, genus Ehrlichia, Entamoeba histolytica, genus Enterococcus, genus Enterovirus, enteroviruses, especially virus Coxsackie A and enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr virus (EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, flaviviruses, Francisella tularensis, genus Fusobacterium, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, genipavirus (vir c Gendra and Nipah virus), hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, hepatitis virus, herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum , HIV (human immunodeficiency virus), Hortaea werneckii, human bocavirus (HBoV), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), human metapenummonia virus (hMPV), human papillomavirus (HPV) , human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), virus Machupo, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, families Orthomyxoviridae (influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, parvovirus Pastmeurella, genus B19, genus Plasmeurella, genus B19 , Pneumocystis jirovecii, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley virus, rotavirus, rubella virus, Sabia virus, Salmonella genus , Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, To xoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, varicella zoster virus (VZV), Variola major or Variola minor, prion vCJD, Venezuelan equine encephalitis virus, Vibrio cholerae , West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, yellow fever virus, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis.

In this context, antigens from pathogens selected from influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, virus Dengue, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus (HBV), Mycobacterium tuberculosis, rabies virus and yellow fever virus.

Tumor antigens:

In another embodiment of the invention, the artificial nucleic acid molecule of the present invention may encode a protein or peptide that contains a peptide or protein containing a tumor antigen, fragment, variant or derivative of said tumor antigen, where preferably the tumor antigen is melanocyte-specific antigen, cancer-testicular antigen or tumor-specific antigen, preferably CT-X antigen, non-X-CT antigen, CT-X antigen binding partner or non-X-CT antigen binding partner, or tumor- a specific antigen, more preferably a CT-X antigen, a non-X-CT antigen binding partner, or a tumor-specific antigen, or a fragment, variant or derivative of said tumor antigen; and where each of the nucleotide sequences encodes a different peptide or protein; and where at least one of the nucleotide sequences encodes 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m , alpha-methylacyl-coenzyme A-racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collagen XXIII, COX-2, CT-9/ BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6- AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT- V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HP V-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallecrein-2, kallecrein-4, Ki67, KIAA0205, KIAA0205/ m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE- C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammoglobin A, MART-1/melan-A, MART-2, MART-2 /m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, class I/m myosin, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/ m, protein, proteins Nazu-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART- 3, SCC, SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, Survivin, Survivin-2B, SYT-SSX-1, SYT-SSX- 2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP- p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, WT1 and an immunoglobulin idiotype of a lymphoid blood cell or an idiotype of a T-cell receptor, or a fragment, variant or derivative of said tumor antigen; preferably survivin or a homologue thereof, a MAGE family antigen or a binding partner or fragment, variant or derivative thereof of said tumor antigen. Most preferred in this context are the tumor antigens NY-ESO-1, 5T4, MAGE-C1, MAGE-C2, survivin, Muc-1, PSA, PSMA, PSCA, STEAP and PAP.

In a preferred embodiment of the invention, the artificial nucleic acid molecule encodes a protein or peptide that contains a therapeutic protein or a fragment, variant or derivative thereof.

In the context of the present description, therapeutic proteins are peptides or proteins that have a beneficial effect in the treatment of any congenital or acquired disease or that improve the condition of the individual. In particular, therapeutic proteins are important for the development of therapeutic agents that can, among other functions, modify and repair genetic errors, destroy cancer cells or pathogen-infected cells, treat immune system disorders, and treat metabolic or endocrine disorders. For example, erythropoietin (EPO), a protein hormone, can be used to treat patients with red blood cell deficiency, which is a common cause of renal complications. In addition, the term "therapeutic proteins" includes adjuvant proteins, therapeutic antibodies, as well as hormone replacement therapy, which is used, for example, in the treatment of women during menopause. More recent approaches use the patient's somatic cells to reprogram them into pluripotent stem cells, replacing controversial stem cell therapies. Such proteins used for reprogramming somatic cells or used for stem cell differentiation are also considered therapeutic proteins in the context of the present disclosure. In addition, therapeutic proteins can be used for other purposes, such as wound healing, tissue regeneration, angiogenesis, and so on. In addition, in the context of the present description, antigen-presenting B-cell receptors and their fragments and variants are considered as therapeutic proteins.

Thus, therapeutic proteins can be used for various purposes, including the treatment of various diseases, such as, for example, infectious diseases, neoplasms (for example, cancer or tumor diseases), diseases of the blood or blood-forming organs, endocrine, nutritional or metabolic diseases, diseases nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, whether they are congenital or acquired.

In this context, the most preferred therapeutic proteins that can be used, among others, for the treatment of metabolic or endocrine disorders are (in brackets the specific disease for which the therapeutic protein is used is indicated): acid sphingomyelinase (Niemann-Pick disease), adipotide (obesity ), agalsidase-beta (human galactosidase A) (Fabry disease; interferes with lipid accumulation, which can lead to kidney and cardiovascular complications), alglucosidase (Pompe disease (glycogenosis (accumulation of glycogen) type II)), alpha-galactosidase A ( alpha-GAL A, agalsidase-alpha) (Fabry disease), alpha-glucosidase (glycogenosis (GSD), Pompe disease), alpha-L-iduronidase (mucopolysaccharidosis (MPS), Hurler syndrome, Scheye syndrome), alpha-N-acetylglucosaminidase (Sanfilippo syndrome), amphiregulin (cancer, metabolic disorder), angiopoietin ((Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7) (angiogenesis, stabilizes cos uds), betacellulin (metabolic disorder), beta-glucuronidase (Sly syndrome), bone morphogenetic protein BMP (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15) (regenerative action, conditions, bone-related, chronic kidney disease (CKD)), CLN6 protein (disease caused by mutations at the CLN6 locus - atypical late infantilism, late-onset variant, early juvenile, neuronal ceroid lipofuscinosis (NCL)), epidermal growth factor (EGF ) (wound healing, regulation of cell growth, proliferation and differentiation), epigen (metabolic disorder), epiregulin (metabolic disorder), fibroblast growth factor (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF- 5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23) (wound healing, angiogenesis, endocrine disruption, tissue regeneration), galsulfase (mucopolysaccharidosis type VI), ghrelin (irritable bowel syndrome (IBS), obesity, Prader-Willi syndrome, type II diabetes mellitus), glucocerebrosidase (Gaucher disease), GM-CSF (regenerative action, leukocyte production, cancer), heparin-binding EGF-like growth factor (HB-EGF) (healing wounds, cardiac hypertrophy and cardiac development and function), hepatocyte growth factor HGF (regenerative action, wound healing), hepcidin (iron metabolism disorders, beta-thalassemia), human albumin (decreased albumin production (hypoproteinemia), increased albumin loss (nephrotic syndrome ), hypovolemia, hyperbilirubinemia), idursulfase (iduronate-2-sulfatase) (mucopolysaccharidosis type II (Hunter syndrome)), αVβ3, αVβ5 and α5β1 integrins (bind matrix macromolecules and proteinases, angiogenesis), iduronate sulfatase (Hunter syndrome), laronidase (mucopolysaccharidosis type I, Hurler and Hurler-Shaie forms), N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase, arylsulfatase A (ARSA), arylsulfatase B (ARSB)) (arylsulfatase B deficiency, Maroteau-Lami syndrome, mucopolysaccharidosis type VI), N-acetylglucosamine-6-sulfatase (Sanfilippo syndrome), nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4/5) (regenerative action, cardiovascular disease, coronary atherosclerosis, obesity, type 2 diabetes, metabolic syndrome, acute coronary syndromes, dementia , depression, schizophrenia, autism, Rett syndrome, anorexia nervosa, bulimia nervosa, wound healing, skin ulcers, corneal ulceration, Alzheimer's disease), neuregulin (NRG1, NRG2, NRG3, NRG4) (metabolic disorder, schizophrenia), neuropilin (NRP- 1, NRP-2) (angiogenesis, axonal guidance, survival, cell migration), obestatin (irritable bowel syndrome (IBS), obesity, Prader-Willi syndrome, type II diabetes mellitus), platelet growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D) (regenerative effect, for wound healing, angiogenesis disorder, arteriosclerosis, fibrosis, cancer), TGF-beta receptors (endoglin, TGF-beta 1 receptor, TGF-beta 2 receptor, TGF-beta 3 receptor) (renal fibrosis, kidney disease, diabetes, end-stage renal diseases (ESRD), angiogenesis), thrombopoietin (THRO) (megakaryocyte growth and development factor (MGDF)) (platelet disorders, platelets for donation, recovery of platelet levels after myelosuppressive chemotherapy), transforming growth factor (TGF (TGF-alpha, TGF -beta (TGFbeta1, TGFbeta2 and TGFbeta3))) (regenerative action, wound healing, immunity, cancer, heart disease, diabetes, Marfan syndrome, Loes-Dietz syndrome), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF) (regenerative action, angiogenesis, wound healing, cancer, permeability), nesritide (acute decompensated congestive heart failure), trypsin (decubital ulcer, varicose ulcer, eschar debridement, spontaneous opening wound, sunburn, mek onium ileus), adrenocorticotropic hormone (ACTH) (Addison's disease, small cell sarcoma, adrenoleukodystrophy, congenital adrenal hyperplasia, Cushing's syndrome, Nelson's syndrome, infantile spasms), atrial natriuretic peptide (ANP) (endocrine disorders), cholecystokinin (various lengths), gastrin (hypogastrinemia), leptin (diabetes, hyperglycinemia, obesity), oxytocin (stimulates breastfeeding, non-delivery), somatostatin (symptomatic treatment of carcinoid syndrome, acute variceal bleeding and acromegaly, polycystic liver and kidney disease, acromegaly and symptoms caused by neuroendocrine tumors ), vasopressin (antidiuretic hormone) (diabetes insipidus), calcitonin (postmenopausal osteoporosis, hypercalcemia, Paget's disease, bone metastases, phantom limb pain, spinal stenosis), exenatide (type 2 diabetes resistant to metformin and sulfonylurea treatment), growth hormone (GH), som atotropin (growth failure due to GH deficiency or chronic liver failure, Prader-Willi syndrome, Turner syndrome, AIDS-related wasting or cachexia during antiviral therapy), insulin (diabetes mellitus, diabetic ketoacidosis, hyperkalemia), insulin-like growth factor 1 IGF-1 ( growth retardation in children with GH gene deletion or severe primary IGF1 deficiency, neurodegenerative disease, cardiovascular disease, heart failure), mecasermine rinfabate, IGF-1 analogue (growth failure in children with GH gene deletion or severe primary IGF1 deficiency, neurodegenerative disease , cardiovascular disease, heart failure), megasermin, IGF-1 analogue (growth failure in children with GH gene deletion or severe primary IGF1 deficiency, neurodegenerative disease, cardiovascular disease, heart failure), pegvisomant (acromegaly), pramlintide ( diabetes mellitus, in combination with insulin m), teriparatide (human parathyroid hormone, residues 1-34) (severe osteoporosis), becaplermin (adjuvant in the debridement of diabetic ulcers), dibotermin-alpha (bone morphogenic protein 2) (surgery to fix the spine (Spinal Fusion), recovery bones when damaged), histrelin acetate (gonadotropin-releasing hormone; GnRH) (precocious puberty), octreotide (acromegaly, symptomatic attenuation of VIP-secreting adenoma and metastatic carcinoid tumors) andalifermin (keratinocyte growth factor; KGF) (severe oral mucositis in patients undergoing chemotherapy, wound healing).

These and other proteins are considered therapeutic since they are intended to treat the individual by replacing the disturbed endogenous production in his body of a functional protein in sufficient quantities. Therefore, such therapeutic proteins are typically mammalian proteins, especially human proteins.

Therapeutic proteins such as alterplase (tissue plasminogen activator; tPA) (pulmonary embolism, myocardial infarction, acute ischemic stroke ("stroke"), occlusion of central venous access devices), anistreplase (thrombolysis), antithrombin III (AT-III) (hereditary deficiency of AT-III, thromboembolism), bivalirudin (reduces the risk of blood clotting in coronary angioplasty and heparin-induced thrombocytopenia), darbepoetin- alpha (treatment of anemia in patients with chronic renal failure and chronic renal impairment (+/- dialysis)), drotrecogin-alpha (activated protein C) (serious sepsis with high risk of death), erythropoietin, epoetin-alpha, erythropoietin, erythropoietin (anemia in chronic disease, myelodysplasia, anemia in renal failure or in chemotherapy, preoperative preparation), factor IX (hemophilia B), factor VIIa (hemorrhage in patients with hemophilia A or B and the use of factor VIII or factor IX inhibitors), factor VIII (hemophilia A), lepirudin (heparin-induced thrombocytopenia), protein C concentrate (venous thrombosis, fulminant purpura), reteplase (mutein deletion in tPA) (treatment of acute myocardial infarction, improvement of ventricular function), streptokinase (acute transmural myocardial infarction, pulmonary embolism, deep vein thrombosis, arterial thrombosis or embolism, occlusion arteriovenous cannula), tenesteplase (acute myocardial infarction), urokinase (pulmonary embolism), angiostatin (cancer), anti-CD22 immunotoxin (recurrent acute myeloid CD33 ⁺ leukemia), denileukin diftitox (cutaneous T-cell lymphoma (CTCL)), immunocyanin (bladder and prostate cancer), MPS (metalopanstimulin) (cancer), aflibercept (non-small cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), hormone-resistant metastatic prostate cancer, wet macular degeneration), endostatin (cancer, inflammatory diseases such as rheumatoid arthritis, as well as Crohn's disease, diabetic retinopathy, psoriasis and endometriosis), collagenase (treatment of chronic skin ulcers and areas of severe burns, Dupuytren's contracture, Peyronie's disease), human deoxyribonuclease I, dornase (cystic fibrosis; reduction of respiratory tract infections in selected patients with an FVC greater than 40% predicted), hyaluronidase (used as an adjuvant to increase the absorption and dispersion of injected drugs, primarily anesthetics for eye surgery and certain imaging agents), papain ( debridement of necrotic tissue or liquefaction of sloughing necrotic masses in acute and chronic injuries such as bedsores, varicose and diabetic ulcers, burns, postoperative wounds, pilonidal cystic wounds, carbuncles and other wounds), L-asparaginase (acute lymphocytic leukemia requiring exogenous asparagine for proliferation), peg-asparaginase (acute lymphocytic leukemia requiring exogenous asparagine for proliferation), rasburicase (childhood patients with leukemia, lymphoma, and solid tumors undergoing anticancer therapy that can cause tumor lysis syndrome), human chorio gonadotropin (HCG) (improves reproductive function), human follicle stimulating hormone (FSH) (improves reproductive function), lutropin-alpha (infertility accompanied by luteinizing hormone deficiency), prolactin (hypoprolactinemia, serum prolactin deficiency, ovarian dysfunction in women, state of fear , arteriogenic erectile dysfunction, premature ejaculation, oligozoospermia, asthenospermia, reduced function of seminal vesicles, hypoandrogenism in men), alpha-1-proteinase inhibitor (congenital antitrypsin deficiency), lactase (gas formation, bloating, cramps and diarrhea due to inability to break down lactose), pancreatic enzymes (lipase, amylase, proteinase) (cystic fibrosis, chronic pancreatitis, pancreatic insufficiency, post-Billroth II gastric bypass surgery, pancreatic duct obstruction, steatorrhoea, maldigestion, gas, bloating), adenosine deaminases a (bovine pegademase, PEG-ADA) (diseases with severe combined immunodeficiency due to adenosine deaminase deficiency), abathecept (rheumatoid arthritis (primarily refractory to TNF-alpha inhibition)), alefacept (plaque psoriasis), anakinra (rheumatoid arthritis), ethanecept (rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, ankylosing spondylitis), interleukin-1 (IL-1) receptor antagonist, anakinra (inflammation and cartilage degradation associated with rheumatoid arthritis), thymulin (neurodegenerative diseases, rheumatism, anorexia nervosa), a TNF-alpha antagonist (autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitis suppurativa, refractory asthma), enfuvirtide (HIV-1 infection), and thymosin α1 (hepatitis B and C)

(in parentheses is the specific disease for which the therapeutic protein is used).

The next object of the present invention is a vector containing

a. an open reading frame (ORF) and/or a cloning site, for example, for inserting an open reading frame or a sequence containing an open reading frame; and

b. at least one 3'-untranslated region element (3'-UTR element) and/or at least one 5'-untranslated region element (5'-UTR element), wherein said artificial nucleic acid is characterized by high translation efficiency .

Typically, the vector of the present invention may contain an artificial nucleic acid molecule of the present invention as described above. In particular, the preferred embodiments of the invention described above in the description of the artificial nucleic acid molecule of the present invention are also applicable to the artificial nucleic acid molecule of the present invention contained in the vector of the present invention. For example, in the vector of the invention, at least one 3'-UTR element and/or at least one 5'-UTR element and the ORF are the structures indicated above when describing the artificial nucleic acid molecule of the present invention, including preferred embodiments of the invention. For example, in a vector of the present invention, a stable mRNA from which at least one 3'UTR element and/or at least one 5'UTR element is derived can preferably be characterized in that it cleaves the mRNA wherein the ratio of the amount of said mRNA at the second time point to the amount of said mRNA at the first time point is at least 0.5 (50%), at least 0.6 (60%), at least 0.7 (70% ), at least 0.75 (75%), at least 0.8 (80%), at least 0.85 (85%), at least 0.9 (90%), or at least 0 .95 (95%).

The cloning site can be any sequence suitable for inserting an open reading frame or a sequence containing an open reading frame, such as one or more restriction sites. Thus, the vector contains a cloning site, preferably suitable for inserting an open reading frame into the vector, preferably for inserting an open reading frame from the 3' side of the 5'-UTR element and/or from the 5' side of the 3'-UTR- element. Preferably, the cloning site or ORF is/is located 3'-side of the 5'-UTR element and/or 5'-side of the 3'-UTR element, preferably in close proximity to the 3'-end of the 5'-UTR- element and/or the 5' end of the 3' UTR element. For example, the cloning site or ORF may be directly attached to the 3'end of the 5'UTR element and/or to the 5'end of the 3'UTR element, or they may be attached via a nucleotide segment, e.g. consisting of 2, 4, 6, 8, 10, 20, etc. nucleotides as described above in the description of the artificial nucleic acid molecule of the present invention.

Preferably, the vector of the present invention can be used to produce an artificial nucleic acid molecule of the present invention, preferably the production of an artificial mRNA of the present invention, for example, optionally by inserting an open reading frame or a sequence containing an open reading frame into the vector and transcribing the vector. Thus, preferably the vector contains elements necessary for transcription, such as a promoter, for example, an RNA polymerase promoter. Preferably, the vector is suitable for transcription using eukaryotic, prokaryotic, viral or phage transcription systems such as eukaryotic cells, prokaryotic cells or in vitro eukaryotic, prokaryotic, viral or phage transcription systems. Thus, for example, the vector may contain a promoter sequence that is recognized by a polymerase, such as an RNA polymerase, for example, a eukaryotic, prokaryotic, viral, or phage RNA polymerase. In a preferred embodiment, the vector contains a phage RNA polymerase promoter, such as the SP6, T3 or T7 phage promoter, preferably the T7 phage promoter. Preferably, the vector is suitable for in vitro transcription using a phage-based in vitro transcription system, such as an in vitro transcription system based on phage T7 RNA polymerase.

In another preferred embodiment of the invention, the vector can be used directly to express the encoded peptide or protein in cells or tissue. For this purpose, the vector contains specific elements necessary for expression in said cells/tissue, for example specific promoter sequences such as the CMV promoter.

The vector may also contain the poly(A) sequence and/or polyadenylation signal described above for the artificial nucleic acid molecule of the present invention.

The vector may be an RNA vector or a DNA vector. Preferably the vector is a DNA vector. The vector may be any vector known to the person skilled in the art, such as a viral vector or a plasmid vector. Preferably the vector is a plasmid vector, preferably a plasmid DNA vector.

In a preferred embodiment of the invention, the vector of the present invention contains an artificial nucleic acid molecule of the present invention.

Preferably, the DNA vector of the invention contains a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 10, 15, 20, 30 or 40% identical, preferably at least about 50% identical, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the nucleotide sequence of the 3'-UTR of the gene transcript, for example, the nucleotide sequences shown in SEQ ID NOs: 152-204.

Preferably, the DNA vector of the invention contains a nucleotide sequence that is at least about 1, 2, 3, 4, 5, 10, 15, 20, 30 or 40% identical, preferably at least about 50% identical, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99%, most preferably 100% of the nucleotide sequence of the 5'-UTR of the gene transcript, for example, the nucleotide sequences shown in SEQ ID NO: 1-151.

Preferably, the DNA vector of the present invention contains a sequence selected from the group consisting of DNA sequences corresponding to the 5'-UTR elements shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (alternatively SEQ ID NO: 3), SEQ ID NO: 138 (alternatively SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (alternatively SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (alternatively SEQ ID NO: 31), SEQ ID NO: 32 , SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (alternatively SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO : 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69 , SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 144 (alternatively SEQ ID NO: 42), SEQ ID NO : 58, SEQ ID NO: 62; SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (alternatively SEQ ID NO: 109), SEQ ID NO: 148 (alternatively SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 149 (alternatively SEQ ID NO: 119 ), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (alternatively SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 150 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125, and 3'UTR elements shown in SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173; SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 204 (alternatively SEQ ID NO: 192), SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 177, or sequence identity at least about 40%, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%; even more preferably at least about 99% of one of these sequences, or a fragment thereof, as described above, preferably a functional fragment thereof.

Preferably, the RNA vector of the present invention contains a sequence selected from the group consisting of sequences representing RNA sequences corresponding to the DNA sequences indicated above when describing the DNA vector of the present invention.

Preferably the vector is a circular molecule. Preferably the vector is a double-stranded molecule, such as a double-stranded DNA molecule. Such a circular, preferably double-stranded, DNA molecule can be used as a convenient storage form for the artificial nucleic acid molecule according to the invention. In addition, it can be used to transfect cells, such as cultured cells. It can also be used for in vitro transcription to produce an artificial RNA molecule according to the invention.

Preferably the vector, preferably a circular vector, can be linearized, for example by restriction enzyme digestion. In a preferred embodiment of the invention, the vector contains a cleavage site such as a restriction site, preferably a unique cleavage site immediately adjacent 3' to the ORF, or immediately adjacent 3' to the 3' UTR element, if present, or located 3' to the poly(A) sequence or polyadenylation signal, if present, or 3' to the poly(C) sequence, if present, or 3' to the histone stem-loop structures, if present. Thus, preferably, the product resulting from vector linearization ends at the 3' end with the 3' end of the ORF or the 3' end of the 3' UTR element, if present, or the 3' end of the poly(A) sequence, or the polyadenylation signal, if present, or the 3' end of the poly(C) sequence, if present. In an embodiment in which the vector of the present invention comprises the artificial nucleic acid molecule of the present invention, the restriction site, preferably a unique restriction site, is preferably directly adjacent 3' to the 3' end of the artificial nucleic acid molecule .

The next object of the present invention relates to a cell containing an artificial nucleic acid molecule of the present invention, or a vector of the present invention. The cell can be any cell, such as a bacterial cell, an insect cell, a plant cell, a vertebrate cell, for example a mammalian cell. Such a cell can be used, for example, to replicate the vector of the present invention, for example, in a bacterial cell. In addition, the cell can be used to transcribe the artificial nucleic acid molecule or vector of the present invention and/or to translate the open reading frame of the artificial nucleic acid molecule or vector of the present invention. For example, the cell can be used to produce a recombinant protein.

The cells of the present invention can be obtained, for example, using standard nucleic acid transfer methods, such as standard transfection, transduction or transformation methods. For example, an artificial nucleic acid molecule or a vector of the present invention can be transferred into a cell by electroporation, lipofection, for example, using cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, transfection based on the use of viruses or based on cationic polymers such as DEAE-dextran or polyethyleneimine, etc.

Preferably, the cell is a mammalian cell, such as a human cell, a domestic animal cell, a laboratory animal cell, such as a mouse or rat cell. Preferably the cell is a human cell. The cell may be a widely used cell line such as CHO, BHK, 293T, COS-7, HELA, HEK, etc. or the cell may be a primary cell such as a human dermal fibroblast (HDF) cell, etc., preferably a cell isolated from the body. In a preferred embodiment of the invention, the cell is an isolated cell of a mammal, preferably a human. For example, the cell may be an immune cell such as a dendritic cell, a cancer or tumor cell, or any somatic cell, etc., preferably a mammal, preferably a human.

Another object of the present invention is a pharmaceutical composition containing an artificial nucleic acid molecule of the present invention, a vector of the present invention, or a cell of the present invention. The pharmaceutical composition according to the invention can be used, for example, as a vaccine, for example, for genetic vaccination. Thus, the ORF may, for example, encode an antigen to be administered to a patient for the purpose of vaccination. Thus, in a preferred embodiment of the invention, the pharmaceutical composition of the present invention is a vaccine. In addition, the pharmaceutical composition of the present invention can be used, for example, for gene therapy.

Preferably, the pharmaceutical composition also contains one or more pharmaceutically acceptable excipients, diluents and/or excipients and/or one or more adjuvants. In the context of the present invention, a pharmaceutically acceptable excipient typically includes a liquid or non-liquid base proposed in the invention of the pharmaceutical composition. In one of the embodiments of the invention, the pharmaceutical composition is in liquid form. In this case, preferably, the carrier is an aqueous carrier, for example, may be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, such as solutions buffered with phosphate, citrate, etc. The buffer may be hypertonic, isotonic, or hypotonic with respect to the specific reference medium, i.e. the salt content of the buffer may be greater than, identical to, or less than that of the particular reference medium, and concentrations of the aforementioned salts may preferably be used which do not result in cell damage due to osmosis or other concentration-related influences. Reference media can be, for example, liquids used in in vivo methods, such as blood, lymph, cytosolic fluids, or other body fluids, or, for example, liquids that can be used as reference media in in vivo methods. vitro" such as conventional buffers or liquids. These conventional buffers or liquids are known to the person skilled in the art. The most preferred liquid base is lactated Ringer's solution.

One or more compatible solid or liquid excipients or diluents or encapsulating compounds that can be used for administration to a patient can also be used in the pharmaceutical composition of the invention. The term "compatible" in the context of the present description means that these components of the pharmaceutical composition according to the invention can be mixed with the artificial nucleic acid according to the invention, the vector or cells indicated in the present description, so that no interaction occurs that could significantly reduce the pharmaceutical efficacy of the pharmaceutical composition according to the invention under normal conditions of use.

The pharmaceutical composition of the present invention may optionally also contain one or more additional pharmaceutically active ingredients. In this context, a pharmaceutically active ingredient is a compound that has a therapeutic effect in relation to the treatment, alleviation or prevention of a particular indication or disease. Such compounds include, but are not limited to, peptides or proteins, nucleic acids, (therapeutically active) small organic or inorganic compounds (molecular weight less than 5000, preferably less than 1000), sugars, antigens or antibodies, therapeutic agents already known from the existing level of technology, antigenic cells, antigenic cell fragments, cell fractions; cell wall components (eg polysaccharides), modified, attenuated or deactivated (eg chemically or by radiation) pathogens (viruses, bacteria, etc.).

In addition, the pharmaceutical composition according to the invention may contain a carrier for an artificial nucleic acid molecule or a vector. Such a carrier may be useful for facilitating dissolution in physiologically acceptable fluids, transport and uptake by the cell of a pharmaceutically active artificial nucleic acid molecule or vector. Therefore, such a carrier may be a component that may be suitable for the storage and delivery of an artificial nucleic acid molecule or vector proposed/proposed in the invention. Such components may be, for example, cationic or polycationic carriers or compounds which may serve as transfection or complexing agents.

In a preferred embodiment of the invention, the artificial nucleic acid molecule or vector is complexed with one or more cationic or polycationic compounds, preferably cationic or polycationic polymers, cationic or polycationic peptides or proteins, for example protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids.

According to a preferred embodiment of the invention, the artificial nucleic acid molecule or vector can be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Therefore, in one of the embodiments of the invention, proposed in the invention, the pharmaceutical composition contains liposomes, lipoplexes or lipid nanoparticles that contain an artificial nucleic acid molecule or vector, preferably RNA, more preferably mRNA.

There is increasing evidence that lipid-based formulations should be considered as one of the most promising delivery systems for nucleic acids, primarily RNA, due to their biocompatibility and ease of large-scale production. Cationic lipids have been extensively studied as synthetic materials for RNA delivery. After mixing, nucleic acids condense with cationic lipids to form lipid/nucleic acid complexes known as lipoplexes. These lipid complexes can protect the genetic material from the effects of nucleases and be delivered to cells by interacting with a negatively charged cell membrane. Lipoplexes can be obtained directly by mixing positively charged lipids at physiological pH with negatively charged nucleic acids.

Conventional liposomes are composed of a lipid bilayer, which may be composed of cationic, anionic, or neutral (phospho)lipids, and cholesterol, which surrounds an aqueous core. Both the lipid bilayer and the water space may include hydrophobic or hydrophilic compounds, respectively. The in vivo characteristics and behavior of liposomes can be modified by adding a hydrophilic polymeric coating, such as polyethylene glycol (PEG), to the surface of the liposomes to impart steric stabilization. In addition, liposomes can be used for specific targeting by attaching ligands (eg, antibodies, peptides and carbohydrates) to their surface or to the ends of attached PEG chains (Front Pharmacol., 6, December 1, 2015, p. 286).

Liposomes are lipid-based and surfactant-based colloidal delivery systems consisting of a phospholipid bilayer surrounding an aqueous compartment. They may be in the form of spherical bubbles and may range in size from 20 nm to several microns. Liposomes based on cationic lipids can form a complex with negatively charged nucleic acids through electrostatic interactions, which leads to the formation of complexes characterized by biocompatibility, low toxicity and the possibility of their large-scale production necessary for clinical applications in vivo. Liposomes can fuse with the plasma membrane for entry; Once inside the cell, the liposomes are processed through the endocytic pathway, and then the genetic material is released from the endosome/carrier into the cytoplasm. Liposomes have long been regarded as drug delivery vehicles due to their very high biocompatibility based on the fact that liposomes are in general analogues of biological membranes and can be obtained from naturally occurring and synthetic phospholipids (Int J Nanomedicine. 9, 2014, pp. 1833-1843).

Cationic liposomes have traditionally been most widely used as non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligonucleotides, and siRNA/small hairpin RNA (shRNA)). Cationic lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids with the formation of nanoparticles through electrostatic interaction, which ensures high efficiency of transfection in vitro. In addition, lipid-neutral RNA delivery nanoliposomes have been developed, such as neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes (Adv Drug Deliv Rev. 66, February 2014, pp. 110-116).

Thus, in one of the embodiments of the invention, an artificial nucleic acid molecule or a vector, preferably RNA, included in the pharmaceutical composition proposed in the present invention, is complexed with cationic lipids and/or neutral lipids and thereby forms liposomes, lipid nanoparticles, lipoplexes or nanoliposomes based on neutral lipids.

In other embodiments, the most preferred transfection or complexation agents are cationic or polycationic compounds including protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell-penetrating peptides (CPPs), such as HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22-derived peptides or analogs, VP22 HSV (herpes simplex virus), MAP, KALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG peptide(s), Pep-1, L-oligomers, calcitonin peptide(s), Antennapedia-derived peptides (primarily derived from Drosophila antennapedid), pAntp, pIsl, FGF, lactoferrin, transportan, buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP or histones.

In addition, such cationic or polycationic compounds may be cationic or polycationic peptides or proteins that preferably contain or are further modified to contain at least one -SH group. Preferably the cationic or polycationic carrier is selected from cationic peptides having the following general formula (I):

in which l+m+n+o+x=3-100 and l, m, n or o independently of each other denote any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81 -90 and 91-100, provided that the total content of Arg (arginine), Lys (lysine), His (histidine) and Orn (ornithine) is at least 10% of the total amino acid content of the oligopeptide; and Xaa can be any amino acid selected from native (ie, naturally occurring) or non-naturally occurring amino acids with the exception of Arg, Lys, His, or Orn; and x can be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided that the total Xaa content does not exceed 90% of the total amino acid content in the oligopeptide. Any of the amino acids Arg, Lys, His, Orn and Xaa can be placed at any position in the peptide. In this context, cationic peptides or proteins containing 7-30 amino acids are most preferred. Preferred cationic peptides and proteins are also described in international patent application WO 2009/030481, the contents of which are incorporated herein by reference.

представленной выше, который содержит или дополнительно модифицирован так, что он содержал по меньшей мере одну -SH-группу, можно выбирать (но, не ограничиваясь только этим) из соединений подформулы (Ia):In addition, a cationic or polycationic peptide or protein defined by the formula

above, which contains or is further modified so that it contains at least one -SH group, can be selected from (but not limited to) compounds of subformula (Ia):

in which (Arg) ₁ ; (lys) _m ; (His) _n ; (Orn) _o ; and x are as defined above, Xaa' is any amino acid selected from native (i.e., naturally occurring) or non-naturally occurring amino acids with the exception of Arg, Lys, His, Orn, or Cys, and y is any number , selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31 -40, 41-50, 51-60, 61-70, 71-80 and 81-90, provided that the total content of Arg (arginine), Lys (lysine), His (histidine) and Orn (orinithine) is at least 10% of the total amino acid content of the oligopeptide. In addition, the cationic or polycationic peptide can be selected from compounds of subformula (Ib):

представлена в настоящем описании и образует ядро аминокислотной последовательности, представленной (полуэмпирической) формулой (III), и в которой Cys₁ и Cys₂ обозначают остатки цистеина, расположенные проксимально или терминально относительно (Arg)₁;(Lys)_m;(His)_n;(Orn)_o;(Xaa)_x. В этом контексте содержание WO 2011/026641 включено в настоящее описание в качестве ссылки.in which the empirical formula

is presented in the present description and forms the core of the amino acid sequence represented by (semi-empirical) formula (III), and in which Cys ₁ and Cys ₂ denote cysteine residues located proximal or terminal relative to (Arg) ₁ ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x . In this context, the contents of WO 2011/026641 are incorporated herein by reference.

Other preferred cationic or polycationic compounds that can be used as transfection agents may be cationic polysaccharides, eg chitosan, polybrene, cationic polymers, eg polyethyleneimine (PEI), cationic lipids, eg DOTMA:chloride [1-( 2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, STAR, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristooxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac chloride -[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]dimethylammonium, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]trimethylammonium , oligofectamine, or cationic or polycationic polymers, e.g. ep, modified polyamino acids such as β-amino acid polymers or reversible polyamides, etc., modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium) bromide), etc. modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methyl acrylate)) etc. modified amidoamines such as pAMAM (poly(amidoamine)) etc. modified polybetaaminoester (PBAE) e.g. modified diamine terminus copolymers 1,4-butanediol diacrylate and 5-amino-1-pentanol, etc. dendrimers, for example, polypropylamine dendrimers or pAMAM-based dendrimers, etc. polyimine(s), e.g. PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, etc. silane backbone polymers, e.g. PMOXA-PDMS copolymers, etc., block copolymers consisting of a combination of one or more cationic blocks (e.g. selected from the cationic polymer above) and one or more hydrophilic or hydrophobic blocks ( e.g. polyethylene glycol); etc.

According to another embodiment of the invention, the pharmaceutical composition according to the invention may contain an adjuvant to enhance the immunostimulatory properties of the pharmaceutical composition. In this context, an adjuvant can be understood to mean any compound that is suitable for facilitating the administration and delivery of components, such as an artificial nucleic acid molecule or a vector, which/which is contained in the pharmaceutical composition of the invention. In addition, such an adjuvant may (but not limited to) initiate or enhance the immune response of the innate immune system, i. nonspecific immune response. In other words, upon administration, the pharmaceutical composition of the invention typically initiates an adaptive immune response directed to the antigen encoded by the artificial nucleic acid molecule. In addition, the pharmaceutical composition of the invention can elicit a (supportive) innate immune response by adding an adjuvant as described herein to the pharmaceutical composition of the invention.

Such an adjuvant can be selected from any adjuvant known to the person skilled in the art and suitable for the case in question, i. to maintain the induction of an immune response in a mammal. Preferably, the adjuvant can be selected from the group including (but not limited to) TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminum hydroxide, ADJUMER™ (polyphosphazene); aluminum phosphate gel; glucans isolated from algae; algammulin; aluminum hydroxide gel (alum); highly protein-absorbing aluminum hydroxide gel; a low viscosity aluminum hydroxide gel; AF or SPT (emulsion containing Squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate buffered saline, pH 7.4); AVRIDINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-D-glucopyranosyl)-N-octadecylamide hydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy vitamin D3); calcium phosphate gel; CAP™ (calcium phosphate nanoparticles); cholera holotoxin, fusion protein cholera toxin-A1 protein-A-D fragment, cholera toxin subunit B; CRL 1005 (P1205 block copolymer); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA ( dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); complete Freund's adjuvant; incomplete Freund's adjuvant; inulin-gamma; Gerbu's adjuvant (mixture containing: I) N-acetylglucosaminyl-( P1-4)-N-acetylmuramoyl-L-alanyl-D35 glutamine (GMDP), II) dimethyldioctadecylammonium chloride (DDA), III) L-proline zinc salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine); imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRV (immunoliposomes prepared from vesicles obtained by the hydration-rehydration method); interferon-gamma; interleukin-1beta; interleukin-2; interleukin-7; interleukin-12; ISCOMSM; ISCOPREP 7.0.3. TM; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); oral adjuvant LT 5 (labile enterotoxin-protoxin from E. coli); microspheres and microparticles of any composition; MF59TM; (water emulsion of squalene); MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (metabolizable oil adjuvant); MPL™ (3-Q-deacyl-4'-monophosphoryl lipid A); liposomes MTP-PE and MTP-PE (N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))ethylamide, single sodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™ and DMURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGIn-sn-glycerindipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISV (nonionic surfactant vesicles); PLEURAN™ (β-glucan); PLGA, PGA and PLA (homo- and copolymers of lactic acid and glycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (protenoid microspheres); polyethylene carbamate derivatives; poly-rA:poly-rU (polyadenylic acid-polyuridic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Alabama); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-aminootecdimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol); SAF-1™ ("composition of adjuvants from Syntex"); Sendai virus proteoliposomes and lipid matrices containing Sendai virus; Span-85 (sorbitan trioleate); Specol (emulsion Marcol 52, Span 85 and Twin 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosane and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracoshexane); stearyltyrosine (octadecyltyrosine hydrochloride); Theramid® (M-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide™ or [thr 1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Tu-particles (Tu-HPV or virus-like particles); Walter-Read liposomes (liposomes containing lipid A adsorbed on aluminum hydroxide) and lipopeptides, including Pam3Cys, especially aluminum salts such as Adju-phos, alhydrogel, rehydragel, etc., emulsions such as like CFA, SAF, IFA, MF59, provax, TiterMax, montanide, vaxfectin, etc., copolymers like optivax (Optivax, CRL1005), L121, poloxamer 4010) etc., liposomes like "liposomes stealth (Stealth), etc., cochleates such as BIORAL, etc., herbal adjuvants such as QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including tomatine, biopolymers such as PLG, PMM, inulin, etc., adjuvants of microbial origin such as romurtide, DETOX, MPL, CWS, mannose, CpG nucleotide sequences, CpG7909, human TLR 1 ligands -13, ISS-1018, 35 IC31, imidazoquinolines, ampligen, Ribi529, IMOxine, IRIV, HPV, cholera toxin, thermolabile toxin, Pam3Cys, flagellin, GPI (glycosylphosphatidylinositol)-anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150 , RSV fusion protein, cdiGMP; and adjuvants useful as antagonists, including the neuropeptide CGRP.

Suitable adjuvants can also be selected from cationic or polycationic compounds, whereby the adjuvant is preferably prepared by forming a complex comprising an artificial nucleic acid molecule or vector included/included in a pharmaceutical composition and a cationic or polycationic compound. The association or formation of a complex comprising an artificial nucleic acid molecule or vector in/in a pharmaceutical composition and the cationic or polycationic compounds described herein preferably provides inherent adjuvant properties and has a stabilizing effect on the artificial nucleic acid molecule or vector in/in into a pharmaceutical composition. Most preferably, such cationic or polycationic compounds are selected from cationic or polycationic peptides or proteins, including protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins such as poly-L-lysine (PLL), polyarginine, basic polypeptides having the ability cell-penetrating peptides (CPPs), such as HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22-derived peptides or analogs, VP22 HSV (herpes simplex virus), MAP, KALA, or domains protein transduction (PTD), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, calcitonin peptide(s), Antennapedia-derived peptides (primarily derived from Drosophila antennapedia), pAntp, pIsl, FGF, lactoferrin, transportan, buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT derived peptides, SAP or histones. Other preferred cationic or polycationic compounds may be cationic polysaccharides, eg chitosan, polybrene, cationic polymers, eg polyethyleneimine (PEI), cationic lipids, eg DOTMA:[1-(2,3-sioleyloxy)propyl)]-N chloride, N,N-trimethylammonium, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, STAR, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristooxypropyldimethylhydroxyethylammonium bromide , DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2- hydroxyethyl)]dimethylammonium, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. polyamino acids such as β-amino polymers acid, or reversible polyamides, etc., modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium) bromide), etc. modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methyl acrylate)) etc. modified amidoamines such as pAMAM (poly(amidoamine)) etc. modified polybetaaminoester (PBAE) e.g. modified diamine terminus copolymers 1,4-butanediol diacrylate and 5-amino-1-pentanol, etc. dendrimers, for example, polypropylamine dendrimers or pAMAM-based dendrimers, etc. polyimine(s), e.g. PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, etc. silane backbone polymers, e.g. PMOXA-PDMS copolymers, etc., block copolymers consisting of a combination of one or more cationic blocks (e.g. selected from the cationic polymer above) and one or more hydrophilic or hydrophobic blocks ( e.g. polyethylene glycol); etc.

In addition, preferred cationic or polycationic proteins or peptides that can be used as an adjuvant by complexing with an artificial nucleic acid molecule or vector, preferably RNA included/included in the composition, can be selected from the following proteins or peptides having the following general formula (I): (Arg) ₁ ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x in which l+m+n+o+x=8-15 and l, m, n or o independently of each other may represent any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, provided that the total the content of Arg, Lys, His and Orn is at least 50% of the total content of amino acids in the oligopeptide; and Xaa may be any amino acid selected from native (ie, naturally occurring) or non-native amino acids with the exception of Arg, Lys, His, or Orn; and x may be any number selected from 0, 1, 2, 3, or 4, provided that the total Xaa content should not exceed 50% of all amino acids in the oligopeptide. The most preferred oligoarginines in this context are, for example, Arg ₇ , Arg ₈ , Arg ₉ , Arg ₇ , H ₃ R ₉ , R ₉ H ₃ , H ₃ R ₉ H ₃ , YSSR ₉ SSY, (RKH) ₄ , Y( RKH) ₂ R etc.

The ratio between the artificial nucleic acid or vector and the cationic or polycationic compound can be calculated based on the nitrogen to phosphate ratio (N/P ratio) of the entire nucleic acid complex. For example, 1 μg of RNA typically contains approximately 3 nmol of phosphate residues, provided that the RNA is characterized by a statistical distribution of bases. In addition, 1 ml of the peptide typically contains about x nmol of nitrogen residues, depending on the molecular weight and the number of basic amino acids. If, as an example, a calculation is made for (Arg) ₉ (molecular weight 1424 g / mol, 9 nitrogen atoms), then 1 μg of (Arg) ₉ contains approximately 700 pmoles (Arg) ₉ and, therefore, 700 × 9 = 6300 pmoles of basic amino acids, i.e. 6.3 nmol nitrogen atoms. It can be calculated that for a PHK/(Arg) ₉ mass ratio of about 1:1, the N/P ratio should be about 2. If, as an example, the calculation is made for protamine (molecular weight about 4250 g/mol, 21 nitrogen atoms, if protamine from salmon is used) at a weight ratio of about 2:1, then for 2 μg of RNA it gives 6 nmol of phosphate; 1 µg of protamine contains approximately 235 pmoles of protamine molecules and therefore 235×21=4935 pmoles of basic nitrogen atoms, i.e. 4.9 nmol nitrogen atoms. It can be calculated that for an RNA/protamine weight ratio of about 2:1, the N/P ratio would be about 0.81. It can be calculated that for an RNA/protamine weight ratio of about 8:1, the N/P ratio would be about 0.2. In the context of the present invention, regarding the ratio of nucleic acid:peptide in the complex, the N/P ratio is preferably in the range of 0.1-10, preferably in the range of 0.3-4, and most preferably in the range of 0.5-2 or 0.7-2, and most preferably in the range of 0.7-1.5.

Patent application WO 2010/037539, the contents of which are incorporated herein by reference, describes an immunostimulatory composition and methods for preparing an immunostimulatory composition. Thus, in a preferred embodiment of the invention, the composition is prepared by performing two separate steps to achieve both an effective immunostimulatory effect and efficient translation of the artificial nucleic acid molecule of the invention. To do this, in the first stage, the so-called "adjuvant component" is obtained by forming a complex that includes an artificial nucleic acid molecule or a vector, preferably RNA, which is part of the adjuvant component, and a cationic or polycationic compound in a specific ratio to form a stable complex. In this context, it is important that the adjuvant component is free from or retains only negligible amounts of free cationic or polycationic compound after complexation with the nucleic acid. Thus, the ratio between the nucleic acid and the cationic or polycationic compound in the adjuvant component is generally chosen to be in the range such that the nucleic acid is completely incorporated into the complex and there is no or only negligible free cationic or polycationic compound present. Preferably the ratio in the adjuvant component, ie. the ratio between mRNA and cationic or polycationic compound is selected from the range of from about 6:1 (wt./wt.) to about 0.25:1 (wt./wt.), more preferably from about 5:1 (wt. ./wt.) to about 0.5:1 (wt./wt.), even more preferably from about 4:1 (wt./wt.) to about 1:1 (wt./wt.) or from about 3:1 (w/w) to about 1:1 (w/w) and most preferably from about 3:1 (w/w) to about 2:1 (w/w).

According to a preferred embodiment of the invention, an artificial nucleic acid molecule or a vector, preferably an RNA molecule according to the invention, is added in a second step to the complexed nucleic acid molecule, preferably RNA, of the adjuvant component to obtain an (immunostimulatory) composition according to the invention. In this case, an artificial nucleic acid molecule or a vector, preferably an RNA molecule according to the invention, is added in the form of a free nucleic acid, i. nucleic acid that is not complexed with other compounds. Prior to addition, the free artificial nucleic acid molecule or vector is not complexed, and preferably after addition of the adjuvant component, it/it should not undergo any detectable or significant complexation reaction.

Suitable adjuvants can also be selected from nucleic acids having the formula (II): G _l X _m G _n in which: G is guanosine (guanine), uridine (uracil) or a guanosine (guanine) or uridine (uracil) analogue; X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above nucleotides (nucleosides); l denotes an integer from 1 to 40, while when l=1, then G is guanosine or its analogue, when l>1, then at least 50% of the nucleotides are guanosine (guanine) or its analogue; m is an integer and is at least 3; when m=3, then X is uridine (uracil) or its analogue, when m>3, then at least 3 consecutive uridines (uracils) or analogues of uridine (uracil) are present; n denotes an integer from 1 to 40, while when n=1, then G is guanosine (guanine) or its analogue, when n>1, then at least 50% of the nucleotides (nucleosides) is guanosine (guanine) or its equivalent.

Other suitable adjuvants can also be selected from nucleic acids having the formula (III): C _l X _m C _n in which: C is cytidine (cytosine), uridine (uracil) or an analogue of cytidine (cytosine) or uridine (uracil); X is guanosine (guanine), uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine) or an analogue of the above nucleotides (nucleosides); l denotes an integer from 1 to 40, while when l=1, then C is cytidine (cytosine) or its analogue, when l>1, then at least 50% of the nucleotides (nucleosides) are cytidine (cytosine) or its equivalent; m is an integer and is at least 3; when m=3, X is uridine (uracil) or an analogue thereof, when m>3, then at least 3 consecutive uridine (uracil) or analogues of uridine (uracil) are present; n denotes an integer from 1 to 40, while when n=1, then C is cytidine (cytosine) or its analogue, when n>1, then at least 50% of the nucleotides (nucleosides) are cytidine (cytosine) or its equivalent.

The pharmaceutical composition according to the present invention preferably contains in a "safe and effective amount" the components of the pharmaceutical composition, especially the artificial nucleic acid molecule according to the invention, the vector and/or the cells presented in the present description. In the context of the present description, the term "safe and effective amount" means an amount sufficient to significantly induce a positive modification of the disease or disorder specified in the present description. However, at the same time, a "safe and effective amount" must be such as to avoid serious side effects and provide a reasonable balance between benefit and risk. The determination of these limits, as a rule, is within the competence of the treating physician.

Another object of the present invention is an artificial nucleic acid molecule according to the present invention, a vector according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention, intended for use as a medicine, for example, as vaccines (for genetic vaccination), or in gene therapy.

The artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention are most suitable for any medical application that uses the therapeutic action or effect of peptides, polypeptides or proteins, or in which the addition of a particular peptide or protein is required. Thus, the present invention provides an artificial nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention for use in the treatment or prevention of diseases or disorders. treatable by the therapeutic action or effect of the peptides, polypeptides or proteins, or treatable by the addition of a particular peptide, polypeptide or protein. For example, an artificial nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention can be used to treat or prevent genetic diseases, autoimmune diseases, cancer, or related with tumor diseases, infectious diseases, chronic diseases or the like, for example, by genetic vaccination or gene therapy.

In particular, such therapeutic treatments, the benefit of which is determined by the increased amount and prolonged presence of therapeutic peptides, polypeptides or proteins in the body of the individual to be treated, are most suitable as a medical approach in the context of the present invention, since the inventive 3'-UTR- the element provides stable and prolonged expression of the peptide or protein encoded by the inventive artificial nucleic acid molecule or the inventive vector, and/or the inventive 5'-UTR element provides increased expression of the peptide or protein encoded by the inventive artificial nucleic acid molecule acid or proposed in the invention vector. Thus, the most suitable medical application of the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention is vaccination. Thus, the present invention provides an artificial nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention for vaccinating a subject, preferably a mammal, more preferably a human. . The preferred types of vaccination are vaccination against infectious diseases such as bacterial, protozoal or viral infections and antitumor vaccination. Such types of vaccination can be carried out for prophylactic or therapeutic purposes.

You can choose ORS depending on the disease to be treated. For example, the open reading frame may encode a protein that is to be delivered to a patient suffering from a complete loss or at least a partial loss of protein function, such as a patient suffering from a genetic disease. In addition, the open reading frame can be selected from ORFs encoding a peptide or protein that has a beneficial effect on the disease or condition of the patient. In addition, the open reading frame may encode a peptide or protein that down-regulates abnormal overproduction of a naturally occurring peptide or protein, or elimination of cells abnormally expressing the protein or peptide. Such absence, loss of function or overproduction may occur, for example, in the case of tumor and neoplasia, autoimmune diseases, allergies, infections, chronic diseases or the like. In addition, the open reading frame may encode an antigen or immunogen, such as an epitope of a pathogen or tumor antigen. Thus, in preferred embodiments of the invention, the artificial nucleic acid molecule or vector proposed / proposed in the present invention contains an ORF encoding an amino acid sequence containing or consisting of an antigen or immunogen sequence, for example, a pathogen epitope or tumor-associated antigen, 3'- UTR element as above and/or 5' UTR element as above and optionally additional components such as poly(A) sequence and the like.

In the context of medical applications, especially in the context of vaccination, it is preferable that the artificial nucleic acid molecule of the present invention is RNA, preferably mRNA, since DNA carries the risk of inducing an immune anti-DNA response and tends to be integrated into genomic DNA . However, in some embodiments of the invention, for example, when a viral delivery vehicle, such as an adenoviral delivery vehicle, is used to deliver an artificial nucleic acid molecule or vector of the present invention, for example, in the context of various gene therapies, it may be desirably, the artificial nucleic acid molecule or vector is/is a DNA molecule.

The artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation using a spray, topically, rectally, intranasally , buccally, vaginally, using an implanted reservoir, or by bolus injection. In the context of the present description, the term "parenteral" includes the methods of subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, epigastric, intrathecal, intrahepatic, carried out in the area of damage, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial and sublingual injection or infusion. In a preferred embodiment, the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention are administered by needleless injection (e.g., jet injection).

Preferably, the artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention are administered parenterally, such as by parenteral injection, more preferably by subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, epigastric, intrathecal, intrahepatic, carried out in the area of damage, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, sublingual injection or infusion. Most preferred are intradermal and intramuscular injections. Sterile injectable forms of the pharmaceutical composition according to the invention may be an aqueous or oily suspension. Such suspensions may be prepared by methods known in the art using suitable dispersing or wetting agents and suspending agents. Preferably, solutions or suspensions are administered by needleless injection (eg, jet injection).

The artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention may also be orally administered in any oral dosage form, including (but, not limited to) capsules, tablets, aqueous suspensions or solutions.

The artificial nucleic acid molecule of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention can also be applied topically, especially when the target for treatment includes areas or organs readily available for topical application, including, for example, diseases of the skin or any other accessible epithelial tissue. For each of these areas or organs, compositions suitable for topical treatment can be easily prepared. For topical application, an artificial nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention may be formulated into a suitable ointment suspended or dissolved in one or more carriers.

In one embodiment, the use as a drug comprises the step of transfecting mammalian cells, preferably in vitro or ex vivo transfection of mammalian cells, more preferably in vitro transfection of cells isolated from the patient to be treated with the drug. If the use as a drug involves in vitro transfection of the isolated cells, then the use may also involve re-administration of the transfected cells to the patient. The use of the artificial nucleic acid molecules or vector according to the invention as a drug may also include the step of selecting successfully transfected isolated cells. Thus, it may be desirable for the vector to also contain a selection marker. Use as a drug may also include in vitro transfection of isolated cells and purification of the expression product, i.e. encoded peptide or protein from said cells. This purified peptide or protein can then be administered to an individual in need thereof.

The present invention also provides a method for treating or preventing a disease or disorder as defined above, comprising administering an artificial nucleic acid molecule of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition, proposed in the present invention, the individual in need of it.

In addition, a method for treating or preventing a disease or disorder is provided, comprising transfecting a cell with an artificial nucleic acid molecule of the present invention or a vector of the present invention. Said transfection can be carried out in vitro, ex vivo or in vivo. In a preferred embodiment of the invention, the cell is transfected in vitro and the transfected cell is administered to an individual in need thereof, preferably a sick individual. Preferably, the cell to be transfected in vitro is a cell isolated from the body of an individual, preferably a diseased human. Thus, the present invention provides a method of treatment comprising the steps of isolating a cell from an individual, preferably from a sick person, transfecting the isolated cell with an artificial nucleic acid of the present invention or a vector of the present invention. of the invention and administering the transfected cell to an individual, preferably a sick individual.

The method for treating or preventing a disorder according to the present invention is preferably a vaccination method or a gene therapy method as described above.

As described above, the inventive 3'UTR element and/or the inventive 5'UTR element has the ability to prolong and/or increase protein production from mRNA. Thus, a further aspect of the present invention relates to a method for increasing and/or prolonging protein production from an artificial nucleic acid molecule, preferably from an mRNA molecule or a vector, wherein the method comprises associating an open reading frame with a 3'UTR element and /or a 5'-UTR element, where the 3'-UTR element and/or the 5'-UTR element provides a high efficiency of translation of the obtained artificial nucleic acid molecule, obtaining an artificial nucleic acid molecule, preferably an mRNA molecule, proposed in the present invention , which is described above, or the vector proposed in the present invention, which is described above.

Preferably, in a method for increasing and/or prolonging the production of a protein from an artificial nucleic acid molecule, preferably from an mRNA molecule or a vector of the present invention, the 3'-UTR element and/or the 5'-UTR element contains or consists of a nucleotide sequence that is derived from the 3'-UTR and/or 5'-UTR of a gene transcript selected from the group consisting of ZNF460, TGM2, IL7R, BGN, TK1, RAB3B, CBX6, FZD2, COL8A1, NDUFS7, PHGDH, PLK2 , TSPO, PTGS1, FBXO32, NID2, ATP5D, EXOSC4, NOL9, UBB4B, VPS18, ORMDL2, FSCN1, TMEM33, TUBA4A, EMP3, TMEM201, CRIP2, BRAT1, SERPINH1, CD9, DPYSL2, CDK9, TFRC, PSMB3, FASN, PSMB6 , PRSS56, KPNA6, SFT2D2, PARD6B, LPP, SPARC, SCAND1, VASN, SLC26A1, LCLAT1, FBXL18, SLC35F6, RAB3D, MAP1B, VMA21, CYBA, SEZ6L2, PCOLCE, VTN, ALDH16A1, RAVER1, KPNA6, SERINC5, JUP, CPN2 , CRIP2, EPT1, PNPO, SSSCA1, POLR2L, LIN7C, UQCR10, PYCRL, AMN, MAP1S, NDUFS7, PHGDH, TSPO, ATP5D, EXOSC4, TUBB4B, TUBA4A, EMP3, CRIP2, BRAT1, CD9, CD K9, PSMB3, PSMB6, PRSS56, SCAND1, AMN, CYBA, PCOLCE, MAP1S, VTN, ALDH16A1 (preferably all human) and Dpysl2, Ccnd1, Acox2, Cbx6, Ubc, Ldlr, Nudt22, Pcyox1l, Ankrd1, Tmem37, Tspyl4, Slc7a3 , Cst6, Aacs, Nosip, Itga7, Ccnd2, Ebp, Sf3b5, Fasn, Hmgcs1, Osr1, Lmnb1, Vma21, Kif20a, Cdca8, Slc7a1, Ubqln2, Prps2, Shmt2, Aurkb, Fignl1, Cad, An1n, Slfn9, Ncaph, Pole , Uhrf1, Gja1, Fam64a, Kif2c, Tspan10, Scand1, Gpr84, Fads3, Cers6, Cxcr4, Gprc5c, Fen1, Cspg4, Mrpl34, Comtd1, Armc6, Emr4, Atp5d, 1110001J03Rik, Csf2ra, Aarsd1, Kif22, Cth, Tpgs1 , Alkbh7, Ms4a8a, Acox2, Ubc, Slpi, Pcyox1l, Igf2bp1, Tmem37, Slc7a3, Cst6, Ebp, Sf3b5, Plk1, Cdca8, Kif22, Cad, Cth, Pole, Kif2c, Scand1, Gpr84, Tpgs1, Cel17, Alkbh7, Ms4a8a , Mrpl34, Comtd1, Armc6, Atp5d, 1110001J03Rik, Nudt22, Aarsd1 (preferably all mice); preferably from the group consisting of ZNF460-5'-UTR, TGM2-5'-UTR, IL7R-5'-UTR, BGN-5'-UTR, TK1-5'-UTR, RAB3B-5'-UTR, CBX6- 5'-UTR, FZD2-5'-UTR, COL8A1-5'-UTR, NDUFS7-5'-UTR, PHGDH-5'-UTR, PLK2-5'-UTR, TSPO-5'-UTR, PTGS1-5 '-UTR, FBXO32-5'-UTR, NID2-5'-UTR, ATP5D-5'-UTR, EXOSC4-5'-UTR, NOL9-5'-UTR, UBB4B-5'-UTR, VPS18-5' -UTR, ORMDL2-5'-UTR, FSCN1-5'-UTR, TMEM33-5'-UTR, TUBA4A-5'-UTR, EMP3-5'-UTR, TMEM201-5'-UTR, CRIP2-5'- UTR, BRAT1-5'-UTR, SERPINH1-5'-UTR, CD9-5'-UTR, DPYSL2-5'-UTR, CDK9-5'-UTR, TFRC-5'-UTR, PSMB3-5'-UTR , FASN-5'-UTR, PSMB6-5'-UTR, PRSS56-5'-UTR, KPNA6-5'-UTR, SFT2D2-5'-UTR, PARD6B-5'-UTR, LPP-5'-UTR, SPARC-5'-UTR, SCAND1-5'-UTR, VASN-5'-UTR, SLC26A1-5'-UTR, LCLAT1-5'-UTR, FBXL18-5'-UTR, SLC35F6-5'-UTR, RAB3D -5'-UTR, MAP1B-5'-UTR, VMA21-5'-UTR, CYBA-5'-UTR, SEZ6L2-5'-UTR, PCOLCE-5'-UTR, VTN-5'-UTR, ALDH16A1- 5'-UTR, RAVER1-5'-UTR, KPNA6-5'-UTR, SERINC5-5'-UTR, JUP-5'-UTR, CPN2-5'-UTR, CRIP2-5'-UTR, EPT1-5 '-UTR, PNPO-5'-UTR, SSSCA1-5'-UTR, POLR2L-5'-UTR, LIN7C-5'-UTR, UQC R10-5'-UTR, PYCRL-5'-UTR, AMN-5'-UTR, MAP1S-5'-UTR, (preferably all human) and Dpysl2-5'-UTR, Ccnd1-5'-UTR, Acox2- 5'-UTR, Cbx6-5'-UTR, Ubc-5'-UTR, Ldlr-5'-UTR, Nudt22-5'-UTR, Pcyox1l-5'-UTR, Ankrd1-5'-UTR, Tmem37-5 '-UTR, Tspyl4-5'-UTR, Slc7a3-5'-UTR, Cst6-5'-UTR, Aacs-5'-UTR, Nosip-5'-UTR, Itga7-5'-UTR, Ccnd2-5' -UTR, Ebp-5'-UTR, Sf3b5-5'-UTR, Fasn-5'-UTR, Hmgcs1-5'-UTR, Osr1-5'-UTR, Lmnb1-5'-UTR, Vma21-5'- UTR, Kif20a-5'-UTR, Cdca8-5'-UTR, Slc7a1-5'-UTR, Ubqln2-5'-UTR, Prps2-5'-UTR, Shmt2-5'-UTR, Aurkb-5'-UTR , Fignl1-5'-UTR, Cad-5'-UTR, Anln-5'-UTR, Slfn9-5'-UTR, Ncaph-5'-UTR, Pole-5'-UTR, Uhrf1-5'-UTR, Gja1-5'-UTR, Fam64a-5'-UTR, Kif2c-5'-UTR, Tspan10-5'-UTR, Scand1-5'-UTR, Gpr84-5'-UTR, Fads3-5'-UTR, Cers6 -5'-UTR, Cxcr4-5'-UTR, Gprc5c-5'-UTR, Fen1-5'-UTR, Cspg4-5'-UTR, Mrpl34-5'-UTR, Comtd1-5'-UTR, Armc6- 5'-UTR, Emr4-5'-UTR, Atp5d-5'-UTR, 1110001J03Rik-5'-UTR, Csf2ra-5'-UTR, Aarsd1-5'-UTR, Kif22-5'-UTR, Cth-5 '-UTR, Tpgs1-5'-UTR, Ccl17-5'-UTR, Alkbh7-5'-UTR, Ms4a8a-5'-UTR (ex preferably all mice); and NDUFS7-3'-UTR, PHGDH-3'-UTR, TSPO-3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'-UTR, BRAT1-3'-UTR, CD9-3'-UTR, CDK9-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, PRSS56 -3'-UTR, SCAND1-3'-UTR, AMN-3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1- 3'-UTR (preferably all human) and Acox2-3'-UTR, Ubc-3'-UTR, Slpi-3'-UTR, Pcyox1l-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'- UTR, Slc7a3-3'-UTR, Cst6-3'-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Plk1-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR , Cad-3'-UTR, Cth-3'-UTR, Pole-3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Cel17-3'-UTR, Alkbh7-3'-UTR, Ms4a8a-3'-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik -3'-UTR, Nudt22-3'-UTR, Aarsd1-3'-UTR, (preferably all murines).

The term "association of an artificial nucleic acid molecule or vector with a 3'-UTR element and/or with a 5'-UTR element" in the context of the present invention preferably means the functional association or functional association of an artificial nucleic acid molecule or vector with a 3'-UTR- element and/or with a 5'-UTR element. This means that the association or binding of the artificial nucleic acid molecule or vector is carried out with a 3'-UTR element and/or with a 5'-UTR element, preferably with a 3'-UTR element and/or with a 5'-UTR element described above, in such a way that the function of the 3'-UTR element and/or 5'-UTR element is carried out, for example, the function of prolonging and/or increasing the production of RNA and/or protein. This generally means that the 3'UTR element and/or the 5'UTR element is integrated into the artificial nucleic acid molecule or vector, preferably into the mRNA molecule, from the 3' side and/or 5' side of the relatively open frame, respectively, preferably so that it is directly adjacent on the 3' side of the open reading frame and/or directly adjacent on the 5' side of the open reading frame, so that the 3'-UTR element is preferably located between the open reading frame and the poly (A) polyadenylation sequence or signal. Preferably, the 3'UTR element and/or the 5'UTR element is integrated into the artificial nucleic acid molecule or vector, preferably into mRNA, as the 3'UTR and/or 5'UTR, respectively, i. so that the 3'-UTR element and/or 5'-UTR element is the 3'-UTR and/or 5'-UTR respectively of an artificial nucleic acid molecule or vector, preferably mRNA, i. e. so that the 5'-UTR ends immediately before the 5'-end of the ORF and the 3'-UTR extends from the 3' side of the open reading frame to the 5' side of the poly(A) sequence or polyadenylation signal, optionally attached to using a short linker, such as a sequence containing or consisting of one or more restriction sites. Thus, preferably, the term "association of an artificial nucleic acid molecule or vector with a 3'-UTR element and/or 5'-UTR element" means the functional association of a 3'-UTR element and/or 5'-UTR element with an open a reading frame located in an artificial nucleic acid molecule or vector, preferably in an mRNA molecule. 3'-UTR and/or 5'-UTR and ORF are the constructs described above for the artificial nucleic acid molecule of the present invention, for example, preferably ORF and 3'-UTR are heterologous and/or ORF and 5'-UTR are heterologous, respectively, eg derived from different genes, as described above. Association with the 3'-UTR element and/or 5'-UTR element can be accomplished either by de novo association of individual elements or by modification of an already existing nucleic acid (template). In the latter case, by way of example and illustration, the nucleic acid molecule of the present invention can be obtained by (I) using a nucleic acid having the corresponding sequence as shown in FIG. 1A as a template, (ii) modifying said template by replacing at least one (a) underlined 5'-UTR element (underlined in FIG. 1A) or (b) 3'-UTR element (underlined in double line on Fig. 1A) to (a) another 5'-UTR element or (b) another 3'-UTR element, thereby carrying out the association of an artificial nucleic acid molecule with the specified 5'-UTR element or 3'-UTR element .

Another aspect of the present invention relates to the use of a 3'UTR element and/or a 5'UTR element, preferably a 3'UTR element as described above and/or a 5'UTR element as described above, to enhance and /or prolonging the production of a protein from an artificial nucleic acid molecule, preferably from an mRNA molecule or a vector, where the 3'-UTR element and/or the 5'-UTR element contains or consists of a nucleotide sequence derived from the 3'-UTR and/or 5'-UTR of a gene transcript selected from the group consisting of ZNF460, TGM2, IL7R, BGN, TK1, RAB3B, CBX6, FZD2, COL8A1, NDUFS7, PHGDH, PLK2, TSPO, PTGS1, FBXO32, NID2, ATP5D, EXOSC4, NOL9, UBB4B, VPS18, ORMDL2, FSCN1, TMEM33, TUBA4A, EMP3, TMEM201, CRIP2, BRAT1, SERPINH1, CD9, DPYSL2, CDK9, TFRC, PSMB3 5'-UTR, FASN, PSMB6, PRSS56, KPNA6, SFT2D2, PARD6B, LPP, SPARC, SCAND1, VASN, SLC26A1, LCLAT1, FBXL18, SLC35F6, RAB3D, MAP1B, VMA21, CYBA, SEZ6L2, PCOLCE, VTN, ALDH16A1, RAVER1, KPNA6, SERINC5, JUP, CPN2, CRIP2, EPT1, PNPO, SSSCA1, PO LR2L, LIN7C, UQCR10, PYCRL, AMN, MAP1S, NDUFS7, PHGDH, TSPO, ATP5D, EXOSC4, TUBB4B, TUBA4A, EMP3, CRIP2, BRAT1, CD9, CDK9, PSMB3, PSMB6, PRSS56, SCAND1, AMN, CYBA, PCOLCE, MAP1S, VTN, ALDH16A1 (preferably all human) and Dpysl2, Ccnd1, Acox2, Cbx6, Ubc, Ldlr, Nudt22, Pcyox1l, Ankrd1, Tmem37, Tspyl4, Slc7a3, Cst6, Aacs, Nosip, Itga7, Ccnd2, Ebp, Sf3b5, Fasn , Hmgcs1, Osr1, Lmnb1, Vma21, Kif20a, Cdca8, Slc7a1, Ubqln2, Prps2, Shmt2, Aurkb, Fignl1, Cad, Anln, Slfn9, Ncaph, Pole, Uhrf1, Gja1, Fam64a, Kif2c, Tspan10, Scand1, Gpr84, Fads3 , Cers6, Cxcr4, Gprc5c, Fen1, Cspg4, Mrpl34, Comtd1, Armc6, Emr4, Atp5d, 1110001J03Rik, Csf2ra, Aarsd1, Kif22, Cth, Tpgs1, Cel17, Alkbh7, Ms4a8a, Acox2, Ubc, Slpi, Pcyox1l, Igf2bp7 , Slc7a3, Cst6, Ebp, Sf3b5, Plk1, Cdca8, Kif22, Cad, Cth, Pole, Kif2c, Scand1, Gpr84, Tpgs1, Ccl17, Alkbh7, Ms4a8a, Mrpl34, Comtd1, Armc6, Atp5d, 1110001J03Rik, Nudt22, Aarsd1 (preferably all mice); preferably from the group consisting of ZNF460-5'-UTR, TGM2-5'-UTR, IL7R-5'-UTR, BGN-5'-UTR, TK1-5'-UTR, RAB3B-5'-UTR, CBX6- 5'-UTR, FZD2-5'-UTR, COL8A1-5'-UTR, NDUFS7-5'-UTR, PHGDH-5'-UTR, PLK2-5'-UTR, TSPO-5'-UTR, PTGS1-5 '-UTR, FBXO32-5'-UTR, NID2-5'-UTR, ATP5D-5'-UTR, EXOSC4-5'-UTR, NOL9-5'-UTR, UBB4B-5'-UTR, VPS18-5' -UTR, ORMDL2-5'-UTR, FSCN1-5'-UTR, TMEM33-5'-UTR, TUBA4A-5'-UTR, EMP3-5'-UTR, TMEM201-5'-UTR, CRIP2-5'- UTR, BRAT1-5'-UTR, SERPINH1-5'-UTR, CD9-5'-UTR, DPYSL2-5'-UTR, CDK9-5'-UTR, TFRC-5'-UTR, PSMB3 5'-UTR, FASN-5'-UTR, PSMB6-5'-UTR, PRSS56-5'-UTR, KPNA6-5'-UTR, SFT2D2-5'-UTR, PARD6B-5'-UTR, LPP-5'-UTR, SPARC -5'-UTR, SCAND1-5'-UTR, VASN-5'-UTR, SLC26A1-5'-UTR, LCLAT1-5'-UTR, FBXL18-5'-UTR, SLC35F6-5'-UTR, RAB3D- 5'-UTR, MAP1B-5'-UTR, VMA21-5'-UTR, CYBA-5'-UTR, SEZ6L2-5'-UTR, PCOLCE-5'-UTR, VTN-5'-UTR, ALDH16A1-5 '-UTR, RAVER1-5'-UTR, KPNA6-5'-UTR, SERINC5-5'-UTR, JUP-5'-UTR, CPN2-5'-UTR, CRIP2-5'-UTR, EPT1-5' -UTR, PNPO-5'-UTR, SSSCA1-5'-UTR, POLR2L-5'-UTR, LIN7C-5'-UTR, UQC R10-5'-UTR, PYCRL-5'-UTR, AMN-5'-UTR, MAP1S-5'-UTR (preferably all human) and Dpysl2-5'-UTR, Ccnd1-5'-UTR, Acox2-5 '-UTR, Cbx6-5'-UTR, Ubc-5'-UTR, Ldlr-5'-UTR, Nudt22-5'-UTR, Pcyox1l-5'-UTR, Ankrd1-5'-UTR, Tmem37-5' -UTR, Tspyl4-5'-UTR, Slc7a3-5'-UTR, Cst6-5'-UTR, Aacs-5'-UTR, Nosip-5'-UTR, Itga7-5'-UTR, Ccnd2-5'- UTR, Ebp-5'-UTR, Sf3b5-5'-UTR, Fasn-5'-UTR, Hmgcs1-5'-UTR, Osr1-5'-UTR, Lmnb1-5'-UTR, Vma21-5'-UTR , Kif20a-5'-UTR, Cdca8-5'-UTR, Slc7a1-5'-UTR, Ubqln2-5'-UTR, Prps2-5'-UTR, Shmt2-5'-UTR, Aurkb-5'-UTR, Fignl1-5'-UTR, Cad-5'-UTR, Anln-5'-UTR, Slfn9-5'-UTR, Ncaph-5'-UTR, Pole-5'-UTR, Uhrf1-5'-UTR, Gja1 -5'-UTR, Fam64a-5'-UTR, Kif2c-5'-UTR, Tspan10-5'-UTR, Scand1-5'-UTR, Gpr84-5'-UTR, Fads3-5'-UTR, Cers6- 5'-UTR, Cxcr4-5'-UTR, Gprc5c-5'-UTR, Fen1-5'-UTR, Cspg4-5'-UTR, Mrpl34-5'-UTR, Comtd1-5'-UTR, Armc6-5 '-UTR, Emr4-5'-UTR, Atp5d-5'-UTR, 1110001J03Rik-5'-UTR, Csf2ra-5'-UTR, Aarsd1-5'-UTR, Kif22-5'-UTR, Cth-5' -UTR, Tpgs1-5'-UTR, Ccl17-5'-UTR, Alkbh7-5'-UTR, Ms4a8a-5'-UTR (pre preferably all mouse); and NDUFS7-3'-UTR, PHGDH-3'-UTR, TSPO-3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'-UTR, BRAT1-3'-UTR, CD9-3'-UTR, CDK9-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, PRSS56 -3'-UTR, SCAND1-3'-UTR, AMN-3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1- 3'-UTR (preferably all human) and Acox2-3'-UTR, Ubc-3'-UTR, Slpi-3'-UTR, Pcyox11-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'- UTR, Slc7a3-3'-UTR, Cst6-3'-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Plk1-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR , Cad-3'-UTR, Cth-3'-UTR, Pole-3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3'-UTR, Ms4a8a-3'-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik -3'-UTR, Nudt22-3'-UTR, Aarsd1-3'-UTR, (preferably all murines).

The uses of the present invention preferably include the association of an artificial nucleic acid molecule, vector or RNA with a 3'UTR element as described above and/or a 5'UTR element as described above.

Compounds and ingredients proposed in the invention of the pharmaceutical composition can also be manufactured and sold separately from each other. Thus, the invention also relates to a kit or kit of components comprising an artificial nucleic acid molecule according to the invention, a vector according to the invention, a cell according to the invention, and/or a pharmaceutical composition according to the invention. Preferably, such kit or kits of components may further comprise instructions for use, transfection cells, an adjuvant, means for administering the pharmaceutical composition, a pharmaceutically acceptable carrier, and/or a pharmaceutically acceptable solution for dissolving or reconstituting the artificial nucleic acid molecule, vector, cells, or pharmaceutical composition.

Method for identifying an element of a 3'-untranslated region (3'-UTR element) and/or an element of a 5'-untranslated region (5'-UTR element)

Another object of the present invention is a method for identifying a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element), comprising the following steps, in which:

a) analyze the stability of the mRNA, carrying out the following sub-steps, in which:

I. determine the amount of the specified mRNA at the first time point in the process of cleavage of the specified mRNA,

II. determining the amount of said mRNA at a second time point in the process of cleavage of said mRNA, and

III. calculate the ratio of the amount of said mRNA determined in step (I) to the amount of said mRNA determined in step (II);

b) A stable mRNA is selected, characterized in that the ratio calculated in substep (III) is at least 0.5 (50%), at least 0.6 (60%), at least 0.7 (70 %), at least 0.75 (75%), at least 0.8 (80%), at least 0.85 (85%), at least 0.9 (90%), or at least 0.95 (95%); and

c) The nucleotide sequence of the 3'- and/or 5'-UTR element of said stable mRNA is determined.

While the stability of the mRNA is preferably assessed under standard conditions, for example, under standard conditions (standard environment, incubation, etc.) for a specific used cell line or used cell type.

To analyze the stability of the mRNA, the cleavage process of said mRNA is evaluated by determining the amount or concentration of said mRNA at the first and second time points during the cleavage of said mRNA (see steps a) I. and a) II.).

To determine the amount or concentration of mRNA during in vivo or in vitro RNA digestion as defined above (i.e. in vitro refers specifically to (“live”) cells and/or tissue, including tissue from a living individual; cells include in particular cell lines, primary cells, cells in tissue or in the body of individuals, mammalian cells such as human cells and mouse cells are preferred, and most preferably human HeLa, HEPG2 and U-937 cell lines and mouse NIH3T3 cell lines are used. , JAWSII and L929, moreover, most preferred are primary cells, in the most preferred embodiments of the invention, human dermal fibroblasts (HDF)), various methods known to the person skilled in the art can be used. Examples of such methods include, but are not limited to, general transcription inhibition, e.g., with a transcription inhibitor such as actinomycin D, the use of inducible promoters to specifically enhance short-term transcription, e.g., the serum-inducible c-fos promoter system and the regulatory promoter system. Tet-off, and kinetic labeling methods such as pulse labeling.

For example, if transcription inhibitor-mediated transcription termination in step a) is used to determine the amount or concentration of mRNA during in vivo or in vitro RNA cleavage as above, then transcription inhibitors such as actinomycin D (ActD), 5, 6-dichloro-1-D-ribofuranosylbenzimidazole (DRB) or -amanitin (α-Am). In this case, in order to assess mRNA cleavage, generally, transcription inhibitors are added to cells and thereby generally inhibit transcription, and RNA cleavage can be detected without being interfered with by ongoing transcription.

Alternatively, in step a), inducible promoters can be used to specifically enhance short-term transcription, in which case the idea is to provide a stimulus that activates transcription and leads to a burst of mRNA synthesis, after which the stimulus is removed to “turn off” transcription and mRNA cleavage monitoring. At the same time, the use of an inducible promoter allows for strict control, due to which the induction and termination of transcription occur in a narrow time window. In the case of mammalian cells, the c-fos promoter is known to be useful for this purpose because it is amenable to rapid and short-term induction in response to the addition of serum, which provides a reliable and simple way to achieve a short-term burst of transcription. The Tet-off promoter system represents another possibility that further expands the application of the transcriptional burst-amplification approach to studying mRNA turnover in mammalian cells.

However, in the present invention, in step a), kinetic labeling methods are preferably used to determine the amount of mRNA during in vivo or in vitro RNA cleavage as described above. In kinetic labeling methods, as a rule, labeling of RNA is carried out, while labels include primarily labeled nucleotides and labeled nucleosides, labeled uridine and labeled uracil are most preferred. Examples of preferred labels include 4-thiouridine (4sU), 2-thiouridine, 6-thioguanosine, 5-ethynyluridine (EU), 5-bromuridine (BrU), biotin-16-aminoallyluridine, 5-aminoallyluridine, 5-aminoallylcytidine, etc. ., with 4-thiouridine (4sU), 5-ethynyluridine (EU) or 5'-bromouridine (BrU) being more preferred. Most preferred is 4-thiouridine (4sU). 4-thiouridine (4sU) is preferably used at a concentration of 100-500 μM. In addition, radiolabeled nucleotides, for example uridine- ^3H , can also be used. Combinations of the above labeled nucleotides can also be used, with the combination of 4-thiouridine and 6-thioguanosine being most preferred.

Kinetic labeling typically labels the resulting RNA, for example by incorporating a labeled uridine or uracil during transcription. After some time, labeling is stopped, and thereafter RNA cleavage can be detected by assessing specifically labeled RNA without overall inhibition of transcription.

To determine the amount of mRNA during RNA cleavage in step a), pulse labeling is preferably used, with label displacement methodology being most preferred. In the context of the present description, the term "pulse labeling" refers to a method in which a label is used, for example, the labels described above, to measure the rates of synthesis and/or cleavage of compounds in living cells. Typically, cells are brought into contact with a small amount of label for a short period of time, so the term "pulse" is used. In a post-pulse labeling dislodge methodology, typically after the required period of contact time with the label, a much larger amount of unlabeled compound corresponding to the "pulse" (compound used for pulse labeling) is added (e.g., unlabeled uridine if labeled was used as the pulse). uridine). The effect of competition between labeled and unlabeled compounds should reduce further absorption of the labeled compound to a negligible level, hence the concept of "crowding out".

To determine the amount or concentration of mRNA, as a rule, it is necessary to isolate the mRNA. The person skilled in the art is aware of various methods for isolating RNA, for example, by extraction with guanidine-thiocyanate-phenol-chloroform or by extraction using a silica gel column. Commercially available kits can also be used, such as the RNeasy Kit from Qiagen.

In addition, an extraction step may be required, in particular when a kinetic labeling method is used (as opposed to a transcription inhibitor where the total RNA is a "cleaving" RNA, since overall transcription inhibition is performed). In the extraction step, labeled RNA (i.e., representing "cleaving" RNA) is extracted from the total isolated RNA. Thus, extraction means can be selected depending on the label used. For example, immunopurification using anti-label antibodies can be used.

In addition, for example, to extract a thio-labeled, for example, 4-thiouridine (4sU) labeled RNA, one can incubate HPDP-biotin disulfide bond) with isolated "total RNA". This reagent reacts specifically with reduced thiols (-SH) in 4-thiouridine (4sU) labeled RNA to form reversible disulfide bonds. Biotinylation allows binding of a thio-labeled, for example, 4-thiouridine (4sU) labeled RNA to streptavidin and can therefore be extracted from total RNA by disulfide reduction with dithiothreitol or beta-mercaptoethanol (or any other reducing agent).

In the case of biotin labeled nucleotides, for example biotin-16-aminoallyluridine, streptavidin can be used directly to extract labeled RNA from total RNA.

For example, to extract novel transcribed 5-ethynyluridine (EU) labeled cellular RNAs from total RNA, EU biotinylation in a copper-catalyzed cycloaddition reaction (often referred to as "click chemistry") can be used, followed by purification based on affinity for streptavidin. This method is performed using the commercially available Click-iT Nascent RNA Capture Kit (catalog number C10365, Invitrogen). The manufacturer's instructions recommend using a pulse labeling time of 30 to 60 min for a dose of 0.5 mM EU or 1 to 24 h for a dose of 0.1 or 0.2 mM EU.

For example, BrU-labeled RNA molecules can be extracted by immunopurification using an anti-bromodeoxyuridine antibody (eg, clone 2B1, catalog number MI-11-3, MBL) and Sepharose G protein.

The amount or concentration of mRNA can then be measured, i. e. the level (number of copies) of the transcript, using various methods known to the person skilled in the art. Examples of such methods include, but are not limited to, microarray analysis, Northern blot analysis, quantitative PCR, or next generation sequencing technology (high throughput sequencing). Most preferred are microarray analysis and next generation sequencing technology. In addition, whole genome/whole transcriptome based approaches are most preferred, e.g. in microarray based analysis, whole genome microarray analysis, e.g. Affymetrix Human Gene 1.0 ST or 2.0 ST, or Affymetrix Mouse Gene 1.0 ST or 2.0 ST, or full transcript analysis by next generation sequencing.

At substages I. and II. step a) determine the amount of mRNA at the first and second time points in the process of mRNA cleavage. Typically, this means that the mRNA is first isolated at the first and second time points in the mRNA cleavage process to determine the appropriate amounts. Therefore, "first time" and "second time" are specific times in the RNA cleavage process at which RNA is isolated to determine the amount of RNA. As a rule, the "second time point" occurs later in the RNA cleavage process than the "first time point".

Preferably, the first time point is chosen such that only the mRNA undergoing the cleavage process is considered, ie. to avoid the resulting mRNA, for example, during the transcription process. For example, if kinetic labeling methods are used, such as pulsed labeling, the first time point is preferably chosen such that incorporation of the label into the mRNA is complete, i.e. there was no more incorporation of the label into the mRNA. Thus, if kinetic labeling is used, the first time point may be at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least at least 60 minutes, at least 70 minutes, at least 80 minutes, or at least 90 minutes after the end of the experimental labeling procedure, for example, after the end of incubation of the labeled cells.

For example, the first time point may preferably occur 0-6 hours after transcription has been terminated (e.g., with a transcription inhibitor), promoter induction has been terminated when using inducible promoters, or after the pulse or label has been terminated, e.g., after labeling has ended. More preferably, the first time point may occur after a period of 30 minutes to 5 hours, even more preferably 1 to 4 hours, and most preferably about 3 hours after transcription has ceased (for example, with a transcription inhibitor), promoter induction has ceased at in the case of using inducible promoters or after the pulse or label is stopped, for example, after the end of labeling.

Preferably, the second time point is chosen so that it occurs as late as possible in the mRNA cleavage process. However, if multiple mRNA species are considered, the second time point is preferably chosen such that there is still a significant number of mRNA species, preferably at least 10% of the mRNA species present in a detectable amount, i.e. in an amount greater than 0. Preferably, the second time occurs after at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, or at least 15 hours after transcription is stopped (for example, with a transcription inhibitor), cessation of promoter induction in the case of inducible promoters or after the termination of the pulse or label, for example, after the end of labeling.

For example, the second time point may preferably occur 3-48 hours after transcription has been terminated (e.g., with a transcription inhibitor), promoter induction has been terminated when using inducible promoters, or after the pulse or label has been terminated, e.g., after labeling has ended. More preferably, the second time point may occur after a period of 6 minutes to 36 hours, even more preferably 10 to 24 hours, and most preferably about 15 hours after transcription has ceased (for example, with a transcription inhibitor), promoter induction has ceased. in the case of the use of inducible promoters or after the termination of the pulse or label, for example, after the end of labeling.

Thus, the time interval between the first time and the second time should preferably be as long as possible within the above limits. Therefore, the time interval between the first time point and the second time point should preferably be at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours. , at least 10 hours, at least 11 hours, or at least 12 hours, with about 12 hours being most preferred. Typically, the second time is at least 10 minutes later than the first time.

At substage III. step a) calculate the ratio of the amount of mRNA determined in stage (I) to the amount of mRNA determined in stage (II). For this purpose, the mRNA amount (transcript copy number) determined as above at the second time point is divided by the mRNA amount (transcript copy number) determined as above at the first time point. The use of this ratio prevents that stable mRNA species, which are present only in very small amounts already at the first time point, will not be taken into account when compared with mRNA species that are present in large quantities.

In step b), an mRNA is selected for which the ratio calculated in substep (III) of step a) is at least 0.5 (50%), at least 0.6 (60%), at least 0, 7 (70%), at least 0.75 (75%), at least 0.8 (80%), at least 0.85 (85%), at least 0.9 (90%), or at least 0.95 (95%). Such mRNA is considered according to the present invention as the most stable mRNA.

In step c) the nucleotide sequence of the 3'- and/or 5'-UTR element of said mRNA is determined, i. mRNA selected in step b). For this purpose, various methods known to the person skilled in the art can be used, such as sequencing or selection from publicly available databases such as, for example, NCBI (National Center for Biotechnology Information). For example, you can search for the mRNA sequence selected in step b) mRNA in the database and then you can select 3'-and/or 5'-UTR from the mRNA sequence available in the database.

In particular, in the method described above for identifying a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element), the concept of "mRNA" and/or "stable mRNA" respectively, may also refer to the types of mRNA presented in the present description, and/or stable types of mRNA, respectively.

In addition, in the context of the present description, preferably "stable mRNA" may be characterized by slower cleavage compared to the average cleavage of the mRNA, which is preferably assessed in vivo or in vitro as described above. Meanwhile, "average mRNA cleavage" can be assessed by examining mRNA cleavage for a plurality of mRNA species.

Therefore, another object of the present invention is a method for identifying a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element), comprising the following steps, in which:

a) analyze the stability of multiple types of mRNA, carrying out the following sub-steps, in which:

I. determine the amount of each mRNA species from the specified set of mRNA species at the first time point in the process of cleavage of the specified mRNA species,

II. determine the amount of each mRNA species from the specified set of mRNA species in the second time in the process of cleavage of the specified mRNA species and

III. calculate for each mRNA species from the specified set of mRNA species the ratio of the number of the specified mRNA species, determined in stage (I), to the number of the specified mRNA species, determined in stage (II);

b) ranking mRNA species from a plurality of mRNA species depending on the magnitude of the ratio calculated for substep (III) for each mRNA species;

c) selecting one or more mRNA species having the highest ratio or highest ratios calculated in substep (III); and

d) determining the nucleotide sequence of the 3'- and/or 5'-UTR element of said mRNA.

The concept of "types of mRNA" in the context of the present description corresponds to the genomic transcription unit, i.e. gene. Thus, in one "type of mRNA" there may be different transcripts, for example, due to the processing of mRNA. mRNA species, for example, can be represented as spots on a microarray. Therefore, the microarray is the preferred "tool" for quantifying multiple mRNA species, for example, at a specific point in time during mRNA cleavage. However, other methods known to one of skill in the art can also be used, such as RNA sequencing (also referred to as shotgun sequencing of the entire transcriptome, which is a technology that uses the power of next-generation sequencing to detect based on instantaneous pictures of the presence and amount of RNA in the genome at a given time), quantitative PCR, etc.

Preferably, "multiple mRNA species" refers to at least 100, at least 300, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 11000, at least 12000, at least 13000, at least 14000, at least 15000, at least 16,000, at least 17,000, at least 18,000, at least 19,000, at least 20,000, at least 21,000, at least 22,000, at least 23,000, at least 24,000, at least 25,000, at least 26,000, at least 27,000, at least 28,000, at least 29,000, at least 30,000 mRNA species. It is most preferable to evaluate the entire transcriptome or the maximum possible number of mRNA species in the transcriptome. This can be done, for example, using a microarray that covers all transcripts.

In this method, step a), including its sub-steps I.-III., practically corresponds to step a), including its sub-steps I.-III., of the previously described method of the invention, but differs only in that the amount of each type of mRNA from the plurality of mRNA species are determined at the first and second time points, and that the ratio value is calculated for each mRNA species. Therefore, the methods detailed above and the preferred embodiments of the invention apply here as well, and the ratio value for the individual mRNA species (and each of the individual mRNA species, respectively) can be determined according to the procedure described above for "mRNA".

However, in contrast to the above method, mRNA stability is assessed not by the absolute value of the ratio, but by ranking mRNA species from a plurality of mRNA species, depending on the ratio value calculated in substep (III) of step a) for each of the mRNA species. Then one or more mRNA species are selected in sub-step c) having the highest ratio or the highest ratio values calculated in sub-step (III) of step a).

In this context, most preferably in step c) 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% of the most stable mRNA species. Alternatively, or additionally, mRNA species can be selected in step c) which are characterized by the magnitude of the ratio calculated in substep III. step a), which corresponds to at least 100% of the average ratio calculated for all analyzed mRNA species. More preferably, mRNA species are selected that have a ratio of at least 150%, even more preferably at least 200%, and most preferably at least 300% of the average ratio calculated for all analyzed mRNA species.

In step d), the nucleotide sequence of the 3'- and/or 5'-UTR element of the mRNA selected in step c) is determined according to the procedure described above for step c) of the previously described method of the invention.

Preferably, in both of the methods described above for identifying a 3'-UTR element and/or a 5'-UTR element according to the present invention, the time period between the first time point and the second time point is at least 5 hours, preferably at least 6 hours. h, preferably at least 7 h, more preferably at least 8 h, more preferably at least 9 h, even more preferably at least 10 h, even more preferably at least 11 h and most preferably at least 12 h .

Preferably, in both of the methods described above for identifying a 3'UTR element and/or a 5'UTR element according to the present invention, mRNA stability is analyzed by pulse labeling, preferably using label displacement methodology.

Method for identifying an element of a 3'-untranslated region (3'-UTR element) and/or an element of a 5'-untranslated region (5'-UTR element)

Another object of the present invention is also a method for identifying a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element), which provides high translation efficiency of an artificial nucleic acid molecule, including the following steps:

a) identifying a 3'-UTR element and/or a 5'-UTR element derived from a stable mRNA using a method for identifying a 3'-UTR element and/or a 5'-UTR element corresponding to one of the methods that described above;

b) synthesize an artificial nucleic acid molecule containing at least one open reading frame and at least one 3'-UTR element and/or at least one 5'-UTR element that corresponds to or is contained in the 3'-UTR element and/or 5'-UTR element identified in step a);

c) analyzing the expression of a protein encoded by at least one open reading frame (ORF) of the artificial nucleic acid molecule synthesized in step b);

d) analyzing the expression of a protein encoded by at least one open reading frame (ORF) of an artificial reference nucleic acid molecule lacking a 3'-UTR element and/or a 5'-UTR element;

e) comparing the expression of the protein from the artificial nucleic acid molecule analyzed in step c) with the expression of the protein from the artificial reference nucleic acid molecule analyzed in step d); and

e) selecting a 3'-UTR element and/or a 5'-UTR element if the expression of the protein from the artificial nucleic acid molecule analyzed in step c) is prolonged and/or increased compared to the expression of the protein from the artificial nucleic acid reference molecule acid analyzed in step d).

In this method, the first 3'-UTR element and/or 5'-UTR element is identified using the method described above, proposed in the present invention. This allows the synthesis of the 3'- and/or 5'-UTR element using methods known to the person skilled in the art, for example, using PCR amplification. The primers used for such PCR may preferably contain restriction sites for cloning. Alternatively, the 3'- and/or 5'-UTR element can be synthesized, for example, by chemical synthesis or oligonucleotide annealing. Thus, in step b), an artificial nucleic acid molecule is synthesized containing at least one open reading frame and at least one 3'-UTR element and/or at least one 5'-UTR element that corresponds to or is contained in in the 3'-UTR element and/or 5'-UTR element identified in step a). In particular, at least one 3'-UTR element and/or at least one 5'-UTR element is typically combined with an open reading frame, resulting in an artificial nucleic acid containing 3'- and/ or a 5'-UTR element according to the invention, if the 3'- and/or 5'-UTR element satisfies the relevant requirements, namely, if they prolong and/or increase protein expression. To test this, the 3' and/or 5' UTR element identified in step a) or the PCR fragment or synthesized sequence, respectively, can be cloned into a particular vector, preferably an expression vector, to assess the expression of the protein with the corresponding ORF.

Then, as described above, in step c), protein expression from an artificial nucleic acid molecule containing at least one 3'-UTR element and/or at least one 5'-UTR element is evaluated and compared with that evaluated in step d) expression of a protein from a corresponding artificial reference nucleic acid molecule that does not contain a 3'-UTR element and/or a 5'-UTR element, as described in step e).

Thereafter, in step e), a 3'-UTR element and/or a 5'-UTR element is selected that increases the expression of the protein from the artificial nucleic acid molecule analyzed in step c) as compared to the expression of the protein from the artificial reference molecule. nucleic acid analyzed in step d). Comparison of protein expression from the inventive nucleic acid molecule with protein expression from the reference nucleic acid molecule is carried out according to the procedure presented in the present description, primarily in the context of the artificial nucleic acid molecule of the invention.

In addition, the present invention proposes the most preferred method for identifying a 3'-untranslated region element (3'-UTR element) and/or a 5'-untranslated region element (5'-UTR element), which provides high translation efficiency of the artificial molecule nucleic acid, including the following stages, in which:

a) cells are fed/incubated with the labeled nucleotide for its incorporation into new transcribed RNA molecules (labeling with label displacement);

b) separating total RNA from the cells at a first time point and at least one second later time point;

c) extract the labeled RNA molecules from the total RNA isolated in step b);

d) measuring the number/number of copies of the transcript of the various types of mRNA present in the labeled RNA;

e) calculate the ratio of the number/number of copies of the mRNA species transcript present at least one second later time point to the number/number of copies of the mRNA species transcript present at the first time point;

e) ranking the types of mRNA depending on the value of the ratio determined in step e);

g) selecting the most stable types of mRNA;

h) determining the nucleotide sequence of the 3'- and/or 5'-UTR of the most stable mRNA species selected in step g);

i) synthesizing the 3'- and/or 5'-UTR element contained in the 3'- and/or 5'-UTR determined in step h);

j) combining the 3'- and/or 5'-UTR element synthesized in step i) with an open reading frame to produce the nucleic acid of the invention as described herein; and

k) optionally comparing the expression of an open reading frame present in the nucleic acid of the invention with the expression of an open reading frame present in a reference nucleic acid lacking the 3' and/or 5' UTR element described herein.

In this case, the detailed description and preferred embodiments of the invention presented above for the methods proposed in the invention can be applied in this case, subject to the appropriate restrictions indicated for stages a)-l).

In particular, the following labeled nucleotides are preferably used for feeding in step a) of the method according to the invention: 4-thiouridine (4sU), 2-thiouridine, 6-thioguanosine, 5-ethynyluridine (EU), 16-aminoallyluridine, 5-aminoallyluridine, 5-aminoallylcytidine, etc. Most preferred is 4-thiouridine (4sU). 4-thiouridine is preferably used at a concentration of 100-500 μM. Alternatively, radioactively labeled nucleotides, for example uridine- ^3H , may be used. Combinations of the above labeled nucleotides may be used. Most preferred is a combination of 4-thiouridine and 6-thioguanosine.

The incubation of cells with the labeled nucleotide in step a) can be varied. Most preferably, the duration of incubation (feeding time) is from 10 minutes to 24 hours. The most preferred time period is from 2 to 6 hours, more preferably from 2 to 3 hours.

Cells that can be used in the method of the invention include, in particular, cell lines, primary cells, cells in a tissue or an individual's body. In specific embodiments of the invention suitable for use according to the present invention may be cell types that allow the cultivation of cells. Most preferred are mammalian cells, eg human cells and mouse cells. In the most preferred embodiments of the invention, human cell lines HeLa, HEPG2 and U-937 and mouse cell lines NIH3T3, JAWSII L929 are used. In addition, primary cells are most suitable, in the most preferred embodiments of the invention, human dermal fibroblasts (HDF) can be used. Alternatively, the individual's tissue can also be treated with labeled nucleotides and, after an incubation period, RNA can be isolated from the tissue according to step c).

To determine the most stable mRNAs in a cell (type), total RNA is extracted at the first time point above, e.g. 0-6 hours after labeling, preferably 3 hours after labeling, and at the second later time point above, , 3-48 hours after labeling, preferably 10-24 hours, most preferably 15 hours after labeling. The second later time occurs at least 10 minutes after the first time.

In step e), mRNA species are ranked according to the ratio calculated in step e). In this context, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% are most preferably selected. , 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% of the most stable mRNA species.

In this context, it is also preferable to select such mRNA species for which the number of transcript copies/number of mRNA species at the second later time point compared to the first time point is at least 50% (0.5-fold), at least 60% (0.6-fold), at least 70% (0.7-fold), at least 90% (0.9-fold) or at least 95% (0.95-fold). This embodiment of the invention is most preferred if the RNA is recovered 3 hours (first time point) and 15 hours (second time point) after labeling.

Alternatively or additionally, mRNA species are selected that have a ratio calculated in step e) corresponding to at least 100% of the average ratio calculated for all analyzed mRNA species. More preferably, mRNA species are selected that have a ratio of at least 150% and more preferably at least 200%, and most preferably at least 300% of the average ratio calculated for all analyzed mRNA species.

In the next step of the method according to the invention, the nucleotide sequence of the 3'- and/or 5'-UTR of the most stable mRNA species selected in step g) is determined, and the 3'- and/or 5'-UTR element is synthesized in step i), for example, using PCR amplification. Primers used for PCR may preferably contain restriction sites for cloning. Alternatively, the 3' and/or 5' UTR element can be synthesized (eg by chemical synthesis or oligonucleotide annealing).

In step j) of the method of the invention, the PCR fragment or synthesized sequence is combined with an open reading frame to produce an artificial nucleic acid containing the 3' and/or 5' UTR element of the invention. Preferably, the PCR fragment or sequence can be cloned into a vector.

The most preferred embodiment of the invention relates to a method comprising steps a) to l) for identifying elements of the 3'-untranslated region (3'-UTR elements) and/or elements of the 5'-untranslated region (5'-UTR elements), where 3 '-UTR elements and/or 5'-UTR elements prolong protein production from an artificial nucleic acid molecule containing at least one of the 3'-UTR elements and/or at least one of the 5'-UTR elements.

Another object of the present invention also relates to a method for creating an artificial nucleic acid molecule, in which an artificial nucleic acid molecule is synthesized containing at least one open reading frame and at least one 3'-UTR element and/or at least one 5' -UTR element identified using the proposed method of the present invention to identify the 3'-UTR element and/or 5'-UTR element specified in the present description. The synthesis of an artificial nucleic acid molecule is generally carried out using methods known to the person skilled in the art, for example, cloning methods, for example, well-known methods or presented in the present description.

Preferably, the vector of the present invention as described herein is used in such an inventive method for generating an artificial nucleic acid molecule.

Preferably, the artificial nucleic acid molecule produced by such a method for producing an artificial nucleic acid molecule is the nucleic acid molecule of the present invention as described herein.

In addition, the present invention also provides an artificial nucleic acid molecule, which can be obtained using the method proposed in the present invention for creating an artificial nucleic acid molecule presented in the present description.

The following drawings, sequences and examples are intended to further illustrate the invention. They are not intended to limit the essence of the invention.

The drawings show:

in fig. 1 - sequences encoding mRNA (ie artificial nucleic acid molecules), which can be obtained by transcription in vitro;

The following abbreviations are used:

• PpLuc (GC): high GC (GC-rich) mRNA sequence encoding Photinus pyralis luciferase; ORF PpLuc (GC) is in italics;

• A64: poly(A) sequence containing 64 adenylates;

• C30: poly(C) sequence containing 30 cytidylates;

• hSL: histone stem-loop sequence taken from Cakmakci, Lerner, Wagner, Zheng and William F. Marzluff, Mol. cell. Biol. 28(3), 2008, pp. 1182-1194;

• 32L4: An artificial variant of the 5'-untranslated region of the 5'-UTR of the human ribosomal large subunit protein 32, which lacks the 5'-oligopyrimidine tract. The 5'-UTR is derived from the human 32 ribosomal large subunit protein, which lacks the 5'-oligopyrimidine tract;

• albumin7: an artificial variant of the 3'-untranslated region (3'-UTR) of human albumin containing three single point mutations introduced to remove the T7 termination signal, as well as the HindIII and XbaI restriction sites.

in fig. 1A - reference construct, SEQ ID NO: 205, i.e. mRNA sequence 32L4 - PpLuc(GC) - albumin7 - A64 - C30 - hSL (R3111);

A description of the human-derived UTR elements that were experimentally tested is shown in Table 3. A description of the mouse-derived UTR elements that were experimentally tested is shown in Table 4;

In FIG. 1B - Artificial nucleic acid (SEQ ID NO: 210), said artificial nucleic acid contains the tested 5'-UTR corresponding to SEQ ID NO: 1 (underlined by one line). All elements of the sequence shown in this drawing, with the exception of the one-underlined element, are identical to SEQ ID NO: 205. Thus, SEQ ID NO: 210 differs from SEQ ID NO: 205 in that it contains a different 5'-UTR element.

[5'-UTR corresponding to SEQ ID NO: 1] - PpLuc(GC) - albumin7 - A64 - C30 - hSL;

In FIG. 1B - Artificial nucleic acid (SEQ ID NO: 211), said artificial nucleic acid contains the tested 3'-UTR corresponding to SEQ ID NO: 152 (double underlined). All elements of the sequence shown in this figure, with the exception of the double-underlined element, are identical to SEQ ID NO: 205. Thus, SEQ ID NO: 211 differs from SEQ ID NO: 205 in that it contains a different 3'-UTR element.

32L4 - PpLuc(GC) - [3'-UTR corresponding to SEQ ID NO: 152] - A64 - C30 -hSL;

In FIG. 2-9 - average levels of expression of luciferase and SEM for mRNA, which were analyzed in triplicate; those. relative levels of PpLuc expression standardized against RrLuc (mean values from three independent experiments and standard error of the mean (SEM) are shown). The influence of 5'-UTR elements and 3'-UTR elements of human origin (Table 3) and 5'-UTR elements and 3'-UTR elements of mouse origin (Table 4) on the expression of luciferase with mRNA was studied in comparison with the expression luciferase with mRNA shown in Fig. 1 (reference design). Data on constructs containing the tested 5'-UTR elements and 3'-UTR elements described in tables 3 and 4 are presented in example 3, especially in sections 3.3 and 3.4. For this purpose, the cell lines indicated in the drawings were transfected with various mRNAs by lipofection (Fig. 2: HDF cells were transfected with mRNAs having 5'-UTR elements and 3'-UTR elements of human origin indicated in Table 3; in Fig. Fig. 3: HDF cells were transfected with mRNA having 5'-UTR elements and 3'-UTR elements of mouse origin shown in Table 4. Fig. 4: L929 cells were transfected with mRNA having 5'-UTR elements and 3'- Human origin UTR elements shown in Table 3. Figure 5: L929 cells transfected with mRNA having 5'-UTR elements and murine 3'-UTR elements shown in Table 4. Figure 6: HEPG2 cells mRNA having 5'-UTR elements and 3'-UTR elements of human origin are transfected in Fig. 7: HEPG2 cells are transfected with mRNA having 5'-UTR elements and 3'-UTR elements of mouse origin indicated in Table 4. Fig. 8: HeLa cells were transfected with mRNA having 5'- UTR elements and 3'-UTR elements of human origin listed in Table 3; in fig. 9: HeLa cells were transfected with mRNA having 5'-UTR elements and 3'-UTR elements of murine origin shown in Table 4). More details are provided in example 3, especially in section 3.5. On each of the FIGS. 2-9 show 96 bars corresponding to 96 wells in the assay described in Example 3. FIG. 2-9 96 columns are located one after another in the following order A1, B1, C1, D1, […], F12, G12, H12.

RLU = relative light units (used when measuring luciferase activity).

Examples

Example 1: Candidate UTR Elements

mRNA expression in human and mouse cells was evaluated experimentally. This made it possible to identify the mRNAs that were expressed in the respective cells. The nucleotide sequence of the 5'- and/or 3'-UTR of the mRNA species was determined by database searching, checked and corrected based on cell transcriptome data obtained by high-throughput sequencing, and amplified by PCR or synthesized by oligonucleotide annealing.

Since the identified mRNAs originate from their native cellular environment, these mRNAs were considered as wild-type mRNAs:

SEQ ID NOs: 1-72 correspond to wild-type human 5'UTR elements;

SEQ ID NO: 73-136 correspond to wild-type murine 5'UTR elements;

SEQ ID NOs: 152-173 correspond to wild-type human 3'UTR elements;

SEQ ID NO: 174-203 correspond to wild-type murine 3'UTR elements.

Although several of these UTR elements were known (for example, they are present in their wild-type environment), this approach also allowed the identification of new 5'-UTR elements and new 3'-UTR elements, i.e. 5'-UTR elements and 3'-UTR elements that were found at the time of the invention to be expressed in human cells and mouse cells, respectively, but which were not known from public databases (NCBI):

In particular, in the group consisting of human 5'-UTR elements having SEQ ID NO: 1-72, the following wild-type 5'-UTR elements differ from the 5'-UTR elements known from public databases (NCBI ): SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49.

In the group consisting of mouse 5'-UTR elements having SEQ ID NO: 73-136, the following wild-type 5'-UTR elements differ from the 5'-UTR elements known from public databases (NCBI): SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 98, SEQ ID NO : 106, SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135.

In the group consisting of mouse 3'-UTR elements having SEQ ID NO: 174-203, the following wild-type 3'-UTR elements differ from 3'-UTR elements known from public databases (NCBI): SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 197, SEQ ID NO: 200.

The differences between the newly identified 5'UTR elements and the newly identified 3'UTR elements from the known 5'UTR elements and the known 3'UTR elements, respectively, are shown in Tables 1 and 2. In particular, these newly identified elements do not have been previously described as 3'-UTR elements or 5'-UTR elements, respectively.

Artificial 5'-UTR elements and artificial 3'-UTR elements

In some cases, artificial 5'-UTR elements and artificial 3'-UTR elements were obtained. This was done by modifying the 5'-UTR elements and wild-type 3'-UTR elements using a PCR-based approach using modified primers as follows:

(I) Some ATG triplets in the 5'UTR sequences (if present) were converted to a TAG triplet. (II) In addition, the presence of a certain specific restriction enzyme cleavage site (if present) was considered undesirable when it was believed that said specific cleavage site would interfere with subsequent cloning experiments. It was believed that a cleavage site present in the wild-type sequence would interfere with subsequent cloning experiments (such as described in Examples 2 and 3) if the restriction enzyme used in subsequent cloning experiments could recognize and cleave the wild-type 3'-UTR element. type or wild-type 5'-UTR element. Since such internal cleavage of 5'-UTR elements and 3'-UTR elements was undesirable, the sites cleaved by said specific restriction enzyme were removed by replacing one nucleotide in the site cleaved by said specific restriction enzyme with a complementary nucleotide, thereby removing the site cleaved by the specified specific restriction enzyme.

SEQ ID NO: 137 (based on SEQ ID NO: 3), SEQ ID NO: 138, (based on SEQ ID NO: 9), SEQ ID NO: 139 (based on SEQ ID NO: 14), SEQ ID NO: 140 - (based on SEQ ID NO: 17), SEQ ID NO: 141 (based on SEQ ID NO: 18), SEQ ID NO: 142 (based on SEQ ID NO: 29), SEQ ID NO: 143 (based on SEQ ID NO: 31), SEQ ID NO: 144 (based on SEQ ID NO: 42), SEQ ID NO: 145 (based on SEQ ID NO: 43), SEQ ID NO: 146 (based on SEQ ID NO: 52 ), SEQ ID NO: 147 (based on SEQ ID NO: 109), SEQ ID NO: 148 (based on SEQ ID NO: 110), SEQ ID NO: 149 (based on SEQ ID NO: 119), SEQ ID NO : 150 (based on SEQ ID NO: 120) and SEQ ID NO: 151 (based on SEQ ID NO: 136) are artificial 5'UTR elements. SEQ ID NO: 204 (based on SEQ ID NO: 192) is an artificial 3'UTR element. The exact differences from the corresponding wild-type sequences are noted above.

In general, SEQ ID NOs: 1-136 can be considered wild-type 5'UTR sequences, and SEQ ID NOs: 137-151 can be considered artificial 5'UTR sequences; SEQ ID NO: 152-203 can be considered as wild-type 3'-UTR sequences, and SEQ ID NO: 204 can be considered as artificial 3'-UTR sequence.

In summary, this example presents several 5'-UTR elements and 3'-UTR elements (wild-type and artificial), each of which can be tested for their relative effect on translation efficiency, compared to the reference UTR elements. (as described below in examples 2 and 3).

Example 2: Reference UTR elements and their containing reference construct

In FIG. 1A shows the RNA sequence of the reference construct used in the present invention. A vector for in vitro transcription was also constructed containing a DNA sequence corresponding to the RNA sequence shown in FIG. 1A. The vector contained the T7 promoter, an artificial variant of the 5'-untranslated region (5'-UTR) 32L4 (ribosomal protein of large subunit 32), a GC-rich sequence (open reading frame, ORF) encoding the Photinus pyralis luciferase gene (PpLuc(GC)) , an artificial variant of the 3'-untranslated region (3'-UTR) of human albumin (ALB), albumin7. The poly(A) sequence of A64 followed by C30 and a stem-loop histone sequence was located 3' to albumin7. Behind the histone stem-loop sequence was a restriction site used to linearize the vector prior to in vitro transcription.

PCR fragments corresponding to the sequences described in Example 1 were individually cloned into a vector containing a DNA sequence that corresponded to the RNA sequence shown in FIG. 1. Where the wild-type sequences described in Example 1 contained ATG triplets in the 5'-UTR and/or unwanted restriction sites in the 5'-UTR or 3'-UTR, the corresponding artificial sequences identified in Example 1 were used. (for example, SEQ ID NO: 137 was used instead of SEQ ID NO: 3).

In more detail, to create a construct for testing a candidate 5'UTR element, the single line underlined 5'UTR element shown in FIG. 1A was replaced with the candidate 5'UTR element to be tested (wild-type or artificial) (Example 1) (the corresponding example is shown in FIG. 1B); and to generate a construct for testing a candidate 3'UTR element, the double-lined 3'UTR element shown in FIG. 1A was replaced with the candidate 5'UTR element to be tested (wild-type or artificial) (Example 1) (a corresponding example is shown in FIG. 1B). The resulting vectors or plasmids (containing DNA corresponding to, for example, an RNA sequence, such as the RNA shown in Figures 1A-B) may also be referred to as DNA templates.

DNA templates were linearized and transcribed in vitro using T7 RNA polymerase (WO 2015/101416). The template DNA was then digested by treatment with DNase. The mRNA transcripts contained a 5'-cap structure prepared by adding an excess of N ⁷ -methylguanosine-5'-triphosphate-5'-guanosine to the transcription reaction mixture. The mRNA thus obtained was purified and resuspended in water. The mRNAs thus obtained for testing the UTR elements corresponded to the mRNA shown in FIG. 1A (reference construct), except that the 5'-UTR element or 3'-UTR element of the reference construct has been replaced with the corresponding UTR element to be tested.

Example 3: Translation Efficiency of Candidate UTR Elements Compared to Reference Construct

Experimentally tested 5'-UTR elements of human origin or murine origin or 3'-UTR elements of human origin or murine origin. In the context of this example, "human origin" may refer to either a human wild-type sequence (e.g., SEQ ID NO: 1) or an artificial sequence constructed from a human wild-type sequence by substitution of one or more bases (e.g., SEQ ID NO: 137), the concept of "murine origin" is used accordingly. More details on how the artificial sequences are created based on the wild type sequences are presented in tables 3 and 4 in example 1.

Whether 5' or 3' UTRs (Tables 3 and 4 below) have a beneficial effect on translation efficiency was experimentally tested. This was carried out using a method comprising the steps in which:

(i) transfecting mammalian cells with the artificial nucleic acid molecule and measuring the amounts of expressed protein encoded by the ORF of the artificial nucleic acid molecule at one or two specific time points after transfection (24 and 48 hours),

(II) transfecting mammalian cells with the reference nucleic acid molecule (FIG. 1A) and measuring the amounts of expressed protein encoded by the ORF of the reference nucleic acid molecule at the same one or two time points after transfection (24 and 48 h),

(iii) calculate the ratio of the amount of protein expressed from the artificial nucleic acid molecule to the amount of protein expressed from the reference nucleic acid molecule,

In this case, the value of the ratio calculated in step (III) is ≥1.

This ratio is associated with high translation efficiency.

All UTR sequences experimentally tested in this example were also referred to as "experimental tested UTR elements" or "tested UTR elements" or "experimental tested UTRs" or "tested UTRs".

The artificial nucleic acid molecule referred to in paragraph (I) contained the tested UTR element. In particular, the artificial nucleic acid molecule (mRNA) did not differ in structure from the reference nucleic acid molecule (FIG. 1A), except that either the 5'-UTR element or the 3'-UTR element of the reference molecule nucleic acid is replaced with the tested UTR element (illustrative examples are presented in Fig. 1B and 1C).

3.1 UTR elements of human origin

Details of the UTR elements of human origin that were subjected to experimental testing are shown in Table 3 below. Experimental testing was performed by comparing with the reference construct detailed above and in the Detailed Design section.

3.2 UTR elements of murine origin

Details of the murine UTRs that were experimentally tested are shown in Table 4 below.

Experimental testing was performed by comparing with the reference design, detailed above and in the section "Detailed description of the design".

3.3 Detailed design description

When testing the 5'-UTR, the 5'-UTR of the reference construct (i.e. 32L4 - PpLuc(GC) - albumin7-A64 - C30 - hSL; Fig. 1) was replaced with the "tested" 5'-UTR - i.e. . either to the wild-type 5'-UTR sequence (mRNA sequence corresponding to the DNA sequence presented in Table 3 or 4, first column), or to an artificial 5'-UTR sequence created on its basis (mRNA sequence corresponding to the DNA sequence presented in table 3 or 4, second column). The remaining elements of the reference construct sequence remained unchanged.

In turn, when testing the 3'-UTR, the 3'-UTR of the reference construct (i.e., 32L4 - PpLuc(GC) - albumin7-A64 - C30 - hSL; Fig. 1) was replaced with the "tested" 3'-UTR - i.e. either to the wild-type 3'-UTR sequence (mRNA sequence corresponding to the DNA sequence presented in Table 3 or 4, third column), or to an artificial 5'-UTR sequence created on its basis (mRNA sequence corresponding to the DNA sequence presented in table 3 or 4, fourth column). The remaining elements of the reference construct sequence remained unchanged.

3.4. Luciferase activity assay to determine translation efficiency

Translation efficiency was experimentally tested in a cell transfection based assay using various mammalian (human, mouse) cell lines, namely HDF, L929, HepG2 and Hela (see FIGS. 2-9).

Human dermal fibroblasts (HDF), L929 cells, HepG2 cells and HeLa cells were seeded in 86-well plates at a density of 1×10 ⁴ cells per well. The next day, cells were washed in Opti-MEM and then transfected using 25 ng per well of mRNA encoding PpLuc complexed with Lipofectamine 2000 in Opti-MEM. Untransfected cells served as controls. To control transfection efficiency, along with PpLuc mRNA (1 ng RrLuc mRNA per well), mRNA encoding Renilla reniformis luciferase (RrLuc) was transfected. 90 min after the start of transfection, the Opti-MEM medium was replaced. At 24 and 48 hours after transfection, the medium was removed by aspiration and the cells were lysed in 100 μl of lysis buffer (passive lysis buffer, Promega). The lysates were stored at -80° until the measurement of luciferase activity.

Luciferase activity was measured in relative light units (RLU) using a Hidex Chameleon plate reader. Ppluc and Rrluc activities were measured sequentially in the same sample using a dual luciferase assay. First, PpLuc activity was measured over a 2 second measurement period using 20 μl of lysate and 50 μl of Beetle-Juice buffer system (PJK GmbH). After 1500 ms, RrLuc activity was measured using 50 μl of Renilla-Juice buffer system (PJK GmbH).

This procedure was performed in 96-well plates, and each UTR element to be tested (in the framework of its containing construct, see section 3.3) was placed in the appropriate position on the 96-well plate. Two 96-well plates were used, one for testing human-derived UTR elements and one for testing murine-derived UTR elements. In each plate, the reference construct (corresponding to the mRNA sequence shown in FIG. 1A) was at positions G12 and H12. The tested UTR elements were in the positions indicated in the last column in Tables 3 and 4, respectively. For example, the construct containing the murine Aarsd1 3'UTR (SEQ ID NO: 186) was at position F12 (see Table 4).

Luciferase activity was measured 24 h (1d, 24 h) after cell transfection and then again 48 h (2d, 48 h) after cell transfection.

The results of this analysis are described below in Section 3.5.

3.5 Results - Identification of UTR Elements Associated with High Translation Efficiency

Expression results using this assay (see Section 3.4 above) measured 24 h (1d, 24 h) and again 48 h (2d, 48 h) after transfection in various mammalian cell lines (HDF, L929, HepG2 and Hela) are shown in FIG. 2-9. It should be noted that two sets of 96-well plates were used here, one for human UTRs and one for mouse UTRs.

While the data in FIG. 2-9 refer to specific positions on a 96-well plate (eg, A1), in tables 3 and 4, specific 5' or 3'-UTR elements tested are assigned to specific positions on a 96-well plate. For example, it can be seen from Table 3 that at position A1 of the 96-well plate with human UTR elements tested, the ZNF460-5'-UTR element (SEQ ID NO: 1) was tested, and so on.

A group of the most preferred UTR elements (which is also referred to as the "final selection" group) was determined. This group contained 5'-UTR elements and 3'-UTR elements that had a translation efficiency, measured after 1 day (24 h), equal to or greater, preferably greater than the efficiency of the reference construct (Fig. 1A) , for at least one of the tested cell lines. This group was divided into four subgroups: (I) 5'-UTR elements of human origin (see Table 5), (II) 3'-UTR elements of human origin (see Table 6), (III) 5'-UTR - elements of mouse origin (see table 7), (IV) 3'-UTR elements of mouse origin (see table 8)

Thus, the 5'-UTR of human genes from the group consisting of ZNF460-5'-UTR, TGM2-5'-UTR, IL7R-5'-UTR, COL8A1-5'-UTR, NDUFS7-5'-UTR, PLK2 -5'-UTR, FBXO32-5'-UTR, ATP5D-5'-UTR, TUBB4B-5'-UTR, ORMDL2-5'-UTR, FSCN1-5'-UTR, CD9-5'-UTR, PYSL2- 5'-UTR, PSMB3-5'-UTR, PSMB6-5'-UTR, KPNA6-5'-UTR, SFT2D2-5'-UTR, LCLAT1-5'-UTR, FBXL18-5'-UTR, SLC35F6-5 '-UTR, VMA21-5'-UTR, SEZ6L2-5'-UTR, PCOLCE-5'-UTR, VTN-5'-UTR, ALDH16A1-5'-UTR, KPNA6-5'-UTR, JUP-5' -UTR, CPN2-5'-UTR, PNPO-5'-UTR, SSSCA1-5'-UTR, POLR2L-5'-UTR, LIN7C-5'-UTR, UQCR10-5'-UTR, PYCRL-5'- UTR, AMN-5'-UTR, MAP1S-5'-UTR, were considered to provide high translation efficiency.

It was also considered that 5'-UTRs having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 137 (or its wild-type equivalent of SEQ ID NO: 3), SEQ ID NO: 138 (or its wild-type equivalent SEQ ID NO: 9), SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 140 (or its wild-type equivalent SEQ ID NO: 17), SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 143 (or its wild type equivalent SEQ ID NO: 31), SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 146 (or its wild-type equivalent SEQ ID NO: 52), SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 144 (or its wild-type equivalent SEQ ID NO: 42), SEQ ID NO: 58, SEQ ID NO: 62, provide high translation efficiency.

Thus, the 3'-UTR of human genes from the group consisting of NDUFS7-3'-UTR, PHGDH-3'-UTR, TSPO-3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B -3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'-UTR, BRAT1-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, SCAND1- 3'-UTR, AMN-3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1-3'-UTR were considered to provide high translation efficiency.

It was also considered that 3'-UTRs having SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173 provide high translation efficiency.

Thus, the 5'-UTR of mouse genes from the group consisting of Dpysl2-5'-UTR, Acox2-5'-UTR, Ubc-5'-UTR, Nudt22-5'-UTR, Pcyox1l-5'-UTR, Ankrd1 -5'-UTR, Tspyl4-5'-UTR, Slc7a3-5'-UTR, Aacs-5'-UTR, Nosip-5'-UTR, Itga7-5'-UTR, Ccnd2-5'-UTR, Ebp- 5'-UTR, Sf3b5-5'-UTR, Fasn-5'-UTR, Hmgcs1-5'-UTR, Osr1-5'-UTR, Lmnb1-5'-UTR, Vma21-5'-UTR, Kif20a-5 '-UTR, Cdca8-5'-UTR, Slc7a1-5'-UTR, Ubqln2-5'-UTR, Prps2-5'-UTR, Shmt2-5'-UTR, Fignl1-5'-UTR, Cad-5' -UTR, Anln-5'-UTR, Slfn9-5'-UTR, Ncaph-5'-UTR, Pole-5'-UTR, Uhrf1-5'-UTR, Gja1-5'-UTR, Fam64a-5'- UTR, Tspan10-5'-UTR, Scand1-5'-UTR, Gpr84-5'-UTR, Cers6-5'-UTR, Cxcr4-5'-UTR, Gprc5c-5'-UTR, Fen1-5'-UTR , Cspg4-5'-UTR, Mrpl34-5'-UTR, Comtd1-5'-UTR, Armc6-5'-UTR, Emr4-5'-UTR, Atp5d-5'-UTR, Csf2ra-5'-UTR, Aarsd1-5'-UTR, Cth-5'-UTR, Tpgs1-5'-UTR, Ccl17-5'-UTR, Alkbh7-5'-UTR, Ms4a8a-5'-UTR were considered to provide high translation efficiency.

Also 5'UTRs having SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO : 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102 , SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 147 (or its wild-type equivalent SEQ ID NO: 109), SEQ ID NO: 148 (or its wild-type equivalent SEQ ID NO: 110), SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO : 149 (or its wild-type equivalent SEQ ID NO: 119), SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 , SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 151 (or its wild-type equivalent SEQ ID NO: 136), SEQ ID NO: 96, SEQ ID NO: 111, SEQ ID NO: 1 50 (based on SEQ ID NO: 120), SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 125 were considered to provide high translation efficiency.

Thus, the 3'-UTR of mouse genes from the group consisting of Acox2-3'-UTR, Ubc-3'-UTR, Slpi-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'-UTR, Slc7a3 -3'-UTR, Cst6-3'-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR, Cad-3'-UTR, Pole- 3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3'-UTR, Ms4a8a-3 '-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik-3'-UTR, Nudt22-3'-UTR, were considered to provide high translation efficiency.

Also 3'UTRs having SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 204 (or its wild type equivalent SEQ ID NO: 192), SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200 , SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 177, were considered to provide high translation efficiency.

Claims (41)

1. An artificial nucleic acid molecule to ensure high translation efficiency of a polypeptide or peptide, containing a. at least one open reading frame - ORF; b. at least one 3'-untranslated region element -3'-UTR element which contains a nucleotide sequence derived from the 3'-UTR of a gene transcript selected from the group consisting of NDUFS7-3'-UTR, PHGDH-3' -UTR, TSPO-3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3'- UTR, BRAT1-3'-UTR, CD9-3'-UTR, CDK9-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, PRSS56-3'-UTR, SCAND1-3'-UTR , AMN-3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1-3'-UTR - preferably all human, and Acox2- 3'-UTR, Ubc-3'-UTR, Slpi-3'-UTR, Pcyox1l-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'-UTR, Slc7a3-3'-UTR, Cst6-3 '-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Plk1-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR, Cad-3'-UTR, Cth-3' -UTR, Pole-3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3'- UTR, Ms4a8a-3'-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik-3'-UTR, Nudt22-3'-UTR , Aarsd1-3'-UTR - preferably all mice others; and in. optionally at least one 5'-untranslated region element-5'-UTR element in which at least one open reading frame is heterologous with respect to the 3'-UTR and/or 5'-UTR element, and where the specified the artificial nucleic acid molecule has a high translation efficiency compared to the translation efficiency of the reference nucleic acid molecule, which is identical to the artificial nucleic acid molecule except for the 3'-UTR element, and the translation efficiency of the artificial nucleic acid molecule and the translation efficiency of the reference nucleic acid molecule are compared using a method comprising the steps of: (I) transfecting mammalian cells with the artificial nucleic acid molecule and measuring the amount of expressed protein encoded by the ORF of the artificial nucleic acid molecule after transfection, (ii) transfecting mammalian cells with the reference nucleic acid molecule and measuring the amounts of expressed protein encoded by the ORF of the reference nucleic acid molecule at the same time point after transfection, (III) calculate the ratio of the amount of protein expressed from the artificial nucleic acid molecule to the amount of protein expressed from the reference nucleic acid molecule, where the value of the ratio calculated in step III is > 1. 2. An artificial nucleic acid molecule according to claim 1, in which at least one 3'-UTR element contains or consists of the nucleotide sequence of SEQ ID NO: 164 or the corresponding RNA sequence. 3. An artificial nucleic acid molecule according to claim 1, in which at least one 3'-UTR element contains or consists of a nucleotide sequence that is at least 95%, even more preferably at least 99% identical to the nucleotide sequence of SEQ ID NO: 164 or the corresponding RNA sequence. 4. The artificial nucleic acid molecule according to claim 1, wherein the 3'-UTR element of the reference nucleic acid molecule has the sequence shown in SEQ ID NO: 207 or the corresponding RNA sequence. 5. An artificial nucleic acid molecule according to any one of paragraphs. 1-4, in which at least one 3'-UTR element and/or at least one 5'-UTR element increases the translation efficiency of said artificial nucleic acid molecule by at least 1.2 times, preferably at least 1.5-fold, more preferably at least 2-fold, even more preferably at least 2.5-fold, compared to the production of a protein from a reference nucleic acid molecule lacking a 3'-UTR and/or at least at least one 5'-UTR, respectively, and/or in which at least one 3'-UTR element and/or at least one 5'-UTR element provides a high translation efficiency of said artificial nucleic acid molecule, exceeding at least 1.5-fold, preferably at least 2-fold, more preferably at least 2.5-fold protein production from a nucleic acid reference molecule lacking a 3'-UTR and/or at least one 5'- UTR respectively. 6. An artificial nucleic acid molecule according to any one of paragraphs. 1-5, in which at least one 5'-UTR element contains or consists of a nucleotide sequence that is derived from a 5'-UTR of a gene transcript selected from the group consisting of ZNF460, TGM2, IL7R, BGN, TK1, RAB3B, CBX6, FZD2, COL8A1, NDUFS7, PHGDH, PLK2, TSPO, PTGS1, FBXO32, NID2, ATP5D, EXOSC4, NOL9, UBB4B, VPS18, ORMDL2, FSCN1, TMEM33, TUBA4A, EMP3, TMEM201, CRIP2, BRAT1, SERPINH1, CD9, DPYSL2, CDK9, TFRC, PSMB3 5'-UTR, FASN, PSMB6, PRSS56, KPNA6, SFT2D2, PARD6B, LPP, SPARC, SCAND1, VASN, SLC26A1, LCLAT1, FBXL18, SLC35F6, RAB3D, MAP1B, VMA21, CYBA, SEZ6L2, PCOLCE, VTN, ALDH16A1, RAVER1, KPNA6, SERINC5, JUP, CPN2, CRIP2, EPT1, PNPO, SSSCA1, POLR2L, LIN7C, UQCR10, PYCRL, AMN, MAP1S, NDUFS7, PHGDH, TSPO, ATP5D, EXOSC4, TUBB4B, TUBA4A, EMP3, CRIP2, BRAT1, CD9, CDK9, PSMB3, PSMB6, PRSS56, SCAND1, AMN, CYBA, PCOLCE, MAP1S, VTN, ALDH16A1 - preferably all human, and Dpysl2, Ccnd1, Acox2, Cbx6, Ubc, Ldlr, Nudt22 , Pcyox1l, Ankrd1, Tmem37, Tspyl4, Slc7a3, Cst6, Aacs, Nosip, Itga7, Ccnd2, Ebp, Sf3b5, Fasn, Hmgcs1, Osr1, Lmnb1, Vma21, Kif20a, Cdca8, Slc7a1, Ubqln2, Prps2, Shmt2, Aurkb, Fignl1, Cad, Anln, Slfn9, Ncaph, Pole, Uhrf1, Gja1, Fam64a, Kif2c, Tspan10, Scand1, Gpr84, Fads3, Cers6, Cxcr4, Gprc5c, Fen1, Cspg4, Mrpl34, Comtd1, Armc6, Emr4, Atp5d, 1110001J03Rik, Csf2ra, Aarsd1, Kif22, Cth, Tpgs1, Ccl17, Ms4ah7, Acox2, Ubc, Slpi, Pcyox1l, Igf2bp1, Tmem37, Slc7a3, Cst6, Ebp, Sf3b5, Plk1, Cdca8, Kif22, Cad, Cth, Pole, Kif2c, Scand1, Gpr84, Tpgs1, Ccl17, Alkbh7, Ms4a8a, Mrpl34, Comtd1, Armc6, Atp5d, 1110001J03Rik, Nudt22, Aarsd1 - preferably all mouse. 7. Artificial nucleic acid molecule according to any one of paragraphs. 1-6, in which at least one 5'-UTR element contains or consists of a nucleotide sequence that is at least about 50% identical, preferably at least about 60%, preferably at least about 70% identical, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of a nucleotide sequence selected from the group consisting of of SEQ ID NO: 1-151 or the corresponding RNA sequence, or in which at least one 5'-UTR element contains or consists of a nucleotide sequence fragment that is at least about 40% identical, preferably at least about 50 %, preferably at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at at least about 90%, even more preferably at least about 95%, even more preferably at least about 99% of a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-151 or the corresponding RNA sequence . 8. Artificial nucleic acid molecule according to any one of paragraphs. 1-7, additionally containing d. a poly(A) sequence and/or a polyadenylation signal that is 3' to the 3' UTR element. 9. An artificial nucleic acid molecule according to any one of paragraphs. 1-8, additionally containing a 5' cap structure, a poly(C) sequence, a histone stem-loop structure, and/or an IRES motif. 10. An artificial nucleic acid molecule according to any one of paragraphs. 1–9, in which the nucleotide sequence contains the 5'-TOP UTR. 11. An artificial nucleic acid molecule according to any one of paragraphs. 1-10, in which the G/C content is at least partially modified in the open reading frame, where preferably the G/C content of the open reading frame is increased compared to the wild-type open reading frame and/or in which the open reading frame contains a codon-optimized region , preferably in which the open reading frame has optimized codons. 12. An artificial nucleic acid molecule according to any one of paragraphs. 1-11 which is an RNA, preferably an mRNA molecule. 13. Expression vector of a polypeptide or peptide containing an artificial nucleic acid molecule according to any one of paragraphs. 1-12. 14. Cell to obtain a polypeptide or peptide containing an artificial nucleic acid molecule according to any one of paragraphs. 1-12 or vector according to item 13. 15. Pharmaceutical composition for vaccination containing an effective amount of an artificial nucleic acid molecule according to any one of paragraphs. 1-12, or a vector according to claim 13, or a cell according to claim 14 and one or more pharmaceutically acceptable carriers, diluents and/or excipients and/or one or more adjuvants. 16. Pharmaceutical composition for gene therapy, containing an effective amount of an artificial nucleic acid molecule according to any one of paragraphs. 1-12, or a vector according to claim 13, or a cell according to claim 14 and one or more pharmaceutically acceptable carriers, diluents and/or excipients and/or one or more adjuvants. 17. The use of an artificial nucleic acid molecule according to any one of paragraphs. 1-12 as a vaccine. 18. The use of an artificial nucleic acid molecule according to any one of paragraphs. 1-12 in gene therapy. 19. Use of the vector according to claim 13 as a vaccine. 20. Use of the vector according to claim 13 in gene therapy. 21. Use of a cell according to claim 14 as a vaccine. 22. Use of a cell according to claim 14 in gene therapy. 23. The use of a pharmaceutical composition according to claim 15 as a vaccine. 24. The use of a pharmaceutical composition according to claim 16 in gene therapy. 25. The method of obtaining an artificial nucleic acid molecule according to any one of paragraphs. 1-12, providing for the union a. at least one open reading frame - ORF; b. at least one element of the 3'-untranslated region -3'-UTR element, which contains a nucleotide sequence that is derived from the 3'-UTR of a human gene transcript selected from the group consisting of NDUFS7-3'-UTR, PHGDH- 3'-UTR, TSPO-3'-UTR, ATP5D-3'-UTR, EXOSC4-3'-UTR, TUBB4B-3'-UTR, TUBA4A-3'-UTR, EMP3-3'-UTR, CRIP2-3 '-UTR, BRAT1-3'-UTR, CD9-3'-UTR, CDK9-3'-UTR, PSMB3-3'-UTR, PSMB6-3'-UTR, PRSS56-3'-UTR, SCAND1-3' -UTR, AMN-3'-UTR, CYBA-3'-UTR, PCOLCE-3'-UTR, MAP1S-3'-UTR, VTN-3'-UTR, ALDH16A1-3'-UTR - preferably all human, and Acox2-3'-UTR, Ubc-3'-UTR, Slpi-3'-UTR, Pcyox1l-3'-UTR, Igf2bp1-3'-UTR, Tmem37-3'-UTR, Slc7a3-3'-UTR, Cst6 -3'-UTR, Ebp-3'-UTR, Sf3b5-3'-UTR, Plk1-3'-UTR, Cdca8-3'-UTR, Kif22-3'-UTR, Cad-3'-UTR, Cth- 3'-UTR, Pole-3'-UTR, Kif2c-3'-UTR, Scand1-3'-UTR, Gpr84-3'-UTR, Tpgs1-3'-UTR, Ccl17-3'-UTR, Alkbh7-3 '-UTR, Ms4a8a-3'-UTR, Mrpl34-3'-UTR, Comtd1-3'-UTR, Armc6-3'-UTR, Atp5d-3'-UTR, 1110001J03Rik-3'-UTR, Nudt22-3' -UTR, Aarsd1-3'-UTR - preferably all murine, and in. optionally at least one element of the 5'-untranslated region of the -5'-UTR element, wherein at least one open reading frame is heterologous with respect to the 3'-UTR and/or 3'-UTR element. 26. The method of claim 25, wherein the vector of claim 13 is used. 27. A method for providing improved translation efficiency of a polypeptide or peptide, comprising obtaining an artificial nucleic acid molecule according to any one of paragraphs. 1-12. 28. Kit for vaccination containing an artificial nucleic acid molecule according to any one of paragraphs. 1-12, or a vector according to claim 13, or a cell according to claim 14, or a pharmaceutical composition according to claim 15 and instructions for its use. 29. Set for gene therapy containing an artificial nucleic acid molecule according to any one of paragraphs. 1-12, or a vector according to claim 13, or a cell according to claim 14, or a pharmaceutical composition according to claim 16 and instructions for its use. 30. The kit according to claim 28 or 29, which contains an adjuvant, a means for transfecting a cell, a means for administering a pharmaceutical composition, a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable solution for dissolving or diluting an artificial nucleic acid molecule, or a vector, or cells, or pharmaceutical composition. 31. The use of a 3'-UTR element contained in an artificial nucleic acid molecule according to any one of paragraphs. 1-12 or in the vector according to claim 13, to ensure high translation efficiency of the polypeptide or peptide.

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