RU2774415C1 - Artificial nucleic acid molecules - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: artificial nucleic acid molecule for increasing protein yield is described, containing: a. at least one open reading frame (ORF); and b. at least one element of 3’-untranslated region (hereinafter – 3’-UTR element) that contains a nucleotide sequence originating from 3’-UTR ribosomal protein gene or from an option of 3’-UTR ribosomal protein gene selected from a group that consists of ribosomal protein S9 (RPS9), ribosomal protein S19 (RPS19), ribosomal protein S21 (RPS21), ribosomal protein S27 (RPS27), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29) and ribosomal protein L35a (RPL35A). In addition, a vector containing the specified nucleic acid, and a cell containing the specified nucleic acid or vector are described in the claim. An appropriate method for increasing protein yield is also described.

EFFECT: invention makes it possible to increase, stabilize and/or prolong the expression of a protein with a nucleotide sequence containing 3'-UTR element.

35 cl, 9 dwg, 5 tbl, 1 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 optionally a poly(A) sequence and/or a polyadenylation signal. The invention also relates to a vector containing a 3'-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 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 allow the development of highly specific and individual approaches for the treatment of a wide variety of diseases. Such treatment approaches may primarily target congenital genetic diseases, malignant or tumor-related diseases, and 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, transcription factors, signal 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 that function in a forward direction relative to the disturbed 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 the isolation of specific cells from the body of the patient to be treated, the subsequent ex vivo transfection of these cells and reintroduction of 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. Thus, 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 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 replaced with 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 (also referred to as the poly(A) tail or poly(A) sequence), are usually 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 event 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, Volume 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, Volume 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., Volume 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.

Regardless of the factors affecting mRNA stability, the efficiency of translation of the introduced nucleic acid molecules by the 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 an mRNA, characterized by increased expression of the corresponding protein encoded by said nucleic acid molecule. One of the specific objectives of the invention was to create mRNA, allowing to increase the efficiency of translation of the corresponding encoded protein. 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 problems underlying the present invention are solved with the help of the objects of the invention presented in the claims.

The present invention was made with government support under Agreement No. HR0011-11-3-0001 signed by DARPA (Defense Advanced Research Projects Agency). The government has certain rights to the invention.

For purposes of clarity and readability, the following definitions are given below. All specifications specified in these definitions apply to each and all embodiments of the invention. In the context of these embodiments, further definitions and explanations may be 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 cell types 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 creates 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. Since the rearrangement of a gene results in an irreversible change in the DNA of each cell, then all progeny of such a 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 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 to MHC 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.

Artificial nucleic acid molecule: An artificial nucleic acid molecule is generally understood to mean a nucleic acid molecule, such as DNA or RNA, that does not occur naturally. 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: Bicistronic or polycistronic RNA is generally 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, usually a modified nucleotide structure, that normally "terminates" 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 amino acids or even more, 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 RNA 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) introducing a nucleic acid, preferably an RNA molecule, as defined herein, directly to a patient - by any route of administration - or in vitro into isolated cells/tissues of a patient, resulting in transfection of the cells patient, 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 a fragment 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 mRNA.

Humoral Immunity/Humoral Immune Response: Humoral immunity generally refers to the production of antibodies and not necessarily the secondary 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).

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-I-like receptor ligand, immunostimulatory nucleic acid that is, an immunostimulatory RNA (isRNA), CpG DNA, an antibacterial agent, or an 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 generally an adenine 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 takes place 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.

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 derived 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 the sequence corresponding to the open reading frame and 3' 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): The 3'UTR is typically the portion of an artificial nucleic acid molecule that is located 3'-side (i.e., forward) of the open reading frame and that 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 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", for example, "3'UTR of a ribosomal protein 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.

5'-untranslated region (5'UTR): By 5'UTR, as a rule, understand a specific segment of messenger RNA (mRNA). It is located on the 5' side of the mRNA open reading frame. Typically, the 5'UTR starts at the site of transcription initiation and ends one nucleotide before the start codon of the open reading frame. 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. In the context of the present invention, the 5'UTR corresponds to the mature mRNA sequence located between the 5' cap and the start codon. Preferably, the 5'UTR corresponds to a sequence that extends from the nucleotide located on the 3' side of the 5' cap, preferably from the 3' side of the nucleotide immediately adjacent to the 5' cap, to the nucleotide located on the 5' side 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", for example, "5'UTR of the TOP 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 5'UTR sequence. In embodiments of the invention, the specified 5'-UTR may be at the 5'end of the ORF. Its length is usually less than 500, 400, 300, 250 or less than 200 nucleotides. In other embodiments of the invention, its length may be in the range of at least 10, 20, 30 or 40, preferably up to 100 or 150 nucleotides.

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 at one nucleotide located 5'-direction relative to the first purine nucleotide located upstream 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 refers to 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 segment consisting of pyrimidine nucleotides of 3-30 nucleotides in length. 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 an appropriate sequence such as an mRNA of the invention, the 5'UTR element of an mRNA of the invention in the invention, or a nucleotide sequence that is derived from the 5'UTR of the TOP gene as defined 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, preferably not referred to as "TOP-motive".

TOR gene: TOR genes. typically 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 5'UTR sequence of the mature mRNA derived from the TOP gene, which preferably extends from the nucleotide adjacent 3'-side of the 5'-cap to the nucleotide adjacent 5'- side to the start codon. The 5'UTR of the TOP gene generally does not contain any start codons, preferably no upstream AUG (uAUG) or upstream open reading frames (uOPC). 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 5'UTR lengths of TOP genes can range 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'UTR 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 (e.g. ATG), these sequences are shown in SEQ ID NO: 1-1363 , SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 14221-1363 patent applications PCT/EP 2012/002448 WO 2013/143700 or are homologues or variants thereof, the description of these documents is included in this description as links. 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 term "5'UTR of the TOP gene" preferably refers to the 5'UTR of the naturally occurring TOP gene.

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 of the 3'-untranslated region (3'-UTR element) containing or consisting of a nucleotide sequence that is derived from the 3'-UTR of the ribosomal protein gene.

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. The term "3'UTR element" preferably refers to a nucleotide sequence which is the 3'UTR of an artificial nucleotide sequence, such as an artificial mRNA, or which encodes the 3'UTR of an artificial nucleic acid molecule. 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 a template for transcription of the 3'-UTR of an mRNA. Thus, the 3'UTR element is preferably 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.

The term "ribosomal protein gene" generally refers to a gene encoding a ribosomal protein. In the context of the present description, this concept refers to any ribosomal protein gene, regardless of the species from which it originates. Specifically, the term refers to a eukaryotic ribosomal protein gene. In addition, in the context of the invention, the term "ribosomal protein gene" can also refer to a gene similar to the ribosomal protein gene, either in terms of structure or in terms of function. In particular, the term includes "ribosomal protein gene-like", pseudogenes, and genes having sequence or structure characteristics, primarily in their 3'-UTR region, of a ribosomal protein gene. Preferably, the term refers to a vertebrate ribosomal protein gene, more preferably a mammalian ribosomal protein gene, even more preferably a primate ribosomal protein gene, especially a human ribosomal protein gene. In addition, the term "ribosomal protein gene" also includes a rodent ribosomal protein gene, especially a mouse ribosomal protein gene. Examples of ribosomal protein genes in the context of the invention are (but not limited to) ribosomal protein L9 (RPL9), ribosomal protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosomal protein L6 (RPL6) genes. ), 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 protein L30 (RPL30), ribosomal protein L27a (RPL27A), ribosomal protein L28 (RPL28), ribosomal protein L29 (RPL29), ribosomal protein L31 (RPL31), ribosomal o 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 large subunit protein, 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), ribosomal protein S15 (RPS15), ribosomal protein S15a (RPS15A), ribosomal protein S16 (RPS16), ribosomal protein S19 (RPS19) , ribosomal protein S20 (RPS20), ribosomal ribosomal protein S21 (RPS21), ribosomal protein S23 (RPS23), ribosomal protein S25 (RPS25), ribosomal protein S26 (RPS26), ribosomal protein S27 (RPS27), ribosomal protein S27a (RPS27a), ribosomal protein S28 (RPS28), ribosomal 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 protein SA (RPSA), fusion product 1: ubiquitin residue A-52-ribosomal protein (UBA52), ubiquitously expressed gene (FAU) of mouse sarcoma virus Finkel-Biskis-Reilly (FBR-MuSV), ribosomal protein L22-like 1 (RPL22L1), ribosomal protein S17 (RPS17), ribosomal protein L39-like (RPL39L), ribosomal protein L10-like (RPL10L), ribosomal protein L36a-like (RPL36AL), ribosomal protein L3-like (RPL3L), ribosomal protein S27-like (RPS27L), ribosomal protein L26-like 1 (RPL26L1), ribosomal protein L7-like 1 (RPL7L1), ribosomal protein L13a pseudogene (RPL13AP), ribosomal protein pseudogene 8 L37a (RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomal protein 11 S26 pseudogene (RPS26P11), ribosomal protein L39 pseudogene 5 (RPL39P5), ribosomal protein P0 large subunit pseudogene 6 (RPLP0P6), and ribosomal protein L36 pseudogene 14 (RPL36P14) ). Preferably, the term "ribosomal protein gene" refers to one of the above genes that is derived from mammals, preferably from Homo sapiens or Mus musculus.

Preferably, at least one open reading frame and at least one 3'UTR element are heterologous. In the context of the present description, the term "heterologous" means that two sequence elements contained in an artificial nucleic acid molecule, such as an open reading frame and a 3'-UTR element, do not occur naturally (in nature) in the considered combination. Preferably the 3'-UTR element is from a different gene from the gene from which the open reading frame is derived. For example, the ORF may be derived from a gene other than the gene from which the 3'UTR element is derived, such as encoding a different protein or the same protein but from a different species, and so on. Preferably, the open reading frame does not encode a ribosomal protein. In a preferred embodiment, the ORF does not encode a human ribosomal protein or a plant ribosomal protein (primarily from Arabidopsis), especially 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. In the most preferred embodiment of the invention, 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 is heterologous 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. In the most preferred embodiment of the invention, the artificial nucleic acid molecule contains an ORF, a 3'-UTR and a 5'-UTR, 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'-UTR). In another preferred embodiment of the invention, the 3'UTR is not derived from a 3'UTR of a viral gene or is not derived from a virus.

Preferably, at least one 3'UTR element is operably linked to the ORF. This means that preferably the 3'-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 an encoded peptide or protein, or the function of stabilizing an artificial nucleic acid molecule. Preferably, the ORF and the 3'UTR element are associated in the 5'→3' direction. Thus, preferably, the artificial nucleic acid molecule contains the structure 5'-ORC-(optional)-linker-3'-UTR-element-3', in which 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 contains a nucleotide sequence derived from a 3'-UTR of a eukaryotic ribosomal protein gene, preferably from a 3'-UTR of a vertebrate ribosomal protein gene, more preferably from a 3'-UTR of a mammalian ribosomal protein gene, still more preferably from a 3'UTR of a primate ribosomal protein gene, especially a human ribosomal protein gene, or from a 3'UTR of a rodent ribosomal protein gene, especially a mouse ribosomal protein gene.

In a preferred embodiment of the invention, at least one 3'-UTR element contains or corresponds to a nucleotide sequence that is derived from the 3'-UTR sequence of a transcript selected from the group consisting of

The phrase "a nucleotide sequence which is derived from the 3'UTR of a ribosomal protein gene" preferably refers to a nucleotide sequence based on the 3'UTR of a ribosomal protein gene or a fragment or region thereof. Said phrase includes sequences corresponding to the entire 3'-UTR sequence, i.e. the full-length sequence of the 3'-UTR of the ribosomal protein gene, and sequences corresponding to a fragment of the sequence of the 3'-UTR of the ribosomal protein gene. Preferably, the 3'UTR fragment of the ribosomal protein gene consists of a contiguous segment of nucleotides corresponding to the contiguous segment of nucleotides of the full-length 3'UTR of the ribosomal protein gene, which comprises 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%, even more preferably at least 70%, even more preferably at least 80% and most preferably at least 90% full-length sequence of the 3'-UTR of the ribosomal protein gene. 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 the translation of the ORF linked to the 3'UTR or a fragment thereof. The term "3'UTR of a ribosomal protein gene" preferably refers to the 3'UTR of a naturally occurring ribosomal protein gene.

The terms "variant 3'-UTR of the ribosomal protein gene" and "variant thereof" in the context of the 3'-UTR of the ribosomal protein gene refer to the 3'-UTR variant of the naturally occurring ribosomal protein gene, preferably the 3'-UTR variant of the ribosomal protein gene vertebrates, more preferably the 3'UTR variant of the mammalian ribosomal protein gene, even more preferably the 3'UTR variant of the 3'UTR variant of the primate ribosomal protein gene, especially the human ribosomal protein gene shown above. Such a variant may be a modified 3'-UTR of a ribosomal protein gene. For example, a variant 3'-UTR may have one or more deletions, insertions, additions, and/or substitutions of nucleotides compared to the naturally occurring 3'-UTR from which the variant is derived. Preferably the variant 3'-UTR of the ribosomal protein gene is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80% %, even more preferably at least 90%, most preferably at least 95% identical to the naturally occurring 3'-UTR from which the variant originates. Preferably the variant is a functional variant presented in the present description.

The phrase "a nucleotide sequence which is derived from a ribosomal protein gene 3'-UTR variant" preferably refers to a nucleotide sequence based on a ribosomal protein gene 3'-UTR sequence variant or a fragment or region thereof as shown above. The phrase includes sequences corresponding to the complete sequence of the 3'-UTR variant of the ribosomal protein gene, i.e. the full-length sequence of the 3'-UTR variant of the ribosomal protein gene, and sequences corresponding to a fragment of the 3'-UTR sequence variant of the ribosomal protein gene. Preferably, the ribosomal protein gene variant 3'-UTR fragment consists of a contiguous segment of nucleotides corresponding to the contiguous nucleotide segment of the full-length ribosomal protein 3'-UTR gene sequence variant, which comprises at least 20%, preferably at least 30%, more preferably at least 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 size option 3 '-UTR of the ribosomal protein gene. 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 a 3'-UTR fragment, a 3'-UTR variant, or a 3'-variant fragment The UTR of the ribosomal protein gene performs at least one, preferably more than one, function of the naturally occurring 3' UTR of the ribosomal protein gene 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 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 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 or lacks a 3'-UTR. , and/or the function of increasing, stabilizing and/or prolonging the production of a protein from an mRNA, preferably in a mammalian cell, such as a human cell, compared to an mRNA that contains a reference 3'-UTR or lacks a 3'-UTR, and /or the function of increasing protein production from an mRNA, preferably in a mammalian cell such as a human cell, compared to an mRNA that contains a reference 3'-UTR or lacks a 3'-UTR. The reference 3'-UTR may be, for example, the naturally occurring 3'-UTR in combination with the ORF. In addition, the functional variant, functional fragment or functional variant of the 3'-UTR fragment of the ribosomal protein gene preferably does not significantly reduce the efficiency of translation of mRNA that contains such a variant, fragment or variant of the 3'-UTR fragment, compared to the 3'- The wild-type 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 variant", "functional fragment" or "functional fragment variant" of the 3'-UTR of a ribosomal protein gene is to increase, stabilize and/or prolong protein production by expression of an mRNA carrying a functional variant, a functional fragment or the functional version of the snippet presented above.

Preferably, the efficiency of one or more functions performed by the functional variant, functional fragment, or functional fragment variant, such as the efficiency of stabilizing mRNA and/or protein production and/or the efficiency of increasing protein production, 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%, more more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% compared to the mRNA and/or protein production stabilization efficiency and/or protein production enhancement efficiency provided by naturally occurring 3'-UTR of the ribosomal protein gene from which the variant, fragment, or variant is derived t fragment.

In the context of the present invention, a 3'-UTR fragment of a ribosomal protein gene or a 3'-UTR variant of a ribosomal protein gene 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 ribosomal protein 3'UTR gene fragment or ribosomal protein 3'UTR gene variant is a functional fragment as described above. In a preferred embodiment, the 3'-UTR of the ribosomal protein gene, or fragment or variant thereof, is 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 20 to 70 nucleotides.

Preferably, at least one 3'-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 of the ribosomal protein gene.

In a preferred embodiment of the invention, at least one 3'-UTR element of the artificial nucleic acid molecule of the present invention improves the stability of the artificial nucleic acid molecule, for example, increases the stability of the mRNA of the present invention, compared with the corresponding nucleic nucleic reference -acid lacking a 3'UTR or containing a reference 3'UTR, such as the naturally occurring 3'UTR in combination with an ORF. Preferably, at least one 3'-UTR element of the artificial nucleic acid molecule of the present invention improves the stability of protein production from the artificial nucleic acid molecule of the present invention, for example, mRNA of the present invention, compared with an appropriate nucleic acid that lacks a 3'UTR or that contains a reference 3'UTR, such as a naturally occurring 3'UTR in combination with an ORF. Preferably, at least one 3'-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, with the mRNA of the present invention, compared with the corresponding nucleic acid an acid that lacks a 3'UTR or that contains a reference 3'UTR, such as a naturally occurring 3'UTR in combination with an ORF. Preferably, at least one 3'-UTR element of the artificial nucleic acid molecule of the present invention increases protein expression and/or overall protein production from the artificial nucleic acid molecule of the present invention, for example, mRNA of the present invention , compared to a corresponding nucleic acid that lacks a 3'UTR or that contains a reference 3'UTR, such as a naturally occurring 3'UTR in combination with an ORF. Preferably, at least one 3'-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 that lacks the 3'-UTR or contains the reference -3'UTR, such as the naturally occurring 3'UTR in combination with ORF. Even more preferably, the 3'-UTR increases the efficiency of translation compared to the efficiency of translation of the protein encoded by the corresponding ORF in natural conditions.

The term "corresponding nucleic acid molecule" or "reference nucleic acid molecule" in this context means that the reference nucleic acid molecule, except for differences in the 3'-UTR, is comparable, preferably identical to the artificial nucleic acid molecule proposed in the present invention containing 3'-UTR element.

The term "stabilizes and/or prolongs the production of a protein" 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 stabilized and/or prolonged compared to the production of a reference protein. a nucleic acid molecule, such as a reference mRNA, for example, which contains a reference 3'UTR or lacks a 3'UTR, preferably in a mammalian expression system such as HeLa or HDF cells. Thus, a protein produced from an artificial nucleic acid molecule, such as an artificial mRNA, is detectable for a longer period of time than the time during which it can be detected in the case of a protein produced from a reference nucleic acid molecule. In other words, the amount of protein produced from an artificial nucleic acid molecule, such as an artificial mRNA, measured as a function of time, falls below a threshold value at a later point in time than the amount of protein produced from a reference nucleic acid molecule. , such as a reference mRNA measured over time. Such a threshold value can be, for example, the amount of protein measured at the initial phase of expression, for example, 1, 2, 3, 4, 5 or 6 hours after the initiation of expression, for example, after transfection with a nucleic acid molecule.

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

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 1.5 times, preferably at least 2 times, more preferably at least 2.5 times compared to a period of protein production from a reference nucleic acid molecule that lacks a 3'-UTR or contains a reference 3'-UTR.

Said effect of prolonging protein production can be assessed based on (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 HeLa or HDF cells, as a function of 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 hours after initiation of expression, for example, 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 falls below the amount of protein detected, e.g. 1, 2, 3, 4, 5, or 6 hours after initiation of expression, with the indicated time point defined for a nucleic acid molecule that lacks a 3'-UTR or contains a reference 3'-UTR.

Preferably, said stabilizing and/or prolonging 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, is at least equal to the amount of protein, produced from a reference nucleic acid molecule that lacks a 3'-UTR or that contains a reference 3'-UTR, such as a 3'-UTR that occurs naturally with the ORF of the artificial nucleic acid molecule. Thus, the present invention provides an artificial nucleic acid molecule that provides prolonged and/or stabilized protein production in a mammalian expression system, such as mammalian cells, for example, in HeLa or HDF 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 equal to the total amount of protein produced, for example, over a specified period of time, from a reference nucleic acid molecule that lacks 3 '-UTR or which contains a reference 3'-UTR, such as the 3'-UTR naturally occurring with the ORF of an artificial nucleic acid molecule.

Thus, "stabilized protein expression" preferably means that there is a more uniform production of protein from the artificial nucleic acid molecule of the present invention over a predetermined period of time, for example within 24 hours, more preferably within 48 hours, yet more preferably within 72 hours, compared to a reference nucleic acid molecule, for example, which contains a reference 3'-UTR or lacks a 3'-UTR. Therefore, the level of protein production, for example, in a mammalian system, from an artificial nucleic acid molecule containing a 3'-UTR element of the present invention, for example, from an mRNA of the present invention, preferably does not decrease to the level found for the reference a nucleic acid molecule, such as the reference mRNA mentioned above. For example, the amount of protein (encoded by ORF) detected 6 hours after initiation of expression, for example 6 hours after transfection of a cell, such as a mammalian cell, with an artificial nucleic acid molecule of the present invention, may be comparable to the amount of protein detected through 48 hours after initiation of expression, eg 48 hours after transfection. Thus, the ratio of the amount of a protein encoded by an ORF, such as a reporter protein, for example, luciferase, detected 48 hours after initiation of expression, for example, 48 hours after transfection, to the amount of protein detected 6 hours after initiation of expression, for example, after 6 hours after transfection, preferably at least about 0.4, more preferably at least about 0.5, more preferably at least about 0.6, even more preferably at least about 0.7. For a nucleic acid molecule of the present invention, the ratio is preferably from about 0.4 to about 4, preferably from about 0.65 to about 3, more preferably from about 0.7 to 2. For a corresponding reference nucleic acid molecule, for example , mRNA that contains a reference 3'-UTR or lacks a 3'-UTR, said ratio may be, for example, from about 0.05 to about 0.3.

Thus, the present invention provides an artificial nucleic acid molecule containing an ORF and a 3'-UTR element as described above, wherein the ratio of the amount of (reporter) protein, for example, the amount of luciferase detected 48 hours after initiation of expression, to the amount of (reporter) ) protein detected 6 hours after initiation of expression, preferably in a mammalian expression system such as mammalian cells, for example in HeLa cells, preferably greater than about 0.4, more preferably greater than about 0.5, more preferably greater than about 0, 6, even more preferably greater than about 0.7, for example, from about 0.4 to about 4, preferably from about 0.65 to about 3, more preferably from about 0.7 to 2, with the total amount of protein being preferred, produced from said artificial nucleic acid molecule, for example, over a period of 48 hours, at least equal to the total amount of protein produced, for example, during a specified period of time, from a reference nucleic acid molecule that lacks a 3'-UTR or that contains a reference 3'-UTR, such as a 3'-UTR naturally occurring with ORF of an artificial nucleic acid molecule. In a preferred embodiment, the present invention provides an artificial nucleic acid molecule comprising an ORF and a 3'-UTR element as described above, wherein the ratio of the amount of (reporter) protein, e.g., the amount of luciferase detected 72 hours after initiation of expression, to the amount reporter) protein detected 6 hours after initiation of expression, preferably in a mammalian expression system such as mammalian cells, e.g. in HeLa cells, preferably greater than about 0.4, more preferably greater than about 0.5, more preferably greater than about 0 ,6, even more preferably greater than about 0.7, for example, from about 0.4 to 1.5, preferably from about 0.65 to about 1.15, more preferably from about 0.7 to 1.0, with the total amount of protein produced from said artificial nucleic acid molecule, for example, over a period of time its 72 hours is at least equal to the total amount of protein produced, for example, during the specified time period, from a reference nucleic acid molecule that lacks a 3'-UTR or that contains a 3'-UTR, such as a 3'-UTR , which occurs naturally with the ORF of an 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 greater than the level of protein found at the same time point after initiation of expression, e.g. after transfection with a nucleic acid reference molecule, such as a reference mRNA that contains or lacks a reference 3'-UTR 3 '-UTR. 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 or in which there is no 3'-UTR. 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.

In one embodiment, "increased total protein production" or "increased total protein production" from an artificial nucleic acid molecule of the invention refers to increased/increased protein production over the period of time that protein is produced from an artificial nucleic acid molecule. , preferably in a mammalian expression system such as mammalian cells, eg HeLa or HDF cells, compared to a reference nucleic acid molecule lacking a 3'-UTR or containing a reference 3'-UTR. 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 invention provides an artificial nucleic acid molecule that is characterized by increased expression of the encoded protein compared to the corresponding nucleic acid molecule that lacks at least one 3'-UTR element or that contains a 3'-UTR ("nucleic reference acid") containing a nucleotide sequence derived from the 3'-UTR of the ribosomal protein gene or from the variant 3'-UTR of the ribosomal protein gene. To evaluate in vivo protein production from the artificial nucleic acid molecule of the invention, the expression of the encoded protein after injection/transfection of the target cell/tissue with the artificial nucleic acid molecule of the invention is determined and compared with protein expression induced by the reference nucleic 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, 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. A few hours or a few days (eg, 6, 12, 24, 48, or 72 hours) after initiation of expression or after the 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, etc., as is known in the art) and carry out 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, FACS analysis can also determine the amount of protein prior to lysis of harvested cells or in parallel using aliquots.

If you want to measure the total amount of protein over a certain period of time, then you can collect tissue or cells at several points in time after the introduction of an artificial nucleic acid molecule (for example, 6, 12, 24, 48 and 72 hours after the initiation of expression or after the introduction of the nucleic acid molecule acid; usually taken from the body of other experimental animals), and determine the amount of protein at each time point as described 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.

Said increase in the stability of the artificial nucleic acid molecule, said increase in protein production stability, said prolongation of protein production and/or said increase/increase in protein expression and/or total protein production are preferably determined by comparison with a corresponding reference nucleic acid molecule lacking the 3' -UTR, for example, an mRNA that lacks a 3'UTR, or a reference nucleic acid molecule that contains a reference 3'UTR, such as a naturally occurring 3'UTR with an ORF as described above.

Stabilizing effect on mRNA and/or protein production and efficiency, and/or increasing protein production effect and efficiency of variants, fragments and/or fragments of variants of the 3'-UTR gene of the ribosomal protein, as well as stabilizing effect on mRNA and/or protein production and efficiency , and/or the protein production-enhancing activity and potency of at least one 3'UTR element of the artificial nucleic acid molecule of the present invention can be determined by any suitable method known to one skilled in the art. For example, one can create artificial mRNA molecules containing the coding sequence/open reading frame (ORF) for a reporter protein such as luciferase and not containing a 3'-UTR, containing a 3'-UTR derived from a naturally occurring ribosomal protein gene, 3'-UTR derived from a reference gene (i.e., a reference 3'-UTR, such as the naturally occurring 3'-UTR with ORF), as a 3'-UTR variant of the 3'-UTR of the ribosomal gene protein, as a 3'-UTR fragment of a naturally occurring ribosomal protein gene, or as a 3'-UTR fragment of a 3'-UTR variant of a ribosomal protein gene. Such mRNAs can be generated, for example, by in vitro transcription of appropriate vectors, such as plasmid vectors, for example, containing the T7 promoter and a sequence encoding the corresponding mRNA sequences. The generated mRNA molecules can be transfected into cells using a transfection method suitable for mRNA transfection, for example, they can be introduced by electroporation into mammalian cells such as HELA cells, and samples can be analyzed at certain time points after transfection, for example, after 6, 24 , 48 and 72 h after transfection. These samples can be analyzed to estimate mRNA amounts and/or protein amounts using methods well known to those skilled in the art. For example, the amount of reporter mRNA present in the cells at the sampling times 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, FACS analysis, 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 protein level detected 48 hours after transfection to the protein level detected 6 hours after transfection. Preferably, the value of such a ratio is greater than 1, i. e. protein expression at a later time point is greater than protein expression at an earlier time point. If the value of this ratio is less than 1, then the protein is more stable the closer the indicated value is to 1. Such measurements can, of course, also be carried out after 72 hours or more, and to determine the stability of protein expression, the ratio of the protein level detected after 72 h after transfection to the protein level detected 6 h after transfection.

In a preferred embodiment, the 3'UTR element of the artificial nucleic acid molecule of the present invention is derived from the 3'UTR region of a gene encoding a ribosomal protein, preferably from the 3'UTR region of a ribosomal protein L9 (RPL9) gene. , 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 protein L30 (RPL30), ri bosomal 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, PO (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), ribosomal protein S15 (RPS1 5), 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 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 LlOa (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 protein SA (RPSA) or rib osomal protein S17 (RPS17). In an alternative embodiment, the 3'-UTR element may be derived from a gene encoding a ribosomal protein or from a gene selected from the fusion product 1 genes of ubiquitin residue A-52-ribosomal protein (UBA52), a ubiquitously expressed (FAU) mouse sarcoma virus Finkel-Bisquis-Reilly protein 1, ribosomal protein L22 like (RPL22L1), ribosomal protein L39 like protein (RPL39L), ribosomal protein L10 like protein (RPL10L), ribosomal protein L36a like protein (RPL36AL), protein like ribosomal protein L3 (RPL3L), ribosomal protein S27 like protein (RPS27L), ribosomal protein L26 like protein 1 1 (RPL26L1), ribosomal protein 1 like protein L7 (RPL7L1), ribosomal protein n3a pseudogene (RPL13AP), pseudogene 8 ribosomal protein L37a (RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomal protein S26 pseudogene 11 (RPS26P11), ribosomal protein L39 pseudogene 5 (RPL39P5), ri pseudogene 6 bosomal protein, large subunit, PO (RPLP0P6); and ribosomal protein L36 pseudogene 14 (RPL36P14). In addition, the 3'UTR element of the artificial nucleic acid molecule of the present invention is preferably derived from the 3'UTR region of a gene selected from the group consisting of genes for a protein like ribosomal protein S4 (RPS41), a putative 60S subunit. ribosomal protein L13a, putative subunit of the 60S protein like ribosomal protein L37a, putative subunit of the 40S protein like ribosomal protein S10, putative subunit of the 40S protein 1 like ribosomal protein S26, putative subunit of the 60S protein 5 like ribosomal protein L39, or subunit of the 60S protein , similar to the acidic ribosomal protein P0.

In the most preferred embodiment of the invention, the 3'UTR element of the artificial nucleic acid molecule of the present invention is derived from the 3'UTR region of the gene encoding the ribosomal protein, preferably from the 3'UTR region of the ribosomal protein L3 gene (RPL3 ), ribosomal protein L11 (RPL11), ribosomal protein L13 (RPL13), ribosomal protein L23 (RPL23), ribosomal protein L23a (RPL23A), ribosomal protein L26 (RPL26), ribosomal protein L27 (RPL27), ribosomal protein L35a (RPL35A) , ribosomal protein L38 (RPL38), ribosomal protein S4, X-linked (RPS4X), ribosomal protein S8 (RPS8), ribosomal protein S9 (RPS9), ribosomal protein S13 (RPS13), ribosomal protein S19 (RPS19), ribosomal protein S21 (RPS21), ribosomal protein S23 (RPS23), ribosomal protein S27 (RPS27), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), ribosomal protein L36 (RPL36), ribosomal protein L36a (RPL36A), ribosome omal protein S18 (RPS18) or ribosomal protein S17 (RPS17). In another preferred embodiment of the invention, the 3'-UTR element may be derived from a gene encoding a ribosomal protein or from a gene selected from the fusion product 1 genes of the ubiquitin residue A-52-ribosomal protein (UBA52), a ubiquitously expressed (FAU) mouse virus Finkel-Biskis-Reilly sarcoma or protein 1 like ribosomal protein L22 (RPL22L1).

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 1, 2, 3, 4, 5, 10, 15, 20, 30% or 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%, most preferably 100% of the nucleotide sequence of the 3'-UTR of the ribosomal protein gene, such as the nucleotide sequence shown in SEQ ID NO: 10-115 or the corresponding RNA sequence:

Further 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 1, 2, 3, 4, 5, 10, 15%, 20%, 30% or 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 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 a ribosomal protein gene, such as the nucleotide sequence shown in SEQ ID NO: 116-205 or corresponding RNA sequence:

In a preferred embodiment of the invention, 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%, 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 3'-UTR sequence of the ribosomal small subunit protein 9 (RPS9) gene. 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% SEQ ID NO: 1 or SEQ ID NO: 2:

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 a ribosomal protein gene, such as the 3'UTR having the sequence shown in SEQ ID NOs: 10-205, where the fragment is preferably functional fragment or functional fragment of the variant 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 fragments of variants are functional variants, functional fragments or functional fragments of the variants described above, which have at least one function inherent in the nucleotide sequence shown in SEQ ID NO: 10-205, such as stabilization of an artificial molecule nucleic acid according to the invention, stabilization and/or prolongation of protein expression from an artificial nucleic acid molecule according to the invention, and/or increase in protein production, 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% of the stabilization efficiency and/or the efficiency of increasing protein production from the artificial nucleic acid molecule, with containing the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

Preferably, at least one 3'-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 the 3'UTR element may be 500 nucleotides or less, such as 400, 300, 200, 150 or 100 nucleotides. In other embodiments, the upper limit may be selected from the range of 50 to 100 nucleotides. For example, a fragment or variant 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.

In addition, the artificial nucleic acid molecule of the present invention may contain more than one 3'-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 the individual 3'-UTR elements may be the same or 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 derived from the 3'-UTR a ribosomal protein gene, for example, from the sequence shown in SEQ ID NO: 10-205, or from a fragment or variant of the 3'-UTR of the ribosomal protein gene having the nucleotide sequence shown in SEQ ID NO: 1 or 2, its functional variants, its functional fragments or functional fragments of the variants described above.

The invention has surprisingly found that an artificial nucleic acid molecule containing the 3'-UTR element described above can be or allows the creation of an mRNA molecule that provides increased, prolonged and/or stabilized protein production. Thus, the 3'-UTR element provided herein can increase the stability of protein expression from an mRNA molecule and/or increase translation efficiency.

The artificial nucleic acid molecule of the present invention may be RNA such as mRNA, DNA such as vector DNA, or may be a modified RNA or DNA molecule. It may be in the form of 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 also optionally contain a 5'-UTR and/or a 5'-cap. The optional 5' cap and/or 5' UTR are preferably located 5' to the ORF 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 the at least one 3' UTR element, preferably the optional poly(A) sequence is attached to the 3' end of the 3' UTR element. The connection may be made directly or indirectly by means of a segment consisting of 2, 4, 6, 8, 10, 20, etc. nucleotides, such as a linker consisting of 1-50, preferably 1-20 nucleotides, for example containing or consisting of one or more restriction sites.

In one embodiment, the optional polyadenylation signal is located downstream of the 3' side of the 3' UTR element. Preferably, the polyadenylation signal contains the NN(U/T)ANA consensus sequence in which N is 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 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 away, in the forward direction from 3'- end of the 3'-UTR element.

Transcription of an artificial nucleic acid molecule of the present invention, for example, an artificial DNA molecule containing a polyadenylation signal located in the forward direction relative to the 3'-UTR element, should result in an immature RNA containing a polyadenylation signal in the forward direction relative to its 3 '-UTR of element. For example, transcription of a DNA molecule containing a 3'UTR element having SEQ ID NO: 1 should result in an RNA carrying a 3'UTR element having the sequence of SEQ ID NO: 2.

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 a polyadenylation signal, can result in an mRNA molecule containing an open reading frame, 3' -UTR element and 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 a poly(A) sequence.

One embodiment of the invention relates to an artificial nucleic acid molecule, which is an artificial DNA molecule containing an open reading frame and a sequence shown in SEQ ID NO: 1 or a sequence that is at least about 40% or more identical to SEQ ID NO: 1, or its fragment described above. In addition, the invention provides an artificial nucleic acid molecule, which is an artificial RNA molecule containing an open reading frame and a sequence shown in SEQ ID NO: 2 or a sequence that is at least about 40% or more identical to SEQ ID NO: 2 , or its fragment described above.

Thus, the invention also provides an artificial nucleic acid molecule that can serve as a template for an RNA molecule, preferably an mRNA molecule, which is stabilized and optimized for 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.

In another embodiment, the 3'UTR of the artificial nucleic acid molecule of the invention does not contain a polyadenylation signal or 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 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 followed by a ribosomal 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.

For example, an artificial nucleic acid molecule of the present invention may contain a nucleotide sequence corresponding to a DNA sequence:

Transcription of these sequences can lead to the production of artificial nucleic acid molecules containing the sequence:

Such artificial RNA molecules, i.e. artificial nucleic acid molecules containing the sequence shown in SEQ ID NO: 4, can also be obtained in vitro using conventional methods of chemical synthesis, and it is not necessary to carry out transcription from the 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.

Preferably, the open reading frame does not encode a ribosomal protein. In a preferred embodiment of the invention, the open reading frame does not encode the ribosomal protein from which the 3'-UTR element of the inventive nucleic acid is derived, primarily does not encode the mammalian ribosomal protein, provided that the 3'-UTR element is identical to the 3'- Mammalian ribosomal protein UTR. In some other embodiments, the open reading frame does not encode RPS9 or variants thereof, provided that the 3'-UTR element has a sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2.

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.

One embodiment of the invention relates to artificial DNA molecules containing an open reading frame, preferably an open reading frame, which encodes a peptide or protein other than the ribosomal protein from which the 3'-UTR is derived; 3'UTR element containing or consisting of at least about 60% sequence identity, preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity , even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of the sequence shown in SEQ ID NO: 1; polyadenylation signal and/or poly(A) sequence. In addition, the invention provides an artificial DNA molecule comprising an open reading frame, preferably an open reading frame, which encodes any peptide or protein other than the ribosomal protein from which the 3'-UTR is derived; 3'UTR element containing or consisting of at least about 60% sequence identity, preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity , even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of the sequence shown in SEQ ID NO: 3.

In addition, the invention provides an artificial RNA molecule, preferably an artificial mRNA molecule or an artificial viral RNA molecule, comprising an open reading frame, preferably an open reading frame, which encodes a peptide or protein other than the ribosomal protein from which the 3'-UTR is derived; 3'UTR element containing or consisting of at least about 60% sequence identity, preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity , even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of the sequence shown in SEQ ID NO: 2; and a polyadenylation signal and/or a poly(A) sequence. In addition, an artificial RNA molecule, preferably an artificial mRNA molecule or an artificial viral RNA molecule, comprising an open reading frame, preferably an open reading frame, which encodes a peptide or protein other than the ribosomal protein from which the 3'-UTR is derived; 3'UTR element containing or consisting of at least about 60% sequence identity, preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity , even more preferably at least about 95%; even more preferably at least 99%; even more preferably 100% of the sequence shown in SEQ ID NO: 4.

The invention provides an artificial nucleic acid molecule, preferably an artificial mRNA, which may be characterized in that it provides increased stability and prolongation of the expression of the encoded peptide or protein. Without wishing to be bound by any particular theory, it is believed that the increased stability of protein expression and also prolonged protein expression may be due to reduced degradation of an artificial nucleic acid molecule, such as an artificial mRNA molecule of the present invention. Thus, the 3'UTR element of the invention can prevent degradation and cleavage of the artificial nucleic acid.

In some embodiments, the artificial nucleic acid molecule may contain a histone stem-loop structure in addition to a nucleotide sequence derived from the 3'-UTR of the ribosomal protein gene. Such an artificial nucleic acid molecule of the present invention may contain, for example, 5'→3' ORFs, a 3'UTR element, an optional stem-loop histone sequence, an optional poly(A) a polyadenylation sequence or signal; and an optional poly(C) sequence. It may also contain a 5'→3' ORF, a 3'UTR element, an optional poly(A) sequence, an optional poly(C) sequence, and an optional stem-loop histone sequence.

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

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:

stem1 or stem2 boundary elements N _1-6 denote a continuous sequence consisting of 1-6, preferably 2-6, more preferably 2-5, even more preferably 3-5, most preferably 4-5 or 5 N, where N each independently selected from a nucleotide selected from A, U, T, G, and C, or a nucleotide analogue thereof;

"stem1" [n _0-2 GN _3-5 ] denotes a sequence reversely complementary or partially reversely complementary to the element "stem2" and is a contiguous sequence of 5-7 nucleotides;

in which N _0-2 denotes a contiguous sequence consisting of 0-2, preferably 0-1, more preferably 1 N, where N is each independently selected from a nucleotide selected from A, U, T, G and C or its nucleotide analog; in which N _3-5 denotes a contiguous sequence consisting of 3-5, preferably 4-5, more preferably 4 N, where N is each independently selected from a nucleotide selected from A, U, T, G and C or its a nucleotide analog, and in which G is guanosine or an analog thereof and may optionally be replaced by cytidine or an analog thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine;

the sequence "loop" [N _0-4 (U/T)N _0-4 ] is located between the elements "stem1" and "stem2" and denotes a continuous sequence of 3-5 nucleotides, more preferably 4 nucleotides;

in which N _0-4 each independently of the other denotes a continuous sequence consisting of 0-4, preferably 1-3, more preferably 1-2 N, where N is each independently selected from a nucleotide selected from A, U, T, G and C or its nucleotide analogue; and wherein U/T is uridine or optionally thymidine;

"stem2" [N _3-5 CN _0-2 ] denotes a sequence reversely complementary or partially reversely complementary to the element "stem1" and is a contiguous sequence of 5-7 nucleotides;

in which N _3-5 denotes a contiguous sequence consisting of 3-5, preferably 4-5, more preferably 4 N, where N is each independently selected from a nucleotide selected from A, U, T, G and C or its nucleotide analog; in which N _0-2 denotes a contiguous sequence consisting of 0-2, preferably 0-1, more preferably 1 N, where N is each independently selected from a nucleotide selected from A, U, T, G or C or its nucleotide analog; and

in which C denotes cytidine or its analogue and may optionally be replaced by guanosine or its analogue, provided that its complementary nucleotide guanosine in "stem1." replaced by cytidine;

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, by Watson-Crick base-pairing of nucleotides A and U /T or G and C, or by non-Watson-Crick base-pairing, e.g., by "wobble" base-pairing ("wobble" base-pairing), the reverse of Watson-Crick base-pairing, by Hoogsteen base-pairing, by the reverse of Hoogsteen base-pairing bases, or they may base-pair with each other to form a partially reversed complementary sequence, with incomplete base-pairing occurring between stem1 and stem2, due to the fact that one or more bases in one stem do not have complementary base in the reverse complementary sequence of each oh stem.

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 (Ia) or (IIa):

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

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

wherein:

имеют указанные выше значения.

have the above meanings.

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:

имеют указанные выше значения.

have the above meanings.

The most preferred stem-loop histone sequence is that shown in SEQ ID NO: 5 CAAAGGCTCTTTTCAGAGCCACCA, or more preferably the RNA sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 5.

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

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

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

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

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

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

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

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

5'-cap - 5'-UTR - ORF - 3'-UTR element - poly(A)/(C)-sequence - poly(A)/(C)-sequence - histone stem-loop structure; 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 in the case when the nucleic acid is in the form of mRNA or encodes mRNA, can be modified with a sequence consisting of at least 10 cytosines, preferably at least 20 cytosines, more preferably at least 30 cytosines (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 inventive nucleic acid contains a poly(C) sequence consisting of 30 cytidine bases. Thus, preferably, the artificial nucleic acid molecule of the present invention contains, preferably in the 5'→3' direction of the ORF, at least one 3'-UTR element as described above, a poly(A) sequence or polyadenylation signal, and a 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 5'-UTR, a promoter or a sequence containing a 5'-UTR and a promoter. A promoter may direct or regulate transcription of an artificial nucleic acid molecule of the present invention, such as an artificial DNA molecule of the present invention. In addition, the 5'-UTR may consist of or contain the 5'-UTR of the gene presented in the present description. In addition, the 5'-UTR can interact with the inventive 3,-UTR element and thus can support the stabilizing effect of the inventive 3'-UTR element. Such elements can further support the stability and efficiency of translation. Therefore, in some embodiments, the invention provides artificial nucleic acid molecules, preferably mRNA molecules, containing in the 5'→3' direction at least one of the following structures

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

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

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

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

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

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

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

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

In the most preferred embodiment of the present invention, the artificial nucleic acid molecule contains at least one element of the 5'-untranslated region (5'UTR element) with a length of less than 500, 400, 300, 250, 200, 150 or 100 nucleotides and/or more than 20, 30, 40, 50 or 60 nucleotides, which preferably contains or consists of a nucleotide sequence which is derived from the 5'UTR of the TOP gene, or which 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.

The nucleotide sequence which is derived from the 5'UTR of the TOP gene is derived from a eukaraotic 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 TOP gene.

For example, the 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 a variant thereof, 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 non-human species that are homologous to 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 S'UTR element 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 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 5' UTR element is derived from a nucleotide sequence extending from the nucleotide position immediately 3'-side adjacent to the 5'-TOP to the nucleotide position immediately adjacent 5'-side to the start codon (located on the 3'- the end of the sequences), for example, up 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 5'UTR element 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 pending in WO2 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 5'UTR element 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. subunits (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 ribosomal large subunit 32 (L32) protein gene, most preferably from a human ribosomal large subunit 32 (L32) protein gene, where preferably the 5'-UTR element does not contain the 5'-TOP of said gene.

Therefore, in the most preferred embodiment of the invention, the 5'-UTR element 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: 6 (5'-UTR of the human large subunit 32 ribosomal protein without the 5'-oligopyrimidine tract:

GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to SEQ ID NO: 1368 presented in patent application WO 2013/143700) or preferably the corresponding RNA sequence, or where 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 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: 6, 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 S'-UTR element 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 RPL27 , RPLPO, RPLP1, RPLP2, RPLP3, RPLPO, RPLP1, RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, 2TUBB1, UBA5 , NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or from a homologue or variant thereof, where preferably the 5'-UTR element does not contain the TOP motif or 5'-TOP of said genes, and where optionally the 5'-UTR element begins with it 5'-end from the nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 forward relative to the 5'-oligopyrimidine tract (TOP) and where optionally also the 5'-UTR -element 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 comes.

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 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 favorable in terms of RNA stability and/or the most 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 said columns 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;

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

the Gln 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

codons encoding Thr 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;

codons encoding Val 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 codons) 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 the so-called "rare codons" are present in the coding region of the artificial nucleic acid molecule according to the invention, the level of translation of the corresponding modified nucleotide sequence is lower than when there are codons that code for 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 such a modification, all codons in the wild-type open reading frame that encode a tRNA that is relatively rare in the cell can be replaced with codons that encode a tRNA that is relatively common in the cell, and which in each case "carries » the same amino acid as a relatively 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 the most frequently occurring codons in the open reading frame is performed according to the most frequently occurring mammalian codons, more preferably according to the most frequently occurring human codons. Preferably, both codon optimization and G/C content modification are performed in the open reading frame.

Further modifications to the artificial nucleic acid molecule can be made to further enhance resistance to cleavage, such as resistance to in vivo cleavage by exo- or endonuclease, and/or to further improve the stability of protein expression from the artificial nucleic acid molecule of the present invention, further modifications to the artificial nucleic acid molecule can be made, 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 modified 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 (ribose) component, and/or the phosphate component forming the backbone (see above) of the RNA sequence. Guanosine, uracil, adenosine, and cytosine analogs include, but are not limited to, any naturally occurring or non-naturally occurring guanosine, uracil, 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-aminoadenosine, 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-chloroaracytosine, 5-fluorouridine, 5-yoduridine, 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-methyladenosine, N7- methylxanthosine, N-uracil-5-hydroxyacetic acid methyl ester, puromycin, queosin, 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 according to 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 mRNA modification.

According to a specific embodiment of the invention, the mRNA in the composition 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;

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 derived from a ribosomal protein 3'-UTR, preferably from the nucleotide sequence shown in any of SEQ ID NOs: 10-115, more preferably RPS9 3'-UTR, more preferably human RPS9 3'-UTR;

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

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

histone stem-loop structure.

In another preferred embodiment of the invention, the artificial nucleic acid molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO: 7 (see FIG. 3) or a complementary DNA 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. A sugar modification according to the present invention is a chemical modification of the sugar of a nucleotide in a nucleic acid molecule as 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 alkoxy 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 major groove chemical modifications 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 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 terminates ("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 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 as 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 the nucleotide adjacent to m7GpppN), CAP2 (methylation of the 2nd nucleotide ribose upstream of m7GpppN), CAP3 (methylation of the 3rd nucleotide ribose upstream of m7GpppN). relative m7GpppN), CAP4 (methylation of the ribose of the 4th nucleotide located in the forward direction relative to 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 an artificial nucleic acid molecule of the invention encodes an antibody.

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, Corynebacterium 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), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, enteroviruses, especially Coxsackievirus A and enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr virus (EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica and Fasciola gigantica, prion FFI, superfamily Filarioidea, flaviviruses, Francisella tularensis, genus Fusobacterium , Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, genipavirus (virus 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 herpes virus 6 (HHV-6) and human herpes virus 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), Machupo virus , 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, M ycoplasma pneumoniae Naegleria fowleri Necator americanus Neisseria gonorrhoeae Neisseria meningitidis Nocardia asteroides Nocardia spp , 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, Tox oplasma 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/CA27-29, 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, HPV -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, protein azu-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-2 V, 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 diseases of the 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.

The following therapeutic proteins can be used to treat blood disorders, circulatory system diseases, respiratory system diseases, cancer or neoplastic diseases, infectious diseases or immunodeficiencies: alterplase (tissue plasminogen activator; tPA) (pulmonary embolism, myocardial infarction, acute ischemic 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 to olorectal 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 (debridement of chronic skin ulcers and areas 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, 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), etanecept (rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, ankylosing spondylitis), interleukin-1 (IL-1) receptor anti/agonist, anakinra (inflammation and cartilage degradation associated with rheumatoid arthritis), thymulin (neurodegenerative diseases, rheumatism, anorexia nervosa), 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 element of the 3'-untranslated region (3'-UTR element) containing a nucleotide sequence that is derived from the 3'-UTR of the ribosomal protein gene.

At least one 3'-UTR element and the ORF are the constructs described above for the artificial nucleic acid molecule of the present invention. The cloning site can be any sequence suitable for the introduction of an open reading frame or a sequence containing an open reading frame, such as one or more restriction sites. Thus, a vector containing a cloning site is preferably suitable for inserting an open reading frame into the vector, preferably for inserting an open reading frame from the 5' side of the 3' UTR element. Preferably, the cloning site or ORF is located 5' to the 3' UTR element, preferably in close proximity to the 5' end of the 3' UTR element. For example, the cloning site or ORF may be directly attached to the 5'end of the 3'UTR element, or they may be attached via a segment of nucleotides, e.g., a segment consisting of 2, 4, 6, 8, 10, 20, etc. nucleotides as described above for 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 promoter, preferably the T7 promoter. Preferably, the vector is suitable for in vitro transcription using a phage based in vitro transcription system, such as a T7 RNA polymerase based in vitro transcription system.

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.

In one embodiment, 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%, 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 ribosomal protein gene, such as the nucleotide sequence shown in SEQ ID NOs: 10-115.

Preferably, the DNA vector of the present invention contains a sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, a sequence complementary to SEQ ID NO: 7, or sequences that are at least about 40% identical, preferably at least 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 by about 95%; even more preferably at least about 99% of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, or a fragment thereof as described above, preferably a functional fragment thereof.

Preferably, the RNA vector of the present invention contains the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, or a 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: 2, SEQ ID NO: 4, SEQ ID NO: 7, or a fragment thereof, preferably a functional fragment thereof.

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 3' UTR element, or 3' to the poly(A) sequence or signal polyadenylation, if present, or 3' to the poly(C) sequence, if present, or 3' to the histone stem-loop structure, if present. Thus, preferably the product resulting from linearization of the vector ends at the 3' end with the 3' end of the 3' UTR element or the 3' end of the poly(A) sequence or polyadenylation signal, if present, or 3 the '-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, a restriction site, preferably a unique restriction site, 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.

According to the present invention, cells 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 cell from a cultured 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. If the pharmaceutical composition according to the invention is in liquid form, but the carrier, as a rule, can be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, such as solutions buffered with phosphate, citrate, etc. The buffer to be injected may be hypertonic, isotonic, or hypotonic with respect to the particular 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 the patient to be treated 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 of the invention can be mixed with the nucleic acid of the invention specified in the present description, so that no interaction occurs that could significantly reduce the pharmaceutical effectiveness of the invention pharmaceutical composition 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 inactivated (eg, chemically or by radiation) pathogens (viruses, bacteria, etc.), adjuvants, preferably as described herein, 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.

полипептиды, обладающие способностью проникать в клетку пептиды (СРР), такие как ВИЧ-связывающие пептиды. Tat ВИЧ-1 (ВИЧ), выведенные из Tat пептиды, пенетратин, полученные из VP22 пептиды или аналоги, VP22 HSV (вирус герпеса простого), MAP, KALA или домены трансдукции белков (PTD), РрТ620, богатые пролином пептиды, богатые аргинином пептиды, богатые лизином пептиды, МРО-пептид(ы), Рер-1, L-олигомеры, пептид(ы) кальцитонина, выведенные из Antennapedia пептиды (прежде всего, выведенные из Drosophila antennapedia), pAntp, pIsl, FGF, лактоферрин, транспортан, буфорин-2, Вас715-24, SynB, SynB(1), pVEC, выведенные из hCT пептиды, SAP или гистоны.In this context, 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,

cell-penetrating peptide (CPP) polypeptides, such as HIV-binding peptides. Tat HIV-1 (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, MPO peptide(s), Pep-1, L-oligomers, calcitonin peptide(s), Antennapedia derived peptides (primarily derived from Drosophila antennapedia), pAntp, pIsl, FGF, lactoferrin, transportan, buforin-2, Vas715-24, 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+0+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.

In addition, a cationic or polycationic peptide or protein defined by the formula {(Arg) _l ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x } (formula (I)) presented above, which contains or further modified such that it contains at least one SH group, may be selected from (but not limited to) compounds of subformula (Ia):

in which (Arg) _l ;(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, 1, 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) make up 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 (1b):

in which the empirical formula {(Arg) _l ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x } (formula (III)) is presented in the present description and forms the core of the amino acid sequence represented by the (semi-empirical) formula (III), and in which Cys ₁ and Cys ₂ denote cysteine residues located proximal or terminal relative to (Arg) _l ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x .

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-035 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-A1a-B-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 Synte x"); 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 penetrate the cell peptides (CPP), such as HIV-binding peptides. Tat HIV-1 (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, MPC peptide(s), Pep-1, L-oligomers, calcitonin peptide(s), Antennapedia derived peptides (primarily derived from Drosophila antennapedid), pAntp, pIsl, FGF, lactoferrin, transportan, buforin-2, Vas715-24, 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) _l ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x , where l+m+n+o+x=8-15 and l, m, n or o independently of each other can be 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.

атомов азота, т.е. 4,9 нмолей атомов азота. Можно рассчитать, что для массового соотношения РНК/протамин, составляющего примерно 2:1, отношение N/P должно составлять примерно 0,81. Можно рассчитать, что для массового соотношения РНК/протамин, составляющего примерно 8:1, отношение N/P должно составлять примерно 0,2. В контексте настоящего изобретения касательно соотношения нуклеиновая кислота:пептид в комплексе N/P-отношение предпочтительно находится примерно в пределах 0,1-10, предпочтительно примерно в пределах 0,3-4, и наиболее предпочтительно примерно в пределах 0,5-2 или 0,7-2, и наиболее предпочтительно примерно в пределах 0,7-1,5.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 an RNA/(Arg) ₉ weight 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 approximately 2:1, then for 2 μg of RNA, RNA provides 6 nmol of phosphate; 1 µg of protamine contains approximately 235 pmoles of protamine molecules and therefore 235×21=4935 pmoles

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.e. 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, uracil or an analogue of guanosine or uracil; X is guanosine, uracil, adenosine, thymidine, cytosine, or an analogue of the above nucleotides; 1 denotes an integer from 1 to 40, while when 1=1, then G is guanosine or its analogue, when 1>1, then at least 50% of the nucleotides are guanosine or its analogue; m is an integer and is at least 3; when m=3, then X is uracil or its analogue, when m>3, then there are at least 3 consecutive uracils or analogues of uracil; n denotes an integer from 1 to 40, while when n=1, then G is guanosine or its analogue, when n>1, then at least 50% of the nucleotides is guanosine or its analogue.

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

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" should 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 of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention intended/intended for use as a drug, for example, as a vaccine (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 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 stable 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 element provides stable and prolonged expression of the peptide or protein encoded by the artificial nucleic acid molecule of the invention or the vector of the invention. 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, protozoan 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 a tumor or neoplasia, in autoimmune diseases, allergies, infections, or chronic diseases, or the like. 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'- The UTR element as above, and optionally other components such as a 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 types of gene therapy, 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, transdermal (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, transdermal (percutaneous), intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial and 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 can be prepared using methods known in the art using suitable dispersing or wetting 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.

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 can also be administered topically, especially in cases where 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 involves in vitro transfection of the isolated cells, then the use may also involve reintroduction of the transfected cells into 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 3'UTR element of the invention has the ability to stabilize an mRNA molecule and/or enhance, stabilize and/or prolong protein production from an mRNA molecule. Thus, a further object of the present invention relates to a method for stabilizing an RNA molecule, preferably an mRNA molecule, comprising the step of associating an RNA molecule, preferably an mRNA molecule, or a vector encoding an RNA molecule, with a 3'-UTR- an element containing or consisting of a nucleotide sequence derived from the 3'UTR of a ribosomal protein gene or from a variant 3'UTR of a ribosomal protein gene, preferably with a 3'UTR element as described above.

In addition, the present invention relates to a method for increasing, stabilizing and/or prolonging the production of a protein from an artificial nucleic acid molecule or from a vector, preferably from an mRNA molecule, and/or stabilizing and/or prolonging the production of a protein from an artificial nucleic acid molecule or from a vector, preferably from an mRNA molecule, comprising the step of associating an artificial nucleic acid molecule or vector, preferably an mRNA molecule, with a 3'-UTR element which contains or consists of a nucleotide sequence derived from the 3'-UTR a ribosomal protein gene or from a 3'UTR variant of a ribosomal protein gene, preferably with a 3'UTR element as described above.

The expression "association of an artificial nucleic acid molecule or vector with a 3'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. This means that an artificial nucleic acid molecule or vector is associated or linked to a 3'UTR element, preferably the 3'UTR element described above, in such a way that the function of the 3'UTR element is performed, for example, the stabilization function production of RNA and/or protein. Generally, this means that the 3'UTR element is integrated into an artificial nucleic acid molecule or vector, preferably into an mRNA molecule, on the 3' side of the open reading frame, preferably so that it is directly adjacent on the 3' side to open reading frame, preferably located between the open reading frame and the poly(A) sequence or polyadenylation signal. Preferably, the 3'UTR element is integrated into an artificial nucleic acid molecule or vector, preferably mRNA, as a 3'UTR, i. e. such that the 3'UTR element is the 3'UTR of an artificial nucleic acid molecule or vector, preferably mRNA, i. e. such that it extends from the 3' side of the open reading frame to the 5' side of the poly(A) sequence or polyadenylation signal, optionally attached via a short linker, such as a sequence containing or consisting of one or more restriction sites. Thus, preferably, the expression "association of an artificial nucleic acid molecule or vector with a 3'UTR element" means the functional association of a 3'UTR element with an open reading frame located in an artificial nucleic acid molecule or vector, preferably an mRNA molecule. The 3'-UTR and ORF are the constructs described above for the artificial nucleic acid molecule of the present invention, eg, preferably the ORF and 3'-UTR are heterologous, eg are from different genes as described above.

Another object of the present invention is the use of a 3'-UTR element, preferably the 3'-UTR element described above, to improve the stability of an RNA molecule, preferably an mRNA molecule, where the 3'-UTR element contains or consists of a nucleotide sequence derived from 3'UTR of a ribosomal protein gene or from a variant 3'UTR of a ribosomal protein gene.

In addition, the present invention provides the use of a 3'-UTR element, preferably the 3'-UTR element described above, to increase protein production from an artificial nucleic acid molecule or vector, preferably from an mRNA molecule, and/or to stabilize and/ or prolonging protein production from an artificial nucleic acid molecule or a vector molecule, preferably from an mRNA molecule, where the 3'-UTR element contains or consists of a nucleotide sequence derived from the 3'-UTR of a ribosomal protein gene or from a variant of the 3'-UTR of a ribosomal gene squirrel.

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.

The compounds and ingredients of the pharmaceutical composition according to the invention can also be produced 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 a kit or set of components may further comprise instructions for use, cells for transfection, an adjuvant, means for administering the pharmaceutical composition, a pharmaceutically acceptable carrier, and/or a pharmaceutically acceptable solution for dissolving or diluting the artificial nucleic acid molecule, vector, cells, or pharmaceutical composition.

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

The drawings show:

In FIG. 1-3 mRNA coding sequences that can be obtained by in vitro transcription. The following abbreviations are used:

rpl32: 5'-UTR of the human ribosomal protein large subunit 32, which lacks the 5'-terminal oligopyrimidine tract;

PpLuc (GC): high GC (GC-rich) mRNA sequence encoding Photinus pyralis luciferase;

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

• ag: central, α-complex-binding portion of the 3'-UTR of human α-globin;

• rps9: 3'UTR element derived from the 3'UTR of the human ribosomal small subunit protein 9;

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

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

albumin: 3'-UTR of human albumin;

in fig. 1 shows the sequence encoding mRNA rpl32 - PpLuc(GC) - A64 - C30 - histone SL (SEQ ID NO: 8);

in fig. 2 shows the sequence encoding mRNA rpl32 - PpLuc(GC) - ag - A64 - C30 - histone SL (SEQ ID NO: 9). The central, α-complex-binding portion of the 3'-UTR of human α-globin is inserted between the ORF and poly(A). The PpLuc(GC) ORF is in italics. The 3'UTR element derived from α-globin is underlined;

in fig. 3 - mRNA sequence rpl32 - PpLuc(GC) - rps9 - A64 - C30 - hSL (SEQ ID NO: 7). The 3'UTR of the human ribosomal small subunit protein 9 is inserted between the ORF and poly(A). The PpLuc(GC) ORF is in italics, the 3' UTR element derived from rps9 is underlined;

in fig. 4 - data demonstrating that the 3'-UTR of the ribosomal protein of the small subunit 9 significantly increases the expression of the protein from mRNA;

The effect of the inventive human ribosomal small subunit 9 3'UTR on luciferase expression from mRNA was studied in comparison to luciferase expression from mRNA lacking the 3'UTR or containing the 3'UTR of human α-globin. For this, human dermal fibroblasts (HDFs) were transfected with various mRNAs by lipofection. Luciferase levels were measured 6, 24, 48 and 72 hours after transfection. Obviously, there was expression of luciferase with mRNA that lacked the 3'-UTR. At the same time, luciferase expression did not increase when the well-known 3'-UTR of human α-globin was included. In contrast, the 3'-UTR of the small subunit 9 ribosomal protein resulted in a significant increase in luciferase expression. Data are plotted as the average of three transfections expressed in relative light units (RLU)±SEM (standard deviation). The results of measurements in RLU are summarized in Example 5.1;

in fig. 5 - sequence encoding mRNA rpl32 - PpLuc(GC) - albumin - A64 - C30 - histone SL (SEQ ID NO: 206). The 3'UTR of human albumin was inserted between the ORF and poly(A). OPC PpLuc(GC) is in italics. The 3' UTR element derived from albumin is underlined;

in fig. 6 - data demonstrating that 3'-UTRs derived from the mouse ribosomal protein genes rps21, rps29, rps9, rps27, rps28, rps19, rpl35a, rpl13, rpl36 and rpl23a increase protein expression with mRNA in HDF cells (human skin fibroblasts). ) at least to the same extent as mRNA containing the 3'-UTR of the albumin gene, which has already been shown to increase protein expression from mRNA (WO 2013/143698);

in fig. 7 - data demonstrating that 3'-UTRs derived from the mouse ribosomal protein genes rps21, rps29, rps9, rps27, rps28, rps19, rpl35a, rpl13, rpl36 and rpl23a increase mRNA protein expression in HeLa cells at least to the same extent as mRNA containing the 3'-UTR of the albumin gene, which has already been shown to increase protein expression from mRNA (WO 2013/143698);

in fig. 8 - data showing that 3'-UTRs derived from the mouse ribosomal protein genes rpl23, uba52, rpl2211, rpl36a, rps4x, rpl27, rpl3, rps23, rps13, rpl26, rps17, rps18, rps8, Fau, rps13, rpl11 and rpl38 upregulates mRNA protein expression in HDF cells (human skin fibroblasts) at least to the same extent as mRNA containing the 3'-UTR of the albumin gene, which has already been shown to increase protein c mRNA expression (WO 2013 /143698);

in fig. 9 - data showing that 3'-UTRs derived from the mouse ribosomal protein genes rpl23, uba52, rpl22l1, rpl36a, rps4x, rpl27, rpl3, rps23, rps13, rpl26, rps17, rps18, rps8, Fau, rps13, rpl11 and rpl38 increase mRNA protein expression in HeLa- at least to the same extent as mRNA containing the 3'-UTR of the albumin gene, which has already been shown to increase protein expression from mRNA (WO 2013/143698).

Examples

1. Obtaining DNA templates

A vector for in vitro transcription was constructed containing the T7 promoter and a GC-rich sequence encoding Photinus pyralis luciferase (PpLuc(GC)). The 5'-untranslated region (5'-UTR) of the 32 large subunit ribosomal protein was inserted 5'-side of PpLuc(GC). A 64-adenine (A64) poly(A) sequence followed by a 30-cytosine (C30) sequence and a stem-loop sequence was inserted 3' to PpLuc(GC). Behind the stem-loop sequence was a restriction site that was used to linearize the vector prior to in vitro transcription. The mRNA obtained from this vector after in vitro transcription was designated as "rpl32 - PpLuc(GC) - A64 - C30 - histone SL".

The specified vector was modified so that it included untranslated sequences from the 3'-side relative to the open reading frame (3'-UTR). In general, vectors were created that contained the following mRNA coding sequences (some of the mRNA coding sequences are shown as examples in Figures 1-3):

rpl32 - PpLuc(GC) - A64 - C30 - histone SL (SEQ ID NO: 8, Fig. 1);

rpl32 - PpLuc(GC) - ag - A64 - C30 - histone SL (SEQ ID NO: 9, Fig. 2);

rpl32 - PpLuc(GC) - rps9 - A64 - C30 - histone SL (SEQ ID NO: 7, Fig. 3);

rpl32 - PpLuc(GC) - rps21 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps29 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps9 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps27 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps28 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps19 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl35a - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl13 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl36 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl23a - A64 - C30 histone SL;

rpl32 - PpLuc(GC) - rpl23 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - uba52 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl22l1 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl36a - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps4x - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl27 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl3 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps23 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps13 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl26 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps17 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps18 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - Fau - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rps13 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl11 - A64 - C30 - histone SL;

rpl32 - PpLuc(GC) - rpl38 - A64 - C30 - histone SL.

2. Transcription in vitro

The template DNA prepared according to the method described in Example 1 was linearized and transcribed in vitro using T7 RNA polymerase. The template DNA was then digested by treatment with DNase. mRNA transcripts containing the 5' cap structure were generated by adding an excess of N7-methyl-guanosine-5'-tryptophosphate-5'-guanosine to the transcription reaction mixture. The resulting mRNA was purified and resuspended in water.

3. Luciferase expression after mRNA lipofection

Human dermal fibroblasts (HDF) or HeLa cells were seeded in 24-well plates three days prior to transfection at a density of 3×10 ⁴ cells per well on medium (RPMI 1640 medium containing L-glutamine and 25 mM Hepes (Lonza, Basel , Switzerland) supplemented with 10% FCS, 1% Pen/Strep, 1% glutamine). Immediately prior to lipofection, the cells were washed in Opti-MEM medium. Cells were lipofected with 25 ng mRNA encoding PpLuc per well, included in the complex with Lipofectamine 2000. To control the efficiency of transfection, along with mRNA PpLuc (2.5 ng mRNA RrLuc per well), mRNA encoding Renilla reniformis luciferase (RrLuc ). 90 min after the start of transfection, the medium was replaced with Opti-MEM. At 6, 24, 48 and 72 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.

4. Measurement of luciferase

Luciferase activity was measured in relative light units (RLU) using a Hidex Chameleon plate reader. PpLuc activity was measured over a 2 second measurement period using 20 μl of lysate and 50 μl of luciferin-containing buffer (Beetle-Juice buffer, PJK GmbH). RrLuc activity was measured over a 2 second measurement period using 20 μl of lysate and 50 μl of buffer containing coelenterazine (Renilla-Juice buffer, PJK GmbH).

5. Results

5.1 The 3'-UTR of ribosomal protein genes increases protein expression.

To study the effect of the 3'-UTR of ribosomal protein genes on protein expression from mRNA, mRNAs containing various UTRs were synthesized: mRNA either did not contain the 3'-UTR or contained the central α-complex-binding portion of the 3'-UTR of human α-globin (ag ) or contained the 3'-UTR of the human ribosomal small subunit protein 9 (rps9). mRNAs encoding luciferase were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured 6, 24, 48 and 72 hours after transfection. Based on these data, the total amount of protein expressed over a period of time from 0 to 72 hours was calculated as area under the curve (AUC) (see Table 1 below and FIG. 4).

Clearly, luciferase was expressed with mRNA lacking the 3'-UTR. However, luciferase expression was not increased in the presence of the well-known α-globin 3'-UTR. In contrast, the 3'-UTR of the small subunit 9 ribosomal protein significantly increased luciferase expression.

5.2 The 3'-UTRs of mouse ribosomal protein genes increase protein expression.

To study the effect of the 3'-UTR of mouse ribosomal protein genes on protein expression from mRNA, mRNAs containing various UTRs were synthesized: mRNAs containing 3'-UTRs of various mouse ribosomal proteins (rps21, rps29, rps9, rps27, rpl13, rpl36 and rpl23a) and for comparison the 3'-UTR of albumin, which is known to increase protein expression from mRNA (WO 2013/143698). mRNAs encoding luciferase were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured 24, 48 and 72 hours after transfection (see Table 2 below and FIG. 6).

The results indicate that mRNA containing the 3'-UTR of mouse ribosomal proteins expressed luciferase in greater amounts than mRNA containing the 3'-UTR of albumin, which has been described to increase protein production.

5.3 The 3'-UTR of the mouse ribosomal protein genes increases protein expression.

To study the effect of the 3'-UTR of mouse ribosomal protein genes on protein expression from mRNA, mRNAs containing various UTRs were synthesized: mRNAs containing 3'-UTRs of various mouse ribosomal proteins (rps21, rps29, rps9, rps27, rpl13, rpl36 and rpl23a) and, for comparison, albumin 3'-UTR. mRNAs encoding luciferase were transfected into HeLa cells. Luciferase levels were measured 24, 48 and 72 hours after transfection (see Table 3 below and Figure 7).

The results indicate that mRNA containing the 3'-UTR of mouse ribosomal proteins expressed luciferase in greater amounts than mRNA containing the 3'-UTR of albumin, which has been described to increase protein production.

5.4 The 3'-UTR of the mouse ribosomal protein genes increases protein expression.

To study the effect of the 3'-UTR of mouse ribosomal protein genes on protein expression from mRNA, mRNAs containing various UTRs were synthesized: mRNAs containing 3'-UTRs of various mouse ribosomal proteins (rpl23, uba52, rpl2211, rpl36a, rps23, rps13, rpl26, rps17, rps18, rps8, Fau, rps13, rpl11 and rpl38) and albumin 3'-UTR for comparison. mRNAs encoding luciferase were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured 24, 48 and 72 hours after transfection (see Table 4 below and FIG. 8).

The results indicate that mRNA containing the 3'-UTR of mouse ribosomal proteins expressed luciferase in greater amounts than mRNA containing the 3'-UTR of albumin, which has been described to increase protein production.

The 5.5 3'-UTR of the mouse ribosomal protein genes increases protein expression.

To study the effect of the 3'-UTR of mouse ribosomal protein genes on protein expression from mRNA, mRNAs containing various UTRs were synthesized: mRNAs containing 3'-UTRs of various mouse ribosomal proteins (rpl23, uba52, rpl22l1, rpl36a, rps23, rps13, rpl26, rps17, rps18, rps8, Fau, rps13, rpl11 and rpl38) and albumin 3'-UTR for comparison. mRNAs encoding luciferase were transfected into HeLa cells. Luciferase levels were measured 24, 48 and 72 hours after transfection (see Table 5 below and Figure 9).

The results indicate that mRNA containing the 3'-UTR of mouse ribosomal proteins expressed luciferase in greater amounts than mRNA containing the 3'-UTR of albumin, which has been described to increase protein production.

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SEQUENCE LIST

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ggcgctgcct acggaggtgg cagccatctc cttctcggca tc 42

<210> 7

<211> 1918

<212> DNA

<213> Artificial sequence

<220>

<223> rpl32 PpLuc(GC) rps9 A64 C30 histoneSL

<400> 7

ggggcgctgc ctacgggaggt ggcagccatc tccttctcgg catcaagctt gaggatggag 60

gacgccaaga acatcaagaa gggcccggcg cccttctacc cgctggagga cgggaccgcc 120

ggcgagcagc tccacaaggc catgaagcgg tacgccctgg tgccggggcac gatcgccttc 180

accgacgccc acatcgaggt cgacatcacc tacgcggagt acttcgagat gagcgtgcgc 240

ctggccgagg ccatgaagcg gtacggcctg aacaccaacc accggatcgt ggtgtgctcg 300

gagaacagcc tgcagttctt catgccggtg ctgggcgccc tcttcatcgg cgtggccgtc 360

gccccggcga acgacatcta caacgagcgg gagctgctga acagcatggg gatcagccag 420

ccgaccgtgg tgttcgtgag caagaagggc ctgcagaaga tcctgaacgt gcagaagaag 480

ctgcccatca tccagaagat catcatcatg gacagcaaga ccgactacca gggcttccag 540

tcgatgtaca cgttcgtgac cagccacctc ccgccgggct tcaacgagta cgacttcgtc 600

ccggagagct tcgaccggga caagaccatc gccctgatca tgaacagcag cggcagcacc 660

ggcctgccga agggggtggc cctgccgcac cggaccgcct gcgtgcgctt ctcgcacgcc 720

cgggacccca tcttcggcaa ccagatcatc ccggacaccg ccatcctgag cgtggtgccg 780

ttccaccacg gcttcggcat gttcacgacc ctgggctacc tcatctgcgg cttccgggtg 840

gtcctgatgt accggttcga ggaggagctg ttcctgcgga gcctgcagga ctacaagatc 900

cagagcgcgc tgctcgtgcc gaccctgttc agcttcttcg ccaagagcac cctgatcgac 960

aagtacgacc tgtcgaacct gcacgagatc gccagcgggg gcgccccgct gagcaaggag 1020

1080

accgagacca cgagcgcgat cctgatcacc cccgaggggg acgacaagcc gggcgccgtg 1140

ggcaaggtgg tcccgttctt cgaggccaag gtggtggacc tggacaccgg caagaccctg 1200

ggcgtgaacc agcggggcga gctgtgcgtg cggggggccga tgatcatgag cggctacgtg 1260

aacaacccgg aggccaccaa cgccctcatc gacaaggacg gctggctgca cagcggcgac 1320

atcgcctact gggacgagga cgagcacttc ttcatcgtcg accggctgaa gtcgctgatc 1380

1440

aacatcttcg acgccggcgt ggccgggctg ccggacgacg acgccggcga gctgccggcc 1500

gcggtggtgg tgctggagca cggcaagacc atgacggaga aggagatcgt cgactacgtg 1560

gccagccagg tgaccaccgc caagaagctg cggggcggcg tggtgttcgt ggacgaggtc 1620

ccgaagggcc tgaccgggaa gctcgacgcc cggaagatcc gcgagatcct gatcaaggcc 1680

aagaagggcg gcaagatcgc cgtgtaagac tagtgtccac ctgtccctcc tgggctgctg 1740

gattgtctcg ttttcctgcc 1800

1860

cccccccccc cccccccccc cccccccccc aaaggctctt ttcagagcca ccagaatt 1918

<210> 8

<211> 1848

<212> DNA

<213> Artificial sequence

<220>

<223> rpl32 PpLuc(GC) A64 C30 histoneSL

<400> 8

ggggcgctgc ctacgggaggt ggcagccatc tccttctcgg catcaagctt gaggatggag 60

gacgccaaga acatcaagaa gggcccggcg cccttctacc cgctggagga cgggaccgcc 120

ggcgagcagc tccacaaggc catgaagcgg tacgccctgg tgccggggcac gatcgccttc 180

accgacgccc acatcgaggt cgacatcacc tacgcggagt acttcgagat gagcgtgcgc 240

ctggccgagg ccatgaagcg gtacggcctg aacaccaacc accggatcgt ggtgtgctcg 300

gagaacagcc tgcagttctt catgccggtg ctgggcgccc tcttcatcgg cgtggccgtc 360

gccccggcga acgacatcta caacgagcgg gagctgctga acagcatggg gatcagccag 420

ccgaccgtgg tgttcgtgag caagaagggc ctgcagaaga tcctgaacgt gcagaagaag 480

ctgcccatca tccagaagat catcatcatg gacagcaaga ccgactacca gggcttccag 540

tcgatgtaca cgttcgtgac cagccacctc ccgccgggct tcaacgagta cgacttcgtc 600

ccggagagct tcgaccggga caagaccatc gccctgatca tgaacagcag cggcagcacc 660

ggcctgccga agggggtggc cctgccgcac cggaccgcct gcgtgcgctt ctcgcacgcc 720

cgggacccca tcttcggcaa ccagatcatc ccggacaccg ccatcctgag cgtggtgccg 780

ttccaccacg gcttcggcat gttcacgacc ctgggctacc tcatctgcgg cttccgggtg 840

gtcctgatgt accggttcga ggaggagctg ttcctgcgga gcctgcagga ctacaagatc 900

cagagcgcgc tgctcgtgcc gaccctgttc agcttcttcg ccaagagcac cctgatcgac 960

aagtacgacc tgtcgaacct gcacgagatc gccagcgggg gcgccccgct gagcaaggag 1020

1080

accgagacca cgagcgcgat cctgatcacc cccgaggggg acgacaagcc gggcgccgtg 1140

ggcaaggtgg tcccgttctt cgaggccaag gtggtggacc tggacaccgg caagaccctg 1200

ggcgtgaacc agcggggcga gctgtgcgtg cggggggccga tgatcatgag cggctacgtg 1260

aacaacccgg aggccaccaa cgccctcatc gacaaggacg gctggctgca cagcggcgac 1320

atcgcctact gggacgagga cgagcacttc ttcatcgtcg accggctgaa gtcgctgatc 1380

1440

aacatcttcg acgccggcgt ggccgggctg ccggacgacg acgccggcga gctgccggcc 1500

gcggtggtgg tgctggagca cggcaagacc atgacggaga aggagatcgt cgactacgtg 1560

gccagccagg tgaccaccgc caagaagctg cggggcggcg tggtgttcgt ggacgaggtc 1620

ccgaagggcc tgaccgggaa gctcgacgcc cggaagatcc gcgagatcct gatcaaggcc 1680

aagaagggcg gcaagatcgc cgtgtaagac tagtagatct aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaatgcatc cccccccccc 1800

cccccccccc cccccccccc aaaggctctt ttcagagcca ccagaatt 1848

<210> 9

<211> 1914

<212> DNA

<213> Artificial sequence

<220>

<223> rpl32 PpLuc(GC) ag A64 C30 histoneSL

<400> 9

ggggcgctgc ctacgggaggt ggcagccatc tccttctcgg catcaagctt gaggatggag 60

gacgccaaga acatcaagaa gggcccggcg cccttctacc cgctggagga cgggaccgcc 120

ggcgagcagc tccacaaggc catgaagcgg tacgccctgg tgccggggcac gatcgccttc 180

accgacgccc acatcgaggt cgacatcacc tacgcggagt acttcgagat gagcgtgcgc 240

ctggccgagg ccatgaagcg gtacggcctg aacaccaacc accggatcgt ggtgtgctcg 300

gagaacagcc tgcagttctt catgccggtg ctgggcgccc tcttcatcgg cgtggccgtc 360

gccccggcga acgacatcta caacgagcgg gagctgctga acagcatggg gatcagccag 420

ccgaccgtgg tgttcgtgag caagaagggc ctgcagaaga tcctgaacgt gcagaagaag 480

ctgcccatca tccagaagat catcatcatg gacagcaaga ccgactacca gggcttccag 540

tcgatgtaca cgttcgtgac cagccacctc ccgccgggct tcaacgagta cgacttcgtc 600

ccggagagct tcgaccggga caagaccatc gccctgatca tgaacagcag cggcagcacc 660

ggcctgccga agggggtggc cctgccgcac cggaccgcct gcgtgcgctt ctcgcacgcc 720

cgggacccca tcttcggcaa ccagatcatc ccggacaccg ccatcctgag cgtggtgccg 780

ttccaccacg gcttcggcat gttcacgacc ctgggctacc tcatctgcgg cttccgggtg 840

gtcctgatgt accggttcga ggaggagctg ttcctgcgga gcctgcagga ctacaagatc 900

cagagcgcgc tgctcgtgcc gaccctgttc agcttcttcg ccaagagcac cctgatcgac 960

aagtacgacc tgtcgaacct gcacgagatc gccagcgggg gcgccccgct gagcaaggag 1020

1080

accgagacca cgagcgcgat cctgatcacc cccgaggggg acgacaagcc gggcgccgtg 1140

ggcaaggtgg tcccgttctt cgaggccaag gtggtggacc tggacaccgg caagaccctg 1200

ggcgtgaacc agcggggcga gctgtgcgtg cggggggccga tgatcatgag cggctacgtg 1260

aacaacccgg aggccaccaa cgccctcatc gacaaggacg gctggctgca cagcggcgac 1320

atcgcctact gggacgagga cgagcacttc ttcatcgtcg accggctgaa gtcgctgatc 1380

1440

aacatcttcg acgccggcgt ggccgggctg ccggacgacg acgccggcga gctgccggcc 1500

gcggtggtgg tgctggagca cggcaagacc atgacggaga aggagatcgt cgactacgtg 1560

gccagccagg tgaccaccgc caagaagctg cggggcggcg tggtgttcgt ggacgaggtc 1620

ccgaagggcc tgaccgggaa gctcgacgcc cggaagatcc gcgagatcct gatcaaggcc 1680

aagaagggcg gcaagatcgc cgtgtaagac tagttataag actgactagc ccgatgggcc 1740

tcccaacggg ccctcctccc ctccttgcac cgagattaat agatctaaaa aaaaaaaaaa 1800

1860

cccccccccc cccccccccc ccccccaaag gctcttttca gagccaccag aatt 1914

<210> 10

<211> 109

<212> DNA

<213> Homo sapiens

<400> 10

gatctaagag ttacctggct acagaaagaa gatgccagat gacacttaag acctacttgt 60

gatatttaaa tgatgcaata aaagacctat tgatttggac cttcttctt 109

<210> 11

<211> 57

<212> DNA

<213> Homo sapiens

<400> 11

tgccaggaac agattttgca gttggtgggg tctcaataaa agttatttttc cactgac 57

<210> 12

<211> 91

<212> DNA

<213> Homo sapiens

<400> 12

actcttaaat ttgattattc cataaaggtc aaatcatttt ggacagcttc ttttgaataa 60

agacctgatt atacaggcag tgagaaacat g 91

<210> 13

<211> 58

<212> DNA

<213> Homo sapiens

<400> 13

acccagcaat tttctatgat tttttcagat atagataata aacttatgaa cagcaact 58

<210> 14

<211> 33

<212> DNA

<213> Homo sapiens

<400> 14

atgtcttaag aacctaatta aatagctgac tac 33

<210> 15

<211> 67

<212> DNA

<213> Homo sapiens

<400> 15

ggtgtctacc atgattattt ttctaagctg gttggttaat aaacagtacc tgctctcaaa 60

ttgaaat 67

<210> 16

<211> 59

<212> DNA

<213> Homo sapiens

<400> 16

atgtacactg ttgagttttc tgtacataaa aataattgaa ataatacaaa ttttccttc 59

<210> 17

<211> 50

<212> DNA

<213> Homo sapiens

<400> 17

attcccgttt ctatccaaaa gagcaataaa aagttttcag tgaaatgtgc 50

<210> 18

<211> 46

<212> DNA

<213> Homo sapiens

<400> 18

gcacaaagga aaacatttca ataaaggatc atttgacaac tggtgg 46

<210> 19

<211> 3787

<212> DNA

<213> Homo sapiens

<400> 19

agccctcctg gggacttgga atcagtcggc agtcatgctg ggtctccacg tggtgtgttt 60

cgtgggaaca actgggcctg ggatggggct tcactgctgt gacttcctcc tgccagggga 120

tttggggctt tcttgaaaga cagtccaagc cctggataat gctttacttt ctgtgttgaa 180

gcactgttgg ttgtttggtt agtgactgat gtaaaacggt tttcttgtgg ggaggttaca 240

gaggctgact tcagagtgga cttgtgtttt ttctttttaa agaggcaagg ttgggctggt 300

gctcacagct gtaatcccag cactttgagg ttggctggga gttcaagacc agcctggcca 360

acatgtcaga actactaaaa ataaagaaat cagccatgct tggtgctgca cacttgtagt 420

tgcagctcct gggaggcaga ggtgagggat cacttaaccc aggaggcaga ggctgcactg 480

agccaggatc acgccactgc actctagcct gggcaacagt gagactgtct caaaaaaaaa 540

aaaagagaca gggtcttcgg cacccaggct ggagtacagt gccacaatca tggctcactg 600

cagtcttgaa ctcatggcct caagcagtcc tccctcagcc tcccaagtag aggggtttat 660

aggcacgaga ccctgcaccc aacctagagt tgcctttttt aagcaaagca gtttctagtt 720

aatgtagcat cttggacttt ggggcgtcat tcttaagctt gttgtgcccg gtaaccatgg 780

tcctcttgct ctgattaacc cttccttcaa tgggcttctt cacccagaca ccaaggtatg 840

agatggccct gccaagtgtc ggcctctcct gttaaacaaa aacattctaa agccattgtt 900

cttgcttcat ggacaagagg cagccagaga gagtgccagg gtgccctggt ctgagctggc 960

atccccatgt cttctgtgtc cgagggcagc atggtttctc gtgcagtgct cagacacagc 1020

ctgccctagt cctaccagct cacagcagca cctgctctcc ttggcagcta tggccatgac 1080

aacccgag aagcagcttc agggaccgag tcagattctg ttttgtctac atgcctctgc 1140

cgggtgccgg tattgaggca cccagggagc tgttactggc gtggaaatag gtgatgctgc 1200

1260

aacatctaaa catctagtag atgacttgca ggctgttggc taccagtttc ctgtctgagg 1320

1380

aggtggggccg ggtgcagtgg ctcacgcctg taatcctaac actgggaggc cgaggcgggt 1440

ggatcacctg atggtgaaac ctcatctcta ctgaaaatac aaaaattagc tgagtggtga 1500

cacacgcctg taatcccagc tacttggtag gcttgaaccc aggaggcaga gattgcagtg 1560

agccgagctg caccactgtg ctccagcctg ggtgacagcg agactcagtc tcaaaaaaag 1620

ttgtacaagg tggatggttg gaagcttgag cctaggctcg aatccctctc acgtgagagg 1680

gcctgaagat ttctggtgga ttccaacctg gctgaagact ggccgtgggg ggtgcagggg 1740

tctccagcgc tctgccctcc agcctgcttc ctccctgccc acaccgcact aggggaaggg 1800

cctttcctgc tgcctgcggg gccgcacctg gagtaggtaa tgccatgtgg tgacgtgaat 1860

ggagcagagg tctgtgcccc atcacaccgc cttgctgttt ttactgtggg acaaaagcac 1920

tctgatctgc gtgttccggg ggccctccta ccagccgact tgacgggaag tcagggttca 1980

ggtatcatct gtgcacctgg ggcggggtag tctgcactga acctgccaga gtcccctcct 2040

catttcactg aaagtcacag tctccagggc tgtgttgcta accttacgtt ctctccgttt 2100

2160

gctgttggcc ggtgctctta gcaagaggtc atttttctag gttgcgctgg gacattgtga 2220

gtttggtgag ggtcatggat gtgggctggg ctgggctggg ctgggccggg ctgcctgctg 2280

cctgctgctc ccctacctga aatgcagcta gtgcggctct gcccttcctg gggctgagga 2340

aggcttctgc aggatagctg gggggctggg caggtggggtg aggcagcctc cctgctgaca 2400

ctcagtcctt gtagctggag caagatctcc tgatccaggt acgggcctgt ctgctccaag 2460

aaagactctg ccaccagatg caaaggggcc ctttgtttta acttagtccc tggggaccgc 2520

ctgattcagc acctgtcggc ccaggatacc ccgctggtgg ggacaagtgc ctgagtgtgg 2580

gccgtgcccg agtgtggcca tccctgagtg gggccgtcct gactaggaag tggcttttca 2640

gttgtgatgt gtgggcctga cctagggggc gctgtggaac ccggggctgga accagccctc 2700

tgtgccaggc cgcagacagg ttccgccggc cctgaggggc agctgccatg gcgtggggtca 2760

ctgggagctg agaggaaggg cccccaccgc acctcaggca aagcggctct gggaacacct 2820

tgatttcgtc catgtgagcc gtcccaggga gggcagccaa gctgtgaagc ctgagaaact 2880

gacctgtgtg cccacgagctt gtggtctgct gcccggtgga ggaagtgcag gtgcgcccag 2940

gctcctcatt ccgttttgca ggattccttc ggggtgtgag catttcctat tcagcctgtc 3000

3060

ttacggcagg ttcatctctg gggcagcctc ccacagtggg tggggctttg ccagcagtgc 3120

ccacgggggt catggggcca ggcgcgctcc ggcgcctgca gaactgatcg gggatagtct 3180

caggaggcgc tagtcacgtg ccccggtgat cggggatagt ctcagaaggc gctagtctcc 3240

tgccccggtg atcggggata gtctcaggag gcacgagtcg cctgcctcgg tgatgcaccg 3300

tttctcacac cggctgctct ggcccgagct aaaggggaag acgtgtgcgg ataggagctg 3360

cacacaattt tcctccatgt attgtttatt ttgctttttc ttttggctag acattaggaa 3420

tttcagtttt cccaagttgt atttttcctt ttctatttta aaattatcat gcagggctgg 3480

gtgaggtcgc tcacgcctat agtctcaaaa ctttgggagg ctgagggggg aggatggcat 3540

gagccgga gtttaaggct gcagtgagcc gagatcgctc cactgtcctc cagcctgcat 3600

gacagagcga gaccctatct caggaaaaaa aaaaacaaaa ctattatgca gtagtttcga 3660

ccctggaaga cgagtgtgca tctttgagtt gtaacacgtg tacctcgccc atccaggcgt 3720

agtttcattt ggaatctggt tatcctgtag ttgctttgtt aaaaatatat gtaattgcaa 3780

atcattt 3787

<210> 20

<211> 63

<212> DNA

<213> Homo sapiens

<400> 20

ttctccagta tatttgtaaa aaataaaaaa aaaaactaaa cccattaaaa agtatttgtt 60

tgc 63

<210> 21

<211> 45

<212> DNA

<213> Homo sapiens

<400> 21

ccctggatcc tactctctta ttaaaaagat ttttgctgac agtgc 45

<210> 22

<211> 61

<212> DNA

<213> Homo sapiens

<400> 22

gtgcagggcc ctcgtccggg tgtgccccaa ataaactcag gaacgccccg gtgctcgccg 60

c 61

<210> 23

<211> 79

<212> DNA

<213> Homo sapiens

<400> 23

aacctcccac tttgtctgta catactggcc tctgtgatta catagatcag ccattaaaat 60

aaaacaagcc ttaatctgc 79

<210> 24

<211> 38

<212> DNA

<213> Homo sapiens

<400> 24

taggtgttaa aaaaaaaaat aaaggacctc tgggctac 38

<210> 25

<211> 1651

<212> DNA

<213> Homo sapiens

<400> 25

atttcattta tctggaaaat tttgtatgag ttcttgaata aaacttggga accaaaatgg 60

tggtttatcc ttgtatctct gcagtgtgga ttgaacagaa aattggaaat catagtcaaa 120

180

aaaatttcaa ttctcgtggt aataccagag tagaaggaga gggtgacttt accgaactga 240

cagccattgg ggaggcagat gcgggtgtgg aggtgtgggc tgaaggtagt gactgtttga 300

ttttaaaaag tgtgactgtc agttgtatct gttgcttttc tcaatgattc agggatacaa 360

atgggcttct ctcattcatt aaaagaaaac gcgacatctt tctaagattc tctgtgggaa 420

aatgactgtc aataaaatgc gggtttctgg gccattcgtc ttactttcat tttttgatta 480

caaatttctc ttgacgcaca caattatgtc tgctaatcct cttcttccta gagagagaaa 540

ctgtgctcct tcagtgttgc tgccataaag gggtttgggg aatcgattgt aaaagtccca 600

ggttctaaat taactaaatg tgtacagaaa tgaacgtgta agtaatgttt ctacaggtct 660

ttgcaacaaa ctgtcacttt cgtctccagc agagggagct gtaggaatag tgcttccaga 720

tgtggtctcc cgtgtggggc ccagcaatgg gggcccctga tgccaagagc tctggaggtt 780

cttgaaagag gggacacgaa ggaggagtga ctgggaagcc tcccatgcca aggagggtggg 840

aggtgccctg gaaatagctg cctcatgcca cttaggccat gactggattt aatgtcagtg 900

gtgtgccaca gtgcagaggc tagacaactg aaaggggcta ccaaggctgg gaaaaaaatg 960

caattgttgc tgtgagtgac tttgaaagac tctggtgcct tgtggtgccc ttctgaaatt 1020

caaacagtaa tgcaaaagtg tctgcattag aatttacggt gtctaaaatt catgttttta 1080

1140

ggaagttcaa tgctcagtcc taccttggct cccattaaac atttggtgct ctgtggattg 1200

agttgaacgt gttgaggctt tgcaatttca cttgtgttaa aggctctggc atttttccat 1260

ttctatgcaa atttctttga agcagaattg cttgcatatt tcttctctgc cgtcacagaa 1320

agcagagttt ctttcaaact tcactgaggc atcagttgct ctttggcaat gtcccttaac 1380

catgattatt aactaagttt gtggcttgag tttacaaatt ctacttgttg cattgatgtt 1440

cccatgtagt aagtcatttt tagtttggtt gtgaaaaaac cctgggctga agttggcatt 1500

tcagttaaaa gaaaaaaaga aactagtccc agatttgaaa acttgtaata aaattgaaac 1560

1620

aaagtggcta acacctggct actcttactg t 1651

<210> 26

<211> 474

<212> DNA

<213> Homo sapiens

<400> 26

actgagtcca gctgcctaat tctgaatata tatatatata tatcttttca ccatatacat 60

gcctgtctgt caatttctgg ttgggctggg aggccacaca cacacactga catgacaggg 120

cttgggcaag actcctgttc tacttatcct tttgaaatac ctcaccctgc cactccacca 180

tgtatgatca ttccagagat ctttgtgact agagttagtg tcctaggaaa accagaactc 240

agaacttgcc tccatggttg agtaacaagc tgtacaagaa ccccttttat ccctggaaga 300

360

gtggattggc tggctctctg gtagcatttt gtcctcacac acccatctac tatgtccaac 420

cggtctgtct gcttccctca ccccttgccc aataaaggac aaggacttca gagg 474

<210> 27

<211> 36

<212> DNA

<213> Homo sapiens

<400> 27

attcagcatt aaaataaatg taattaaaag gaaaag 36

<210> 28

<211> 45

<212> DNA

<213> Homo sapiens

<400> 28

actggcagat tagattttta aataaagatt ggattataac tctag 45

<210> 29

<211> 48

<212> DNA

<213> Homo sapiens

<400> 29

agtaatctta tatacaagct ttgattaaaa cttgaaacaa agagcctg 48

<210> 30

<211> 32

<212> DNA

<213> Homo sapiens

<400> 30

atgctttgtt ttgatcatta aaaattataa ag 32

<210> 31

<211> 90

<212> DNA

<213> Homo sapiens

<400> 31

accttttcac ctacaaaatt tcacctgcaa accttaaacc tgcaaaattt tcctttaata 60

aaatttgctt gttttaaaaa cattgtatct 90

<210> 32

<211> 4065

<212> DNA

<213> Homo sapiens

<400> 32

agccacatgg agggagtttc attaaatgct aactactttt tccttgtggt gtgagtgtag 60

gttcttcagt ggcacctcta catcctgtgt gcattgggag cccaggttct agtacttagg 120

gtatgaagac atggggtcct ctcctgactt ccctcaaata tatggtaaac gtaagaccaa 180

cacagacgtt ggccagttaa acatttctgt ttataaagtc agaataatac ctgttgatca 240

ctgaaaggcc tgcatgtatt gtactctgaa ttttacagtg aatgagagaa tgtaccctaa 300

ttgttcaaca gggctcaaaa ggaaagattc cattttgatg ggtcacattc taaagagggg 360

cagtgtgata ggaatgagat ggtcctttag gacttaagtt ctcagcccaa ggtttttcca 420

cgtggccccc tcatcttttt ttttttttta aacggagtct ctcttgccag gctggagtgc 480

agtggcacga tctcggctca ctgcagcctc cgcctcccag gttaagcgat tctcctgcct 540

cagcttcctg actaactggg attacaggcg cccaccacca tgcccagcta atttttgtat 600

tttcagtaga gatggggttt caccatgttg gccatgctgg tctctaactc ctaacctcaa 660

gtgatctgcc cacatcggcc tccaaaagtt ctgggattat agtgtgagcc actgcgcccg 720

gccatggctc cttaatcttg atccaaatta ttgttacatc cagaatgtga tgaatcaaaa 780

tctcgagatg ggggtccagc aatctgaaat ttcagtatgc cagggctttt ctgtatgtca 840

aagtgggttt gaaatagtta atttttcttc tagtctgaaa tgtatcggga aaatttggaa 900

atcctgaagg ctggaaattg aaataagttt ttctaggatt tgtgtctctt gctattggaa 960

aactgatggt gaccaattca tgtttacaaa taagatcctc atagatctcg gtaaattata 1020

atttgctaca gttttatggt tcttcctgtg attttgagct ttttttgacc caaaataata 1080

cagtctaaaa ctatagacaa ataagatggc acttagactc ctgggtttta gttagtggag 1140

gtttccttag tgcactgtgg ggtcataata agccgagaac catggctgtc tatgggacac 1200

atctgtcagg acaaccttta gaggatgttg gggatcaaat agaaggcaca gagaagcact 1260

gaattggctt acataagaat aggctagaat tacaagtagt gaaacctcga ttcagctgga 1320

caattttaaa caaatgtatc atttggcttg tatcttctgt tgtgctggag aagttagaaa 1380

taagggctct ccagaccagc ctgaccaacc tggagaaacc ttgtctctac taaatacaca 1440

aaattagcca ggcgtggtgg cacatgcctg taatcccagc tactttggag gctgagccag 1500

gagaatctcc aggaggcggga ggttgctgtg agccgagatc gtgccattgc actccagctt 1560

gggcaacaag agtgaaactc tgtccacccc ccccaaaaaa agtaagggct ctccattagg 1620

gcccatagag gacttgtaat atggaacctg aatccaagga tcccacaata agtggtcagt 1680

agttcatgat gaattaaaag actcaatatt tggtcttcac ccaatacctg tgtgactttt 1740

agtcctaatt tcctcatctt taaaatttca gtgaaagtgc ctacctgagg attgtgtaga 1800

ttaaaatgga aaccgtgcac ttaatttttt gttttgtttt gagacggagt ctcgctctgt 1860

cgcccaggct ggagtgcagt ggtgcgatct cagatcactg caagctccgc ctcctaggtt 1920

cagaccattc tcctgcctca gcttcccaag tagctgggac tacaggcgcc cgccactgcg 1980

accgtgttag ccaggatggt 2040

ctcgatctcc tgatctgccc gcctcagcct cccaaagtgc tgggattaca ggcatgagcc 2100

accgcgcccg gcccaggcac ttaatttttg tgtttgactt agtaacttaa gtgcaaacta 2160

ttacgggagc agatggagtc aattggcctt catgtgattg tcagtgggaa attggtccaa 2220

gcagagggaa tactggttca ggaaactggt ttgggaaggt taggcaaacg ggaagtgcta 2280

tggtggagag aaagattact ctggccgggc tgtaaaggac ggctacaatg ggaggctgaa 2340

ggcagaacca agaaaatggg agtgagtatg gaaaaggtac gattcagacg gcataatgga 2400

2460

tagagtttgg ggggaaggat ccctagtccc ttaatggcta ccttcttccc caggagttgt 2520

taggccatcc gatcccctgg cctgggaaag aaacactgat ttcgttgctg gcttgttcac 2580

tcaccagaag ctacagctac taacagttct aaaaactgtt tcatgtgatg aggaacagac 2640

gaaaatagtt ttgagccta agtccgccga ttccagtgct ttcttgaacc cgcatttact 2700

aaaatatttt catgactgcc aagctttgaa tagcctgctg tgttcatgga ggctcatact 2760

ggcgatctct agtggctggc taaagcttga attgcaaaag atctaatttc tggtctaatg 2820

tatatatgcc ttaaatatag ttgcgttcaa acgtgggagc tgcaggtgca acttgatttt 2880

atgacaaatg gctgccacat aatttgcaca agcagtgctc gtcaagggca gctaaatcag 2940

gcgagctttc aatcaaaata aatgtactac taaaccctac ttagcggcta actagcccaa 3000

3060

cacctggtgg ctccatcggc cgcgttcatc agtcagcacg acccgacctc agtggcgtcc 3120

3180

agcttctact tccggagacg atgactcccc cgcgtcccag accggaagaa gcccggcgga 3240

gaccggcctc gctcggccac ttccggcaag ggcggagccg gccagtggtg cgcgagcgca 3300

gtaactccc ctggagaggc gggatgttca actccacccc tggtccttgg gcggccgtgg 3360

gtccccttcg aagcggagga atggccaacc tcgccgcact tcgagcccct ttagggtgcg 3420

tttaagaaca gtgggcgtgg cctttacgta aatcttcgag atgggaacct ccagaatttg 3480

tctcaattgt ctaaaaggta atgagcgtca gcgacattca agggcacttt gggctaaaaa 3540

agaaagtgct tgtacacgga tggaaatatt ctagaagaac ataaaaggaa tttcctctta 3600

ggaggttagg gaaatgagca cgaagtatgt ttttggtgcag ttttttgttc aacccaatgc 3660

gtattttcat attgagaggc aatataaatg gagcgaaagt atcttgagaa aaaaaaaaaa 3720

actaccagaa cttgccgttg ctgaaaagta atattttctc tttcgagagt tttcatggcc 3780

ttttaaatta cacccccacc tccacaggca aataaatttg ttttggaatg cataccacat 3840

catctggctc tagaaacgta ttttgtgtag ctcccctagc aagaatatag gttaaagcgt 3900

3960

acgttgtttc aaataaccta gtttgtgtat ccctgtaagt cattttggta taaagtaggt 4020

tataagtgta catgcgaaaa gatgttttta acaaaaatgt aactg 4065

<210> 33

<211> 3865

<212> DNA

<213> Homo sapiens

<400> 33

gccttgctct gctcccccgc ccccaggcag ccatccgcag ggccagcgcc atcctgcgca 60

gccagaagcc tgtgatggtg aagaggaagc ggacccgccc caccaagagc tcctgagccc 120

cctgccccca gagcaataaa gtcagctggc tttctcacct gcctcgactg ggcctccctt 180

tttgaaacgc tctggggagc tctggccctg tgtgttgtca ttcaggccat gtcatcaaaa 240

ctctgcatgt caccttgtcc atctggaggt gatgtcaatg gctggccatg caggaggggt 300

ggggtagctg ccttgtccct ggtgagggca agggtcactg tcttcacaga aaaagtttgc 360

tgacttgtga ttgagaccta ctgtcccatt gtgaggtggc ctgaagaatc ccagctgggg 420

cagtggcttc cattcagaag aagaaaggcc ttttctagcc cagaagggtg caggctgagg 480

gctggggccct gggccctggt gctgtagcac ggtttgggga cttggggtgt tcccaagacc 540

tgggggacga cagacatcac gggaggaaga tgagatgact tttgcatcca gggagtgggt 600

gcagccacat ttggagggga tgggctttac ttgatgcaac ctcatctctg agatgggcaa 660

cttggtgggt ggtggcttat aactgtaagg gagatggcag ccccagggta cagccagcag 720

gcattgagca gccttagcat tgtcccccta ctcccgtcct ccaggtgtcc ccatccctcc 780

cctgtctctt tgagctggct cttgtcactt aggtctcatc tcagtggccg ctcctgggcc 840

accctgtcac ccaagctttc ctgattgccc agccctcttg tttcctttgg cctgtttgct 900

ccctagtgtt tattacagct tgtgaggcca ggagtttgag accatcctag gcaacataat 960

gagacaccgt ctctaaaata aaattagctg ggtgtggtgg tgcaccgcct gtggtcccag 1020

ctcctcagag gttgagtaga ggctgaggtg agcggagcac ttgagccaag agtatgaggc 1080

1140

tgaggcctgg atggggaaag agtcctgctg ttgatcctca catgtttcct gggcacctaa 1200

ctctgtcagc cactgccagg gaccaaggat ccagcatcca tggcacccct ggttcctgcc 1260

atcctggggt acccgattca aagaaggact ctgctccctg tctgagacca cccccggctc 1320

1380

ctgggagccc tgcgttttga ggcagaccac tgcccttccg acctcagtcc tgtctgctcc 1440

agtcttgccc agctcgaagg agagcagatc tgaccacttg ccagcccctg tctgctgtga 1500

attaccattt cctttgtcct tcccttagtt gggtctatta gctcagattg agaggtgttg 1560

ccttaaaact gagttgggtg acttggtacc tgctcaggac cccccgcact gtcccaatcc 1620

cactcaggcc cacctccagc tggcctcact ccgctggtga cttcgtacct gctcaggagc 1680

ccccactgtc ccagtcccac tcaggcccat ctctggctgg cctcactgcg ctgggactcc 1740

gccttcataa ggagagctca ctgctcacgt tagtagatgg ccccttctcg tgaggcctct 1800

cccctggcac ctgcttcagt tgtcctccac agcactgatt tgcagcccac aagctggcag 1860

1920

atataatgag atgcttgatg aatggtgcat attgaatgta taaagcccac cggtcctgag 1980

agtttgctca ctggagactt tctggagatg gagtctcgct ctgttgccca ggctggcgag 2040

tgcaatggcg cgatcttggc tcactgcagc ctccacctcc tgggttcaag cgattctcct 2100

gcctcagcct cccgagtagc tgggattaca ggtgggtgtc accacaccca gctcagtatt 2160

gtatttttag cagagatggg gtttcaccat tttgcccagg ctggtttgga actcctgact 2220

2280

ctgatgtggc tgctgctgct cagaaggcct tgtccttaac cacctccttg cctgccctgg 2340

aggcttgtgc ctctaggccc caccccctgt ggagtcctgc tggctttctc catccctatc 2400

tgaatcctcc ctgctgtgtg gcctcccctg gtctcatccg taacacagcc cagcttagtg 2460

ggcctctgtt cctgcgggtg gccagcctgt ctgtgtggct gggctgggga ggccacgtct 2520

ggtatctgaa tgctatcggt gggttggggt ggaggaacca ggagaggggct ggagggaggg 2580

agatggtctc agccccacag agtttggagt cctcagtgtg ctgagcaaac gtggagacac 2640

catttccctc ctctagacct catcttggag agagagatgt tggatggggc catctattcc 2700

agcttttattc acacaaatca tgtctgttgg cctggaaatt ggaaaaccag ttaaaccaaa 2760

aacatgatat taagaaaaca ggcaggctca ccatagtaaa aatgctgaaa gccaaagaca 2820

aaattgggag aacaaaagaa aagcgtcttg tcacatacag aaggtccctg ataaagttag 2880

tagctgccct catcagaaac caggcccagg cagtggggac acatccagag tgctgaaaga 2940

acctccccca ggtcatccta tccccaagag tgatgcccgg cagcattccc agctcaggggc 3000

3060

atgcccagct gtggggactt gggcagctcg tttagtagca ccgtgcctca gtttcccata 3120

tgtaaaaggc cattttgagt gcctttcaca gccctgcata aggcaggtgt ctcagtgttc 3180

actgctgtct ctccagctct tagtccagta gctgcatggt gagtgagcgt agggcgcacc 3240

ctggaaggct gccaagccca aagttgtgca gagcgctggg gactccagac tccccacagc 3300

agcagagact cgggactgag gcatcctctg ttcacaggac atgctggcat ctactgggtc 3360

agggctctgc tgctcggtgg ctgtgcaacc ttgggcaagt tcctcaacct ctctgtgtct 3420

tcgtaccctc atctgtaaca tgcgtgtcga tagaccctac tactcagggt tgatgagaag 3480

attaaatgtg caaaacctgc ttgactgtgc ccacaaatcc tgattgtagg aataaattaa 3540

tgacttttta taaatatttt gatcagatgg actcatgatc acagatgtct tcacatgcct 3600

3660

atccatgtgc agtaatgcct ggtggctcca ggtctgcccc gccgtcctgt ggggctgtga 3720

gctttcccag cctcctgccg gtgtttgtga atatcattct gtcctcagct gcatttccag 3780

cccaggctgt ttggcgctgc cgggaatgg tatcaattcc cctgtttctc ttgtagccag 3840

ttactagaat aaaatcatct acttt 3865

<210> 34

<211> 288

<212> DNA

<213> Homo sapiens

<400> 34

ttttttactg tcaggcagga agagcggtaa ctgccatcgc ggcgggcatc cctggcgcca 60

gggtgttggt ctgggtaccg gcttccctct cggccgactt gtcagctctg tgagccgcgc 120

gcgtctgagc ccgtgtcctc acctgtaaag tggagaaatg aaaaaggacc tgaacttcct 180

240

gtcagggtgg ggatggatgg tttcatccca caggtgggaa gattgagg 288

<210> 35

<211> 430

<212> DNA

<213> Homo sapiens

<400> 35

gtttttctca ggtccttgat tggaactgcc tcagagccaa gggtcctttt actcagtggc 60

agcaacaaac gcagtctgtt ggctagtgat cctcctgtct cagggacacg tagtccaggg 120

agcagccaat tgcttggcac ttggggaccc cgttctgggg agtcctgaaa gctttcacct 180

cttggattgc cgaatacatg ggtggccctt cctagactaa gggactggcc tgagtgaggc 240

tgggcctctc agccaagctg atgttgaacc actgctgtgg ggatggggcct ggggttcctg 300

ggaagctgtt catacccatt gccaggagcg tgggctctgg ctggacctgg atcagatcct 360

aactgaagcg gcagctttct ggcatgagaa aggagtgttt tcatggtgga cagaattgggg 420

ctatgagtgt 430

<210> 36

<211> 120

<212> DNA

<213> Homo sapiens

<400> 36

atatctctgc caacatgagg acagaaggac tggtgcgacc ccccaccccc gcccctgggc 60

taccatctgc atggggctgg ggtcctcctg tgctatttgt acaaataaac ctgaggcagg 120

<210> 37

<211> 655

<212> DNA

<213> Homo sapiens

<400> 37

agggagccct cctggaagtg gatgaggcct tgggtctcgg ctcttcattg cttcctgagc 60

tgcagcagat gcctttacaa ccaagctcac cgaggacgtc tgtctcccat attaccctgg 120

cagaggggcca ggcctgttct acacggccgg ggtttcaaca aggtactgat gtcttctgcc 180

cttgcctctt cgacaggcaa gtaataagac ttaagtgaag agaattcttt aggcacacaa 240

attcacattt gatgtaatct cattatactt cctgatctgt gattgaaaac tttcatttcg 300

360

gaattaagtc tatttaaatg aaaaaaactt agatatgagt ctgcatggcc tcaggaaaat 420

gatgttttaa aatagagatt ttaggttgtc tgcactctag cttttttgtc gttttcttaa 480

ggctttttta actgcatcaa aaattcagat acgaaacata cactaaaaaa taatacatca 540

600

cagatatgtt caaaaaggtc tgaacaattg attttctgaa accatgaagg actac 655

<210> 38

<211> 331

<212> DNA

<213> Homo sapiens

<400> 38

agccatttaa attcattaga aaaatgtcct tacctcttaa aatgtgaatt catctgttaa 60

gctagggggtg acacacgtca ttgtaccctt tttaaattgt tggtgtggga agatgctaaa 120

gaatgcaaaa ctgatccata tctgggatgt aaaaaggttg tggaaaatag aatgcccaga 180

240

ctgttacttc ctttacagat ctacagacag tcaatgtgga tgagaactaa tcgctgatcg 300

tcagatcaaa taaagttata aaattgcctt c 331

<210> 39

<211> 1152

<212> DNA

<213> Homo sapiens

<400> 39

gcagctcatg tgcacgtttt ctgtttaaat aaatgtaaaa actgccatct ggcatcttcc 60

ttccttgatt ttaagtcttc agcttcttgg ccaacttagt ttgccacaga gattgttctt 120

ttgcttaagc ccctttggaa tctcccattt ggaggggatt tgtaaaggac actcagtcct 180

tgaacagggg aatgtggcct caagtgcaca gactagcctt agtcatctcc agttgaggct 240

gggtatgagg ggtacagact tggccctcac accaggtagg ttctgagaca cttgaagaag 300

cttgtggctc ccaagccaca agtagtcatt cttagccttg cttttgtaaa gttaggtgac 360

aagttattcc atgtgatgct tgtgagaatt gagaaaatat gcatggaaat atccagatga 420

atttcttaca cagattctta cgggatgcct aaattgcatc ctgtaacttc tgtccaaaaa 480

gaacaggatg atgtacaaat tgctcttcca ggtaatccac cacggttaac tggaaaagca 540

ctttcagtct cctataaccc tcccaccagc tgctgcttca ggtataatgt tacagcagtt 600

tgccaaggcg gggacctaac tggtgacaat tgagcctctt gactggtact cagaatttag 660

tgacacgtgg tcctgatttt ttttggagac ggggtcttgc tctcacccag gctgggagtg 720

cagtggcaca ctgactacag ccttgacctc cccaggctca ggtgatcttc ccacctcagc 780

cttccaagta gctgggacta cagatgcaca cctccaaacc tgggtagttt ttgaagtttt 840

tttgtagagg tggtctagcc atgttgccta ggctcccgaa ctcctgagct caagcaatcc 900

tgcttcagcc tcccaaagta ctgggattac aggcatcttc tgtagtatat aggtcatgag 960

ggatatggga tgtggtactt atgagacaga aatgcttaca ggatgttttt ctgtaaccat 1020

cctggtcaac ttagcagaaa tgctgcgctg ggtataataa agcttttcta cttctagtct 1080

agacaggaat cttacagatt gtctcctgtt caaaacctag tcataaatat ttataatgca 1140

aactggtcct tc 1152

<210> 40

<211> 75

<212> DNA

<213> Homo sapiens

<400> 40

actaacgaaa aatcaataaa taaatgtgga tttgtgctct tgtattttta agtggattaa 60

aaaacttact acctt 75

<210> 41

<211> 1176

<212> DNA

<213> Homo sapiens

<400> 41

gaatgtcaac gattagtcat gcaataaatg ttctggtttt aaaaaataca tatctggttt 60

tggtaaggta tttttaatca attaggcttg tagtatcagt gaaatactgt aggtttaggg 120

actgggctag cttcatatca gatttacttg ttaagtgact gttttggaat gtttactttt 180

ggactgggtt tgtaacacgg ttaaaggcaa tgagaaacaa gcagaattcc aggagtcctt 240

gaagcagagg gcactggaag acaatatagc agattaaaat agcacagctc atgtggcata 300

ggtgggtatt ttagatgttt gagtaaattt gaaagagtat gatgtttaaa ttacctttag 360

caacatgttc atctgctatg ctgtcatgac tagggggatg attattagtc acatagagct 420

tgggagtacc actggaaacg tatgggtagg agtttaggtg gcttctgttt ttcaaaagat 480

gatcttatcc tagtatctgt aatgctcact tggcacacct gacttgtggg ctgtgtgtaa 540

ggtggctagc taagtgaaaa aagcctgcta ggtgtgagtc aacttaagaa tatgtaaata 600

ggtttgagaa aaagtagggc ttgggtgcaa gtaaagattg agcaggaaat aaaggaaaat 660

caagtataat ccctgagatt tgtagactaa aggcaatgat gtgggactac ttggtcgaat 720

ttttttagcc ctcaacttgg taattgggtg tttctgtgtt aaagcactga aacttgctgt 780

cgtgccttcc tagttttcgt ggtttattga cagggttggg ggtttttttt gtttttttaa 840

aatgaaggga caaagtcaac tggactgctg agtgagaggg caggggcagt tgaagggaac 900

atgaattgct ggaacagcta cataaaatag tgatgtagcc aagtcatgct atttaaatta 960

taattctcca ctgtgtttag aataacatct gaggttctta acctggcctt ggaagggtat 1020

cacttttact tgtaacctgg aatggcttta taatgtgcta gctaattgct actctcatct 1080

tgtattttaa ctcctaattt acccttcagg tctcagcttc agaacattca cttataaaga 1140

aaccctgctg attaaatctc tcttgggctt cctccc 1176

<210> 42

<211> 59

<212> DNA

<213> Homo sapiens

<400> 42

acgctcctct actctttgag acatcactgg cctataataa atgggttaat ttatgtaac 59

<210> 43

<211> 49

<212> DNA

<213> Homo sapiens

<400> 43

accagacaca ctgattggaa ctgtattata ttaaaatact aaaaatcct 49

<210> 44

<211> 178

<212> DNA

<213> Homo sapiens

<400> 44

ggaattgcac atgagatggc acacatattt atgctgtctg aaggtcacga tcatgttacc 60

atatcaagct gaaaatgtca ccactatctg gagatttcga cgtgttttcc tctctgaatc 120

tgttatgaac acgttggttg gctggattca gtaataaata tgtaaggcct ttcttttt 178

<210> 45

<211> 73

<212> DNA

<213> Homo sapiens

<400> 45

tcaccaaaaa gcaaccaact tagccagttt tatttgcaaa acaaggaaat aaaggcttac 60

ttctttaaaa agt 73

<210> 46

<211> 38

<212> DNA

<213> Homo sapiens

<400> 46

acctctttta taacatgttc aataaaaagc tgaacttt 38

<210> 47

<211> 40

<212> DNA

<213> Homo sapiens

<400> 47

attcctgctc ccctgcaaat aaagcctttt tacacatctc 40

<210> 48

<211> 1301

<212> DNA

<213> Homo sapiens

<400> 48

cagggtctcc ttggcagctg tattctggag tctggatgtt gctctctaaa gacctttaat 60

aaaattttgt acaaagacac aaggtctgac tagactgttc agtattcaga ctgaggggca 120

tgttggcctc tggagcatta catatcttct tggttttaac catacttgtg gtatttgcaa 180

gggccagaac agtaagaccc aagcagagcc aaccagagaa ataatatttg tgtgatagag 240

aaggctgata gcaagcaagg cagcaccttg attcgttgtc ctgtagttca ggattgtagg 300

360

aaagtgaata taaatggcca ttatattttg tgtcatgctg tgctctaagt gttctttaca 420

tatgtactcg ttaatcaacc tctctaaagt gtaaaggaaa tttgcttgca ccactgaagg 480

cacataaggc tcagaagtaa atttgcctaa gcagtataaa gctatcatta gaatccacat 540

tcctaagttg tgttctctta ggggatcatg gaaccagtca ttggtactac aggctattat 600

gttctggaga actgtgaaga acatttaaat tgtctctgat tttatctatc aatgttttga 660

agtattttct accagtgtct gtacttcaca agaaattcgg cactattttt tcaggcaaaa 720

ctagtgaggg acaggttggc ttgaaaatca tgagactgtt gttaaatcag atgctggttg 780

atcacagagg ggacttccag ggaaagctgt tatcaggtgg ctgcttcctg gtgatgcagc 840

ctggctgatg agataaccct ggctccacag atggcttagc aggtgctgtg atgatttggt 900

tttcttctca attagactga gctgcacatg gtgtttatat tgcttggcac atggtaaggg 960

cttaatattt gaggtaatta tgtagggcgt acactgacaa gtatctgacc cccccttcct 1020

ttttgactca taaattggtc atcttaacca tttaagtgta cacttctata gtgacagagt 1080

tagccctctg tccaagggat ttgcatctgt ggattcaacc aactttggggt caaaaataat 1140

1200

ctatattttc ttgtcactta ttttcttgta cactgcagtt gtaacagcta tgtagcatgt 1260

acattaggta ttaaaagtaa tccagtgaag attgaaagtc t 1301

<210> 49

<211> 576

<212> DNA

<213> Homo sapiens

<400> 49

cagcctcttc catgagtggg gagcccgctg cttgtctcca gctcctagca gtgagtcctg 60

ataatctcaa atttaaggac agtaactttg tctgggatga gtgtgggaaa ggatgtgttt 120

gggaacagac gcgagcctgc agaggtgttt gtaaccatct ctttctaagt ggtgggaagc 180

agacatttta ttctttaact gttaatatat atagtgtgtg ttttttatgc atgaaatatt 240

ttatagtttt taaaaatgcc cacactacta ttttgaaagt aaatgaggta atgtatgtgt 300

cagaacccaa tacccaaagc gatcgtagta agaggtgggg cctttgggaa ggcattaaat 360

tgcttaggga atgagggtgg aaccctcatg aatgagatta gagccttata ggagaggttg 420

gagggagttg cctggcctcc ctctcccatg tgaagactca gcaagaaaac attatttagg 480

aagcagagag ccctcatcaa acaccagatc tgctggccac ctgatctggc actttccagc 540

cttcagaact gtgagaaata aatttctgtt gtctat 576

<210> 50

<211> 57

<212> DNA

<213> Homo sapiens

<400> 50

agttcagact tcaaatagtg gcaaataaaa agtgctattt gtgatggttt gcttctg 57

<210> 51

<211> 1324

<212> DNA

<213> Homo sapiens

<400> 51

agctcacgtt gatgtcaaga ctaccgatgg ttacttgctt cgtctgttct gtgttggttt 60

tactaaaaaa cgcaacaatc agatacggaa gacctcttat gctcagcacc aacaggtccg 120

ccaaatccgg aagaagatga tggaaatcat gacccgagag gtgcagacaa atgacttgaa 180

agaagtggtc aataaattgt aagtgtttct ttgcttcctc acacaacaca accttgagta 240

ttggattatt cctgagatga gagaacgcat atgagacaag gtaaaggtct gttgaaatcc 300

360

tatgattatt attactatta ttatttgagt cagagtcttg ctctgttgcc caggctcgag 420

tgcagtattg tgatcctcct tggctcactg taaccactgc ttcctggggtt caagcagttc 480

ttgagcctca gcctcctgag tatctgggaa tacaggggac tgccaccata cccagctaat 540

ttttttaaat ttttagtaga gatggggttt catcatgttg gccaggctgg tcttgaactc 600

ctgacttcag gtgatctgcc agtactctaa atgataacag ttttttcgtg tttatttatt 660

ttgaatgaag ctgtctcaca gtagatggag ttgaaggaca ggaaatgttt ttccctact 720

tggaaaatac actgaataag ttgagtgggg tgggatgtgc ctggagtccc agctactcag 780

gaggctgagg tggtaggatt gtttgagccc aggagtttga ggccagcctg ggcaatatag 840

ggagaccctg tcccaaaaaa taaaaaatat acgtatatat atatacacac acaaagaaaa 900

aatacactga atagacaaaa cctttcatga ttaatgatgc acgggaataa gtgatgaaaa 960

aagtttcggt cccagatgat ggccagtgat aacaacattt ttctgatgtt cccatgcaat 1020

atacagttag ctaaaggggt gtaatggaaa aagcataagg cttggactca gaagactcta 1080

ctaactttgc cactagctag ctatgtaatt cagatcatct atcctttaca tgtgaaaggt 1140

aaataatggc ttatcttaac aggaggattt atgcaggtta aatgaggtag gtgttatgtg 1200

taggtttatt ccaaggcttc tctactttta aaggaaatgg cttatatctg agaactagga 1260

cttttagaaa aaaatttact gttactggtt tgcaggattc cagacagcat tggaaaagac 1320

atag 1324

<210> 52

<211> 68

<212> DNA

<213> Homo sapiens

<400> 52

aatgggtccc tgggtgacat gtcagatctt tgtacgtaat taaaaatatt gtggcaggat 60

taatagca 68

<210> 53

<211> 60

<212> DNA

<213> Homo sapiens

<400> 53

attgcagtag cagcatatct ttttttcttt gcacaaataa acagtgaatt ctcgtttctt 60

<210> 54

<211> 53

<212> DNA

<213> Homo sapiens

<400> 54

ttttcccagc tgctgcccaa taaacctgtc tgccctttgg ggcagtccca gcc 53

<210> 55

<211> 37

<212> DNA

<213> Homo sapiens

<400> 55

gattttttga gtaacaaata aataagatca gactctg 37

<210> 56

<211> 36

<212> DNA

<213> Homo sapiens

<400> 56

acaaaaatga ctaaataaaa agtatatatt cacagt 36

<210> 57

<211> 55

<212> DNA

<213> Homo sapiens

<400> 57

atccttgttt tgtcttcacc catgtaataa aggtgtttat tgttttgttc ccaca 55

<210> 58

<211> 70

<212> DNA

<213> Homo sapiens

<400> 58

gtccacctgt ccctcctggg ctgctggatt gtctcgtttt cctgccaaat aaacaggatc 60

agcgctttac 70

<210> 59

<211> 50

<212> DNA

<213> Homo sapiens

<400> 59

aattggagag gattcttttg cattgaataa acttacagcc aaaaaacctt 50

<210> 60

<211> 77

<212> DNA

<213> Homo sapiens

<400> 60

ggctggacat cggcccgctc cccacaatga aataaagtta ttttctcatt cccaggccag 60

acttgggatc ttccgcg 77

<210> 61

<211> 21

<212> DNA

<213> Homo sapiens

<400> 61

agaaataaat ctttggctca c 21

<210> 62

<211> 41

<212> DNA

<213> Homo sapiens

<400> 62

atttgtctgt gtactcaagc aataaaatga ttgtttaact a 41

<210> 63

<211> 31

<212> DNA

<213> Homo sapiens

<400> 63

tggctcagct aataaaggcg cacatgactc c 31

<210> 64

<211> 53

<212> DNA

<213> Homo sapiens

<400> 64

ggatgtaata catatattta caaataaaat gcctcatgga ctctggtgct tcc 53

<210> 65

<211> 64

<212> DNA

<213> Homo sapiens

<400> 65

gcccatcgtg actcaaaact cacttgtata ataaacagtt tttgagggat tttaaagttt 60

caag 64

<210> 66

<211> 37

<212> DNA

<213> Homo sapiens

<400> 66

aacaaaccat gctgggttaa taaattgcct cattcgt 37

<210> 67

<211> 386

<212> DNA

<213> Homo sapiens

<400> 67

ctgcattctc ctccgccaaa aaagtgacca agcagagtct ttctctgtca cccaggctgg 60

agtgcaatgg cgtgatctca gctcactgca acctctgcct cctgggttca agtgattctc 120

gtgtctcagc ctcctgagta gctgagacta caggtgtgca ccagtgttcc cagctgatt 180

ttgtatttta tgtagagatg gggttatgcc attttggcca ggctagtctc gaactcctga 240

gctcaggtga tacacacacc tcagcaaatc ttttaaatta tacattctgt gatatttcct 300

360

aaaactcctt tatgatggaa aatttc 386

<210> 68

<211> 281

<212> DNA

<213> Homo sapiens

<400> 68

gtcaactatt ttaataaatt gatgaccagt tgttaacttc tgttggtttt tattcagaat 60

actggcagat tttaggaata taaaggtgta ctatgagact tccacttttc aggtggaata 120

180

gattccgtta actacataat atgtgagacc tgactggttt tattggacac tggcagttta 240

taactttggc atactctaga taaattctga ttggtatggg g 281

<210> 69

<211> 53

<212> DNA

<213> Homo sapiens

<400> 69

ctggagagaa tcacagatgt ggaatatttg tcataaataa ataatgaaaa cct 53

<210> 70

<211> 2785

<212> DNA

<213> Homo sapiens

<400> 70

atattaatgg tgaaaacact gtagtaataa attttcatat gccaaaaaat gtttgtatct 60

tactgtcccc tgttctcacc acgaagatca tgttcattac caccaccacc cccccttatt 120

ttttttatcc taaaccagca aacgcaggac ctgtaccaat tttaggagac aataagacag 180

ggttgtttca ggattctcta gagttaataa catttgtaac ctggcacagt ttccctcatc 240

ctgtggaata agaaaatggg atagatctgg aataaatgtg cagtattgta gtattacttt 300

aagaacttta agggaacttc aaaaactcac tgaaattcta gtgagatact ttctttttta 360

ttcttggtat tttccatatc gggtgcaaca cttcagttac caaatttcat tgcacataga 420

ttatcttagg tacccttgga aatgcacatt cttgtatcca tcttacaggg gcccaagatg 480

ataaatagta aactcaaaat tgctccccac tctgtttatt atttaaaggt gtcaggatct 540

gtgttgtaat gtgtctacat taatgtgttt aggagaatac aggcattgga tcatttagtt 600

gatggaagta tatgccaggc aagggagata aggtatacga caagactgat gttttcagta 660

tcttctcatg aggttgtcag agaccttcat gtcttcaaag actagtcagc aaatgaagtg 720

gtttagtgta gagacaagat tggttgtgtt ttgataattt aagctaggta ttgagtacat 780

gtggattttg ctgtccacaa atacttgttt cagagttttc atggatacag tggcatggtt 840

gaaatgaagc tgtgagcctt ctgctttaaa tctgatgtaa gaaactcctg ttaacaaata 900

gtaagtatgg gttaattagc cctttgatca aagcctagct ttacattgtt taggatcttt 960

ggaaaacaat tggtttggtt gcccactttc cgtaggatca agagcagaac ctttcacatg 1020

gcacagaaga acccaggttg cgcttcatac ctgcatattc cagccttagc ctgccatttc 1080

tctccttggc actttgtgct ccagcaacac tggtctcagt tggtcatcct caaacttggg 1140

ttccatatcc agcctcagga cctctgttcc tgttactatg gttccttgca tgtcgcctgc 1200

tcttactaaa gagctcgtgt gttttccagc acacttcggt ttatctcttg atgatgatgc 1260

1320

cgtgtagctg cagtcagtat ttggctaagc tgttcccagg ggctcaacag atgctttcgg 1380

atgagcctta actgacccaa tcctttgtga tgcggggagag attgctaggc ctcgctcacc 1440

tggccagaac cagggaaaga ggccgcggtt gcagcgcgat tccaggccct gggcgtcagg 1500

1560

caggaaggga gtgggacagc gcgggcgccc agggatgtgg cctggttacc tgccttctct 1620

gatacgtcaa gacaccttca acaatggctt gcagctgtac cctgttggct gcaccagga 1680

cgcccttttc actgctaagc agtcctacct gaggcccagg ggctgccaga ttgacccata 1740

aataatctcc ggcgcctcag atccagaagc tgctgagcct gatcttagtg ccttctcctt 1800

tctctgtgtg gccccccagc ccctttcccc actgccttgt gtccaaggcc ctttccttca 1860

tgtatccatg guggagagac aaaaatacac atcaataaaa taagataggg aatccataaa 1920

tagacattca gaagtatggc caacggattt atcttaaaac caatggagga agaagagttt 1980

caataaatgt tgtggacttc catttgtcaa agaccaaaac aaaggaaccc caaccttaca 2040

tgtaatacaa acttaactca aaatggatca tatatctaaa tgtaaaatgg aaagctataa 2100

aactgaaaac agactatctt tacaacctag gcgtaggtat agtttttaga cattacacca 2160

aaagcacatg ccgtaaaaga aaaaatagat aaattggtgg atttcattaa aattaaaaaa 2220

ctttttctct ctgaaaaatc ctgttaagct gggcgctgtg gttcatgcct gtaatcccag 2280

cactttggga ggctgagttg ggaagaaatt aatagcttga ggccaggagt tcaagatcat 2340

cctgggcagc aaagtcatac actcttgagg gaagagagag accttctcat attgttttat 2400

attgttttat actcagtacc tgttttaaga aaaaaacaag gaagtgaaat caaagacagg 2460

cagccggca ccaggcctga aaccagccct gggcctgcct ggcctaaacc tagtagttaa 2520

aaatcaactt acgacttaga acctgatgtt atccgtagat tccaagcatt gtataaaaaa 2580

attgtgaaac tccctgttgt gttctgtacc agtgcatgaa acccctgtca catatcccct 2640

agattgctca atcaatcacg accctttcat gtgaaatctt tagtgttgtg agcccttaaa 2700

agggacagaa attgtgcact tgaggagctc agattttaag gctgtagctt gccgatgctc 2760

ccagctgaat aaagcccttc cttct 2785

<210> 71

<211> 48

<212> DNA

<213> Homo sapiens

<400> 71

ataggtccaa ccagctgtac atttggaaaa ataaaacttt attaaatc 48

<210> 72

<211> 67

<212> DNA

<213> Homo sapiens

<400> 72

ggagctgagt tcttaaagac tgaagacagg ctattctctg gagaaaaata aaatggaaat 60

tgtactt 67

<210> 73

<211> 62

<212> DNA

<213> Homo sapiens

<400> 73

aagcactctg agtcaagatg agtgggaaac catctcaata aacacatttt ggataaatcc 60

tg 62

<210> 74

<211> 276

<212> DNA

<213> Homo sapiens

<400> 74

60

agaatggttt ttaagcacca aattgatggt cacaccattt ccttttagta gtgctactgc 120

tatcgctgtg tgaatgttgc ctctggggat tatgtgaccc agtggttctg tatacctgcc 180

aggtgccaac cacttgtaaa ggtcttgata ttttcaattc ttagactacc tatactttgg 240

cagaagttat atttaatgta agttgtctaa atataa 276

<210> 75

<211> 139

<212> DNA

<213> Homo sapiens

<400> 75

gcttggctgc tcgctgggtc ttggatgtcg ggttcgacca cttggccgat gggaatggtc 60

tgtcacagtc tgctcctttt ttttgtccgc cacacgtaac

agcgtttgtg tttcaagtt 139

<210> 76

<211> 94

<212> DNA

<213> Homo sapiens

<400> 76

atgctcttcc ttcagaggat tatccggggc atctactcaa tgaaaaacca tgataattct 60

ttgtatataa aataaacatt tgaaaaaacc cttc 94

<210> 77

<211> 1355

<212> DNA

<213> Homo sapiens

<400> 77

tataagtaaa gtttgtaaaa ttcatactta ataaacaatt taggacagtc atgtctgctt 60

120

taagaaagtt aaagtgcaat aatgtttgaa gacaataagt ggtggtgtat cttgtttcta 180

ataagataaa cttttttgtc tttgctttat cttattaggg agttgtatgt cagtgtataa 240

aacatactgt gtggtataac aggcttaata aattctttaa aaggagagaa ctgaaactag 300

ccctgtagat ttgtctggtg catgtgatga aacctgcagc tttatcggag tgatggcaat 360

gctctgctgg tttattttca agtggctgcg tttttttttag tttggcaggt gtagactttt 420

taagttgggc tttgaaaat ctgggttagc ctgaagaaaa ttgcctcagc ctccacagta 480

ccattttaaa ttcacataaa aggtgaaagc tcctggttca gtgccatggc ttcatggcat 540

tcagtgatta gtggtaatgg taaacactgg tgtgttttga agttgaatgt gcgataaaat 600

tattagcctt aagattggta agctagcaat gaatgctagg gtgggaagct ggtgagccag 660

tggccattag ataaatacct ttcaagtgtg agcttagacg tcaaccctaa aatacttaac 720

cgtaatgcta attgtgatca ttatgaatcc cttcagtcac attaggggga aagtagttgg 780

ctataagtac gtcattctta gtccagtcag tcttaaaaac atcttgggtt acccactctg 840

tccactccca taggctacag aaaaagtcac aagcgcatgg tttccaacca tatgtgtttt 900

ctgcagttat ttctcttgtt ctggccaaac aaccctaaaa atccttacca ttccacaaag 960

ttggaccatc acttgtgcac ccactttgac tatgagtata ccaccacatt gcatttctgt 1020

ttgcaccatg tcttccagga gactagacta ctgttgtcca gggtcaattt gagtgtaaag 1080

aaaatgtaga caaggaattg cccaatttta aattctgact ttgctgactt aatttaaatg 1140

ctcgttctga accaattttc tcctatcttc tctaggggtt tcaaaagact cagttaattg 1200

1260

1320

ataaatgtct attaaactaa aacaaatgga ccttc 1355

<210> 78

<211> 2017

<212> DNA

<213> Homo sapiens

<400> 78

gcaattcttc tgcctcggcc tcccaaatag ccaggactac aggcgcacac tgccatgccc 60

agctaagttt tgtattttta gtagagactg ggtttcacta tgttggccag gctggtctcg 120

aactcctgac ctcaagtgat ccacctgcct tggcctccca aagtgctggg attacaggcg 180

tgagccacca cccccagccc aattttttatt ttttgtacag acaggatctc actatgttgc 240

ccaggttggt ctcaaactac tggcctcaag caatcctgcc ttggcctccc aaagtgctgg 300

aattatagga atgagccacc acaccggggcc caaatttact ttagtaataa caacaattgg 360

ctgggtgcgg tggctcacgc ctgcaatccc aacactttcg gtaaccaagg tgggcttgag 420

ctcatgagtt agagagcagc ctgagcaacg tggtgagagc ccatctcaca aaaaataaca 480

aatcagctgg gcatggtgtt gcacgcctgt agtctccgaa atcacaccac tgcactccca 540

tcttgggtga tagagccaga acttgtctca aaaataacaa ttggtttctt acaatcccaa 600

aaggtgcagt tactagtatt aatccttttt tgccaatgag gaaacacaaa gatgaagcaa 660

cttgctcaaa gtcatacagt gacagtctga attcaaatcc tatacactta aagtttattt 720

gttttgtttt ggtttttttt gagatggagt ctcactgtgt cgcaaggctg gagtgcagtg 780

gcacgatctc agctcactgc aacccggggtt caagcgattc tcctgcctca gcctcccgag 840

tagctgggac tacaggcacg caccaccaca cccagctaat ttttgtattt ttagtagaga 900

cggtttcacc atgttggcca ggatggtctc gagctcctga cctcaggtga tcctcccgcc 960

ttggcctccc aaagtgccgg gattacaggt gtcagccact gcacgtggcc aacttaaagt 1020

ttttgataga taatacatta acgttaaaaa ttcaaaagat aagtataggc tctacagtac 1080

aaacccttct gcctcctagt tcctctccct ggaggcaagg tgatcagttt aacaatattt 1140

ttttattttg agacagggtc tcactgttgc ccaggctgga gtgtagtggc gcgttcacaa 1200

cttactgtag cctcaacctc ctggctcaag caatcctccc acctcagcct gtcgagtagc 1260

tggaaccaca ggtgcacacc accatgccag gctaattttt gtattttttg tagagacagg 1320

1380

tgcttcccaa agtgctggga ttacagatgt gggccacggt gcctggccta catatgtatt 1440

ttttcctttt cttccccaag tggtaggata tgatacacat tgttgatttt tttgtttagt 1500

1560

ttgagatgta gttttgctct tatagcccag gttggagtac agtaacacaa tcttggctcg 1620

cagcaacttc tgcctcccag gttcaagcga ttctcctgcc tcagcctccg agtagctagg 1680

attacaggtg cctgccacta catccagcta tttttgtatt ttcagtagag acggggtttc 1740

accattttgg ccaagctggt ctcgaactcc tgacctcagg tgatccgccc atctcagcct 1800

1860

gtataactgc atggtatccc atcatgtgga tgtaccatga ttcattggat gtggaccctc 1920

ctgatggaca tttaaatttc ttccaatctg ttgctattac aaaaagaaaa atgtgtgcat 1980

acatctttat tcatctgtag aataaattct tagaagt 2017

<210> 79

<211> 37

<212> DNA

<213> Homo sapiens

<400> 79

ggtttttata caagaaaaat aaagtgaatt aagcgtg 37

<210> 80

<211> 140

<212> DNA

<213> Homo sapiens

<400> 80

gtggcaatca taaaaagtaa taaaggttct ttttgacctg ttgacaaatg tatttaagcc 60

tttggattta aagcctgttg aggctggagt taggaggcag attgatagta ggattataat 120

aaacattaaataatcagttc 140

<210> 81

<211> 55

<212> DNA

<213> Homo sapiens

<400> 81

acaagattcc tcaaaatatt ttctgttaat aaattgcctt catgtaaact gtttc 55

<210> 82

<211> 1485

<212> DNA

<213> Homo sapiens

<400> 82

gggcttccaa tgtgctgccc ccctcttaat actcaccaat aaattctact tcctgtccac 60

ctatgtcttt gtatctacat tcttgacggg gaaggaactt cctctgggaa ccttttgggtc 120

attgcccttt cacttcagaa acaggttgac aactcagccc tgctcatgag gcagcaaacc 180

ctgcaaaggg ctgggactgg tggccttatg tcagttgtct actctggagc ttgacttgga 240

cctccccagg tcctagggcag taggttgaaa aacactgaag tgcttttcat gaagcacagc 300

tgcagcaaag ccttgcaatc ccaggctggg gtcagcctac agttgtgttg cttattacaa 360

cacatgcgga ccaagagggg cttgtgggct agaggctgac cagcagcgtt tatttagcaa 420

gggtaggtgt gcatcacatt gggcttgttc tcacccatct ggtttggcca ttcctccttg 480

gtgggaatca tccaggtact gctgaggtca cctgcgattt gccccatttc ctatctctag 540

caacctcctg ggccccatgc ccccacccct tctagaacct gcattcccag ggccttcacc 600

acctgaccaa aggtctaggc taacctttgg tcatttgtaa caagacctcg gaacagacac 660

gtgtgtggca tggtttggcc tggggatctt agatgtctga cctgaactat tgtagaacag 720

cgctggcttt tgggggagca gcaaaaatga gaggagtgct aggtgggtgg cctgagcatc 780

tgtatccagg gacaggactc caaaggcttt tggtcccaga gctggggtat gttggcccca 840

gcccccagcc tgtggctccc aaaaggcctc tggttttttg taatctcagt ttacagccat 900

ttcttaggtt tttaattacc tttattttat tttgccaaac atacctggga atacctttta 960

tttttttttt accttggggt gatggttcca aaccataaat gtgattatag ttaacacatg 1020

acccttctag cgtcccagcc agtgtttttc ctgacctctg ttctttggag aggaggatgg 1080

aagggagggg tccggcacgc tgctggcatt ttgctgtgtc ctgcagcccc tttccgggac 1140

acctgggttc acacagcttt ttagcttaca taactggtgc agattttctg tgtggagatg 1200

ttgccttgac cagccttggc tggactttac caggcatgca gaagcctgta ccaacacaga 1260

ctacagcacc caggaggtgc gagtgtggct gctcagcggt tataacaggc ctgactgcat 1320

tgttcaccgg attataatga gccaaaatgt ttcccggtgt ttgctggttt cagggaagga 1380

gtttgatata gcagattaac caccctcctt gtagctattg gggcttaatg gtttcctggt 1440

gattcttacc aatccacaat aaacatggcc cattggcata tctgc 1485

<210> 83

<211> 31

<212> DNA

<213> Homo sapiens

<400> 83

ggcacatttg aataaattct attaccagtt c 31

<210> 84

<211> 39

<212> DNA

<213> Homo sapiens

<400> 84

ggggcgcatt gtcaataaag cacagctggc tgagactgc 39

<210> 85

<211> 486

<212> DNA

<213> Homo sapiens

<400> 85

gcccaataaa gactgttaat tcctcatgcg ttgcctgccc ttcctccatt gttgccctgg 60

aatgtacggg acccaggggc agcagcagtc caggtgccac aggcagccct gggacatagg 120

aagctgggag caaggaaagg gtcttagtca ctgcctcccg aagttgcttg aaagcactcg 180

gagaattgtg caggtgtcat ttatctatga ccaataggaa gagcaaccag ttactatgag 240

tgaaagggag ccagaagact gattggaggg ccctatcttg tgagtggggc atctgttgga 300

ctttccacct ggtcatatac tctgcagctg ttagaatgtg caagcacttg gggacagcat 360

gagcttgctg ttgtacacag ggtatttcta gaagcagaaa tagactggga agatgcacaa 420

ccaaggggtt acaggcatcg cccatgctcc tcacctgtat tttgtaatca gaaataaatt 480

gctttt 486

<210> 86

<211> 44

<212> DNA

<213> Homo sapiens

<400> 86

gcccctcccc tgccctctcc ctgaaataaa gaacagcttg acag 44

<210> 87

<211> 406

<212> DNA

<213> Homo sapiens

<400> 87

gtgtcatctt ttattatgaa gacaataaaa tcttgagttt atgttcactt catttgtttg 60

ctgttcatct tttgggggg aataagctag agccatcaat acaattccgc ttgtggggaa 120

180

catatgttgc aattttctaa tttggcactt caatcactag gggccttatg aggcagtttg 240

tcattatgca atggttattg gttatcatgt gagtagacac atttcaggct aatagggaga 300

agtcagtaac acattcatag tgaatatgag atgtctttgc taagagttaa gtgtcagatc 360

tttgttataa cagttaattt aataaagaat tttggcattg ttcttc 406

<210> 88

<211> 1994

<212> DNA

<213> Homo sapiens

<400> 88

ttgccgtaag gatatgcact tgtctctagt ccacacactt catgatatag gtatagcgtt 60

agtttagcga agttttcact gcactgatat atctagtagg tgatggagct gggaatgcaa 120

180

cccttctgaa gaggcaaaaa attgaggaat gtgccctgct ttcctaagaa ctgaagtgtg 240

agtacactgg taaatccttt catttgcctt gttccttatc tgtcaatatg tctgaatcct 300

360

ctacctgttc tcgtgaagtc ttaaaactct tatagaatag ccatttaggc ctttctgcta 420

gcctcctgaa ttctgtattc tcaggctgag cgagtttctg tttactctca aaccttaggt 480

gatttggcta actcttaaag taattagcac gatgattgga acggagcatt ctctccaaca 540

cagcatttct tttggcactt tgcttcttgt gcagtttagc tccagaaagt attaaggaat 600

gactttagtg ctcatttgga tgcagtaagt ggtttgatct cagggtggca aaaagaatgc 660

tttttttata ccttttcaca ttcggataac ttgtttagaa gacagaggtt ctaactaggt 720

tttggcctat taagaactgc aaactagcag cagcagaact ctggctaaag gggcaagctt 780

attaggaaat tgagtattta aaagttgagc taccatatga tccaacaatc ccactgctgg 840

gtatataccc agaagaaaat cggtatatca aagagatatc tgcactccta tgtttgttgt 900

agcactgttt ataatagcta agatttagaa gcaaccttag tgtccatcgg gatgaatgga 960

taaagaaaat gtacctatac gcggccaggc acggtggctt gtgcctagca ctttggaaag 1020

ccgaggcggg tggatcacct gaggtcagga gttcgagacc agcctggcca agatagtgaa 1080

accccgtctc tagtaaaaat acaaaaatta gccgggcttg tggtgtgggc ctgtaatctc 1140

agccacccgg gaggctgagg caggagaatc gctggaacct gggaggcaga ggctgcagtg 1200

agccgagatc acgccactgt actccagcct gggcgacaga gcaagactcc atctcaaaaa 1260

aaaaaaaaaa aaaaagggaa aaagaaaatg cacctataca cagtggtact attcagccat 1320

1380

agtgaaatag gcacacaaag acaagcatca catgttcttg tttgtgggat ctaaaaatca 1440

aaacaagtgg acttgtcata tagagagtag aaggatggtt accagaagct gagaacttct 1500

ggtggcggga ggtggggatg gttaatggggt acaaaaagaa aaaagaatga attagaccaa 1560

ctatttgata gcacgacagc gtgactaaag tcaataactt agttacatat tttaaaataa 1620

1680

gagaaaccta acaagcacta cctcagccag gtaatcaagg ttaacatcaa cagtcacgag 1740

tcatgttgat atataccctt gataaggtgt gatgaaaatg acacttaaac ctaaaaatcc 1800

ataaccctat ctaatgagaa aaataacaaa tcccaagagg ggcattttac aaaatacttg 1860

accagtagtg cggaaattgt caaggtcatc aaaaaagtct gagaaattgc cacagccaaa 1920

ggagtctaga gacatgatga ctaaatgtta ggtggtgtcc tgcgtggggt cctagaacag 1980

aaaaaggaca ttag 1994

<210> 89

<211> 307

<212> DNA

<213> Homo sapiens

<400> 89

accgctagct tgttgcaccg tggaggccac aggagcagaa acatggaatg ccagacgctg 60

gggatgctgg tacaagttgt gggactgcat gctactgtct agagcttgtc tcaatggatc 120

tagaacttca tcgccctctg atcgccgatc acctctgaga cccaccttgc tcataaacaa 180

aatgcccatg ttggtcctct gccctggacc tgtgacattc tggactattt ctgtgtttat 240

ttgtggccga gtgtaacaac catataataa atcacctctt ccgctgtttt agctgaagaa 300

ttaaatc 307

<210> 90

<211> 38

<212> DNA

<213> Homo sapiens

<400> 90

gtctgtaggc cttgtctgtt aataaatagt ttatatac 38

<210> 91

<211> 133

<212> DNA

<213> Homo sapiens

<400> 91

agtgtctagc agtgagctgg agattggatc acagccgaag gagtaaaggt gctgcaatga 60

tgttagctgt ggccactgtg gatttttcgc aagaacatta ataaactaaa aacttcatgt 120

gtctggttgt ttg 133

<210> 92

<211> 1802

<212> DNA

<213> Homo sapiens

<400> 92

tgtcactgcc atggccgcct tgctgcattt ctgaggatgc ttcatctctc caccttcttc 60

tccactcagc agccagcagg gcactgtgga aatcggagtc acatgagctg gcacctctgt 120

tcagaaccct ccagggctcc acatctctct cacccaaatg ccaaagacct cccccgccc 180

ccacaatccc ccacgacctg gccactggcc tcccaccacc ttccagctcc agcggctcct 240

accacattta aggctttcct tcctagtttt aatttttcct cgtcagcagt tgattttatt 300

360

tttttttggt ctgtgttcct aattagctca ttgctatacc cctggcgccc agaacaatgc 420

cttggacaca gtacgcagta gactaaataa atacttgttg aatgactgac tgacggaatg 480

acggctgtgt ggggagtgga ttgggtcgtg aggcagaggc tgcggtggaa actcaggcag 540

gaggtgatgg tggttcttgg ggctgcggaa tgccaagttt agaagctctt cctctgctgt 600

ggcacatgaa ccggtcactc gagaaggctt ttagatttac tttgcctaat cccctcttag 660

tgcatgtggg gaaactgagg tacacaaaag gaattcccca ccaagttagg ggcagaacct 720

agcccccttg tctcccagat ggatatcttc tttttttttt gagacggagt cttgctctgt 780

tgcccaggct ggagtgcagt ggtaccatct tggctcactg caacctctgc ttcccaggtt 840

caagcgattc tcctgcctca gcctcctgag tgtctgcgat tacaggtgca cacaaccacg 900

cctggctaat ttttgtattt ttagtagaga cggggtttca ccgtgttggt cagggtgacc 960

tcaaactcct gacctcatga tccacccagc tcagcctccc aacgtgctgg gattacaggc 1020

atgagccacc gtgcctggct ggacatcttg ttattaaagc ttcttctctc tttgtagggg 1080

agggggagat gcctctggtg gagaagacca gtgtggcagt gactgtgtct gttagtgaac 1140

ctggtggctg gttgagggtc tgtcgtggtg actgaggaca catacaaagt gcttttctca 1200

gtggtcacct tggtgttggt gaataagggt cagaagatgg ctcctgtcct agggcactgc 1260

cagtcggttt ggaagctgaa atgcctgctt agcagtttga ggaaacacag accttggagg 1320

atcttctggt tgcctcttca agaattcatt ctattcccct tctgctcccc aaatttgctt 1380

ttcttggggt gggtcttggt tggcctaagc caagaaagta tggcatctac tccttccata 1440

gcaatagctc aggaataggc agtgacccag acctgaacca atcagtgcat ggaattaccc 1500

1560

gtggttggag agatcgcact tgctccagag gtcataatgt gcagatctga ggcttggaac 1620

1680

agcagggcag agctgagaga acagcaggga aacagccaga gtcttgctca agcctccctg 1740

aagtatctat acccctggac tctagttatg ggggctaata aatgttatat actgtttaag 1800

gt 1802

<210> 93

<211> 34

<212> DNA

<213> Homo sapiens

<400> 93

tgctgagggc ctcaataaag tttgtgttta tgcc 34

<210> 94

<211> 502

<212> DNA

<213> Homo sapiens

<400> 94

60

tgtgctggag ctacctcacc tcagcttgag agagccagtt gtgtgcatct ctttccagtt 120

ttgcatccag tgacgtctgc ttggcatctt gagattgtta tggtgagagt atttacacct 180

cagcaaatgc tgcaaaatcc tgttttcccc cagagagctg gaggttaaat actaccagca 240

catccctaga tactactcaa gttacagtat atgatcacta atatagtatg ctcttggtac 300

caggagctct gatatatatc tggtacatgt ttgataatga cttgattgtt attataagta 360

cttattaata cttcgattct gtaaaagtt tagggtttga ttttataaaa tccaaaatga 420

gcctttttatt gaatccagtt ctctatgtga ccagttctct gtatgaatgg aagggaaaag 480

aattaaaaat cttgcaaagg gg 502

<210> 95

<211> 502

<212> DNA

<213> Homo sapiens

<400> 95

60

tgtgctggag ctacctcacc tcagcttgag agagccagtt gtgtgcatct ctttccagtt 120

ttgcatccag tgacgtctgc ttggcatctt gagattgtta tggtgagagt atttacacct 180

cagcaaatgc tgcaaaatcc tgttttcccc cagagagctg gaggttaaat actaccagca 240

catccctaga tactactcaa gttacagtat atgatcacta atatagtatg ctcttggtac 300

caggagctct gatatatatc tggtacatgt ttgataatga cttgattgtt attataagta 360

cttattaata cttcgattct gtaaaagtt tagggtttga ttttataaaa tccaaaatga 420

gcctttttatt gaatccagtt ctctatgtga ccagttctct gtatgaatgg aagggaaaag 480

aattaaaaat cttgcaaagg gg 502

<210> 96

<211> 48

<212> DNA

<213> Homo sapiens

<400> 96

attttttctg tagtgctgta ttatttttcaa taaatctggg acaacagc 48

<210> 97

<211> 171

<212> DNA

<213> Homo sapiens

<400> 97

gctgttcttg cataggctct taagcagcat ggaaaaatgg ttgatggaaa ataaacatca 60

gtttctaaaa gttgtcttca tttagtttgc tttttactcc agatcagaat acctgggatt 120

gcatatcaaa gcataataat aaatacatgt ctcgacatga gttgtacttc t 171

<210> 98

<211> 2309

<212> DNA

<213> Homo sapiens

<400> 98

ggtggttctt tccttgaagg gcagcctcct gcccaggccc cgtggccctg gagcctcaat 60

aaagtgtccc tttcattgac tggagcagca attggtgtcc tcatggctga tctgtccagg 120

gaggtggctg aagagtgggc atctccctta gggactctac tcagcactcc attctgtgcc 180

acctgtgggg tcttctgtcc tagattctgt cacatcggca ttggtccctg ccctatgccc 240

ctgactctgg atttgtcatc tgtaaaactg gagtaaaaac ctcagtcgtg taattggtgg 300

gactgaggat cagttttgtc attgctggga tcctgtcagg cactttgagg tgtccctcag 360

gccttggccc tgaagtgtct aggtgtgtgg agatgggtag aaaattaggt acacccaatg 420

gtgtagaacg ttgattctca aattttttta ttttatacaa atggggtctc actatgttgt 480

ccaggctggt cttgaactcc tgggctcaag ccatccgccc atctcagccc ctcaaagtgt 540

tgggattaca agcaagaact gccatgcctg acccagttct cagttttttg tttgtttgtt 600

tgtttgtttg ttttgagacg gagtcttgct ctgtcgccca ggctggagtg cagtggcgca 660

gtctcggctt actacaacct ctgcctccgg ggttcacatc cttctcctgc ctcagcctcc 720

cgagtagctg ggactacagg tgcccgccac aactcctggc taatttttttg tatttttagt 780

agagacgggg tttcactgggg ttagccaggt tggtctcgat ctcctgacct tgtgatccat 840

tcgccttggc ctcccagaat gctggtatta caggcgtgag ccagcacgcc tggcccagtt 900

actcagtttt gaatctgagg ccgtgacatc actcatggtc tgcagtcagt gctctgcccc 960

tgagctgtac cctctcctat gataatcact cttaagaagg gcaacccttg gtgttttccc 1020

cttaaggtca cccaggctgg aatgcagtgg tgtggtcatg gctccctgta ccctggaact 1080

caggcttggg tgatcctctc tcctttgcct ccgaagtagc caggactaca ggtgtgcacc 1140

caccaccaca ctcagataat tgctttggtg tttttaaagc ttgtaatgat cagtaggctg 1200

1260

ttttatttac tgatttaaaa ttttgagaca gtctcactgt cacccaggtt ggggtgcagt 1320

ggtaggatca tagcttgctg cagccttgat ctcccaggat cttgcctcag cctcccgagt 1380

agctgggact gcatgcttgt gccaccacac tcggttaata ttttgtagag atggggtctt 1440

gctatgttgc ccaggctggc ttcaaactcc tgaacttaaa agcctcctgt ttagttttgg 1500

ttttttatca cttttttttt ttttttttga gatggagcct tgctcccatc gtgcaggctg 1560

gagtgcggtg gcgcagtctc ggctcactgc agcttctgcc tctcgggttc aagcgattct 1620

cctttctcag cctcttgagt agctggaatt accagtgtgc gccaccacca ccacgcctgg 1680

ctagtttttc tgtttttagt agagacaggg ttttgctatg ttggccaggc tggtcttgaa 1740

ctactgacct cttgtgatct acctgtcttg gccttccaaa gtgctaggat tacaagcgta 1800

agccacagcg cctggccttg ctacattttt tttttttttt ttttttttac agacatggtc 1860

tcgctatgtt gcccagaatg gttttgcact gggtccaagc agttctgccg cagcctccca 1920

aagtgctggg attacagggg tgaggcacct tgctggcccc tgttttgatt agggtgcagt 1980

gctggtgaag ccggtgcacg aggccagtga tgcatcctaa tgaggggtgg agttggcggg 2040

acttcctggg ccagtttgggg gactttcaca aaagaccccc atgactcagg gttttgagtt 2100

cttaactgat cgaatgaagg attcaaaatt aaccactcca aggggggatt gaaggaagaa 2160

ccactcttaa tggacaaaaa gaaagaaagg ggagggagta acagggatat gagctctagc 2220

cgccaagct agcaatggca acccttctgg gtccccttcc agcatgtgga agctttcctt 2280

tcgcttcatt caataaacag ctgctgctc 2309

<210> 99

<211> 58

<212> DNA

<213> Homo sapiens

<400> 99

gtcttttgta attctggctt tctctaataa aaaagccact tagttcagtc atcgaaaa 58

<210> 100

<211> 1502

<212> DNA

<213> Homo sapiens

<400> 100

gcaaaggctc cccttacagg gctttgctta ttaataaaat aaatgaagta tacatgagaa 60

ataccaagaa attggctttt agtttatcag tgaataaaaa atattatact cttgaacttt 120

tgtctcattt ttttgagtat gctgtttata tgattttgat ttccctctga taactatcaa 180

cagtatttaa atagcttata gctggtataa ttttttccca cgatttccaa aatcttttat 240

gtactcaggt aaaagtagcg ttatatagga aatctttttt ttagacactc tcgttctgtc 300

acccaggctg gagtgcagtg actcagcttc ctaaatagct ggaattacag gtgtgagcca 360

ccatgcccgg ctaatttttt gtacttttag tagagtaggg tttggccatg ttggccaggc 420

tggtttcaaa ctcctgacct caagtgatct acccacctcg gcttcccaaa gtgctgatta 480

tagctgtgaa ccaccatgcc cggccaggaa atcttactgt agaacaattt tttatatagc 540

tgtataaaat gtatatgatt gtcttgacag tctcaaatac tgtttttaat agcttgtaaa 600

tgtaatctca agtgcttaga acagttctta catataagtt gctctgtagt ttgctcttat 660

agttagccca aagactctgg gtgtgaggcc tgctgtaaac caatgttaaa ctgcttatta 720

gaaagcccta accacctgct ttgtaggcac cagaaactca aaaccaaatc tcaactcagc 780

tacagaatct actgtggtcc ttgtctgaaa aaattagttc actcggttgg aatcttgtct 840

cagagcatcc tcatctcttt ctcaaaagcc cctaccccaa caccggcgtg ttggttgtct 900

attgaaactt acaagtggat ggaccctttc tcccgaataa actggccttt gaaagctcta 960

atcgaaatgg tttggcaaaa tccatactgc aggagattag ggaggacaag aatgatgtgc 1020

ctttttgtac tgctgagcct gatggtggtg ccactacttc aggtacttag atgagtcttg 1080

atgctaatag aattgtgtcg ccaaacatat ctggacagtt acaacctaat ctatgcatta 1140

attggtttgg gaattgcttg aaattattgt ttaattcaat gttttaattc gttttcctaa 1200

aaatttaagt gcccccatca tcgtgcaata cctcagtgca gcaactcctt gattcttgga 1260

tgactgaact tcctaacttg gctctgcccc attgttccca tttttcatgt ttttcacaaa 1320

tagttaacca ggtacctact actgtgcacc gctgcagagc attgaggatg tatgtgatga 1380

gtaaaaacac ccagcctgct ctgctgtgtt agtattatga cggaaactga tcaaatcaca 1440

tgtgaacaaa tttactgcta caaaagggag ggcttaataa aaggaatttc atctgggaag 1500

gc 1502

<210> 101

<211> 48

<212> DNA

<213> Homo sapiens

<400> 101

attttttctg tagtgctgta ttatttttcaa taaatctggg acaacagc 48

<210> 102

<211> 193

<212> DNA

<213> Homo sapiens

<400> 102

ggaattgcac atgagatggc acacatattt atgctgtatc aagttcacga tcatcttacg 60

atatcaagct gaaaatgtca ccactacctg gacagttgca catgttttac tgggaatatt 120

tttttctgtt tttctgtatg ctctgtgcta gtagggtgga ttcagtaata aatatgtgaa 180

agcttttgtt tcc 193

<210> 103

<211> 73

<212> DNA

<213> Homo sapiens

<400> 103

aggttttggc agtactgtct ccttgggcca tgctggtctg acttatgctt actaataaat 60

tctgtttact ggc 73

<210> 104

<211> 93

<212> DNA

<213> Homo sapiens

<400> 104

actttgggat atttttcttc aattttgaag agaaaatggt gaagccatag aaaagttacc 60

cgagggaaaa taaatacagt gatattctta cgc 93

<210> 105

<211> 257

<212> DNA

<213> Homo sapiens

<400> 105

gctgtgtggg gtggatgaac cctgaagcgc accgcactgt ctgccccaat gtctaacaaa 60

ggccgggaggc gactcttcct gcgaggtctc agagcgctgt gtaaccgccc aaggggttca 120

ccttgcctgc tgcctagaca aagccgattc attaagacag gggaattgca atagagaaag 180

agtaattcac acagagctgg ctgtgcggga gaccggagtt ttatgtttta ttattactca 240

aatcgatctc tttgagc 257

<210> 106

<211> 692

<212> DNA

<213> Homo sapiens

<400> 106

tgattcaaac agcttcctga attttaattt tgtgttgtct cacagaaagc cttatcataa 60

attccataat tctaattaat ttaccaagat aatgtaatta catttggttt tgtaaggtat 120

acagcagtaa tctcctattt tggtgtcagt ttttcaataa agttttgatt atgggcaaat 180

240

300

tttttgagag agagagtcct gctctgtcac tcaagctgga gtgcagtggt gcgatctcgg 360

ctcactgcaa ccctccgcctc ccaggtccaa gcaattctct tgccttgtcc tcccaagtag 420

ctgggattat aggcacacac caccatgcct ggctaatttg tgtctcattt tcaagagtag 480

aaaccctaaa tattttattt tcattccttt tccaaattgc tatgaatggg attaaaggat 540

tacagatgta aagtctatta tttgtgaatt ctaaatgtag ttctgctgtt gtacctgtgg 600

aaacatctta aagaagtaca tattttgcac gtcctgcacg tgtaccccag aacttaaact 660

ataattaaaa agaatagttt caaaaaaata ca 692

<210> 107

<211> 228

<212> DNA

<213> Homo sapiens

<400> 107

atagaacctg ttgtgcaacc acggtttaac cggagatttt gaggctaggg tgtgtttctt 60

tcgaactttt cggaatgtct ggaacatttc atttcctgtt ttgttacctg tgcctctgta 120

aatctacttt tgcaatttta agtaataatt ttatgaataa aaatgggaaa tgcttcctaa 180

ttccacatag tatttgcatt gttttataaa taaattccac ttactatc 228

<210> 108

<211> 529

<212> DNA

<213> Homo sapiens

<400> 108

acccaggtga ggcagggctg aaaactgccc ttgggctgac ttttgatagg ccatgccttg 60

ccactttaca agttcttttt gcatttacta gtatttaaga gtaaccttga gattgggagg 120

aatagaggag gctggtacaa atagatggag acctgctggg atcagtgaat gcctgattag 180

gacatggggc tatgcatagc ctaagagtta taggcttaaa gatgtcgagt aactaaaaac 240

tgtattgctg gccgggcgcg gtggctcacg cctgtaatcc cagcactttg ggaggccaag 300

gcgggcagac catgaggtca ggagattgag accatcctgg ccaacatggt gaaaccctgt 360

ctctactaaa aatacaaaaa tgagctgggt gtggtggcac gtgcctgtag tcccagctac 420

tcgagaggct aaggcaggaa aatcgcttga acccaggagg cagagattgc agtgagccaa 480

gattgcacca gtgcactcca gctgggcgac agagcgagac tccatctcg 529

<210> 109

<211> 174

<212> DNA

<213> Homo sapiens

<400> 109

gtggaaaaga acatgaaaaa gaaaactgac aaatacacac aggtctcctc aagatccatg 60

gacttctggt ctgagcctaa taaagactgt ttgtttattc ctcaaaaaca aacaaacaaa 120

aaaaaaccct ctgtattata aattattctg tgtaatggtg tgttaccata catt 174

<210> 110

<211> 156

<212> DNA

<213> Homo sapiens

<400> 110

atgctcctct actctttgag acatctctgg cctataacaa atgggttaat ttatgttaaa 60

aaaaaaaaaa gagagagaga gtgaaacaac aatctacaca atcagagaaa atatttgcaa 120

atcttatatc tgattagaaa ttagtatctg gaacat 156

<210> 111

<211> 46

<212> DNA

<213> Homo sapiens

<400> 111

aattggagag gattatttca cattgaataa acttacagcc aaaaaa 46

<210> 112

<211> 67

<212> DNA

<213> Homo sapiens

<400> 112

ggagctgagt tcttaaagac tgaagacagg ctattctctg gagaaaaata aaatggaaat 60

tgtactt 67

<210> 113

<211> 294

<212> DNA

<213> Homo sapiens

<400> 113

ggaattgaac atgagatggc acacatattt atgctgtcta aaggtcacaa tcatgttacc 60

atatcaagct gaaaatgtca ccactatctg gacagttgga catgtttttt tgggaatata 120

ctttttctct ctgaatctgt taggaacttt ctggttggct gggttccgta ataaatacat 180

gagacctttc atttcaaaaa aaagaaaaat aggcctcctt cccaggggct ccggatttca 240

tcagccttct gtgcatgccc agccatacaa accacgcagg gatggctcca agtg 294

<210> 114

<211> 171

<212> DNA

<213> Homo sapiens

<400> 114

tcaccaaaaa gcaaccaact tagccagctt tatttgcaaa acaaggaaat aaaggcttac 60

ttctttaaaa aataaataaa taaataaata aataaataat aaataaataa ataaataaat 120

aaatagataa ataaataaaa agttttctac tcacactgaa gtgacgaagt c 171

<210> 115

<211> 49

<212> DNA

<213> Homo sapiens

<400> 115

gcccccttcc ccctgccctct ccctgaaata aagaatagct tgacagaaa 49

<210> 116

<211> 88

<212> DNA

<213> Mus musculus

<400> 116

ggaggcctca gttcctggcc ccagaaacga gatcctgacc acatgaacaa tttgggctct 60

tttgggagaa taaaagactt atatattg 88

<210> 117

<211> 69

<212> DNA

<213> Mus musculus

<400> 117

ttccaggacc actttgtgca gatggtgggg tctcaccaat aaaatatttc tactcacact 60

ggttttccc 69

<210> 118

<211> 118

<212> DNA

<213> Mus musculus

<400> 118

actattaaaa attgttaaat tccagagagc aagtagagac cgcatatttc aataaatcaa 60

acatgtggtg acaaaccctt gtgtgactct taaattgtgg atgtttccaa gccccttg 118

<210> 119

<211> 1008

<212> DNA

<213> Mus musculus

<400> 119

agcagttttc tatgaagatt ttttcataaa gacaataaac atattgatca agcagctttt 60

tctgtgttaa gctgttatta atgagactat aggaaatagt gtgaaattac aaaagcaaag 120

aagtagatag ttatttaatt aattaaatta attttacctt ttgtgttgca ccataaccta 180

ccactggtgg gattaagggc aagtattacc atgcctagct gagagtcttt ctccaggaaa 240

aaccagctta catgggttcc tgcaaatctc atgagtgttt cttgggtttc tagtcttcct 300

gggaggtgtc cttatctttc agattttcag atctggtaat tagcatgatc atcaggacat 360

ttattacaaa caaattgatt agtgggaaga aagtatctca aggtcaatct tggaagtgaa 420

caactggtgc taatccatgg ctttaaagat ttgagaacaa cggtgaaatt tggtttgagg 480

agaaggggggt gtctaggacg tttcattttt atggtacatg ccagacatga atgtacatag 540

gaaaataact tgaaagggtc aaatattaaa ccttgaatat caggttcact tgggaaagca 600

ttaggtgctt atgcctctta gtaaaatagcc cttcatccca gaaggagcaa gaattgtctt 660

cctgacttaa tccagtctta gctgaggtgc tgtgcatctt tatcatcttt gccttgcctc 720

acagtgtcag gctctgtggt actggggcta cacaggtcag gtaaacagtt aactgcttac 780

ctacatcccc agcaaagata atgtgacgat actaagatga acctatcaga gcttaaagat 840

aatgagtttc agtcacagtg ataactgcat gctaacttca gcatgtagaa tatatgccga 900

agtgtgtaag tacacagtaa 960

atcatgctat attaactgaa ctttttaata aatgagtcat ttgaattt 1008

<210> 120

<211> 306

<212> DNA

<213> Mus musculus

<400> 120

60 attgttaacc taattaaaca

120

ccctttctgg aaagacctgc ttaatctgaa gcatgtgagc taggctagtc cactgggtcc 180

tgctctctgc ccatccccag ctggctttgg attagaggca catacactgc catggctgcc 240

ttttactgtg gctgtggttt tgcccttttt ttttaagcaa atagaaaatg ctgctgacta 300

tactgg 306

<210> 121

<211> 63

<212> DNA

<213> Mus musculus

<400> 121

ggtgtcaccc attgtatttt tgtaatctgg tcagttaata aacagtcaca gcttggcaaa 60

ttg 63

<210> 122

<211> 49

<212> DNA

<213> Mus musculus

<400> 122

atgtacacta aattttctgt acctaaatat aattacaaaa ttatcttga 49

<210> 123

<211> 39

<212> DNA

<213> Mus musculus

<400> 123

acttgatcca aaaagctaat aaaattttct cagaaatgc 39

<210> 124

<211> 47

<212> DNA

<213> Mus musculus

<400> 124

gaagcaacaa gaaaatattc caataaaaga ctatctgata accagtg 47

<210> 125

<211> 42

<212> DNA

<213> Mus musculus

<400> 125

ttctgtgttg gagagctgca ataaattttc cataaagcaa aa 42

<210> 126

<211> 556

<212> DNA

<213> Mus musculus

<400> 126

ttctccagtg tatttgtaaa atatattcat taaagtctct gctctgagag ctggtcttct 60

tgacaccttt tccaatatca gctttgcaga aggaaactta aatttcagtt cagggcatga 120

ccttcatgac cttgcagaac ttcttcactt tccaggttaa gtaaaggcga tctttaggggg 180

ctgtccagat ggatcagcta taaagattca attgtagaag gttcacgtct caatgcccac 240

gtggtagctg taacttcaat taaaaaacaa aaacagccgg gcgtggtggt gcacgccttt 300

aatcccagca cttgggaggc agaggcaggc ggatttctga ggccagcctg atctacagag 360

tgagttccag gacagccagg aatacacaga gaaacccttg tctccaaaaa ccaaaaaaaa 420

caaaacacgc attcttttca ggtctttgct gggaccaggt acacataaca cagataaata 480

ttagagcaaa ccatgcacat atggtaaatt atctttggggt tttggggtccc taaaataaag 540

tggtgtgttc attgtg 556

<210> 127

<211> 47

<212> DNA

<213> Mus musculus

<400> 127

ccctggatct taactgttaa taaaaaaaac attggatgat gatggta 47

<210> 128

<211> 693

<212> DNA

<213> Mus musculus

<400> 128

acacagagac ccactgaata aaaacttgag actgtccttg cttgtttgct tctatgtccc 60

tggagaggtc ccagttggtc ccgtccctaa caacatgcta gccctgctca cctgcctgtc 120

agccttgctc agtggcatct ttccataggt gtgtatcccc ttagattagc ttcagcccca 180

ctacgatttg tctaggacat agcctgagcc ctgcctgtga cactgagggg tagcagtctg 240

tttctggact ccagggtgct gctgtctcag gcctaagaat tccagacatg actataatcc 300

aagcctgggg acctggttga gctttttatc ctgctggctc taagcttcag ctaggtggaa 360

atgaggccag ccaagcccca cagtgagctt gcaagcttta gatggggaca gggttacgct 420

ttggtgaatg atggaggaaa catgggggtt ccttttgttg ggtgcagcca gcacggcatc 480

atcatggtgc ccaatcttga aagggcacag gcctgaagct tcctgggact gttctgtcac 540

agggaggaac ctactgcagt tgcctacaat tgctacctct gagggacttg cctctggccc 600

cttgtagaca tttccatgtc tacacatggc ccagagtact ttcagggata gcaatgtgtg 660

aatggcactt agaagataac atgtgaaagc cat 693

<210> 129

<211> 98

<212> DNA

<213> Mus musculus

<400> 129

agcttccctc gtgtctgtac atagcggcct ggctgtggcc tcatgtggat cagtctttaa 60

aataaaacaa gcctttgtct gttgccctct tgtttagc 98

<210> 130

<211> 1077

<212> DNA

<213> Mus musculus

<400> 130

tgtacacaaa gaaataaaat accagcacca ggactgtgaa gtgttttcct tagactgtag 60

tgtggggttt gctcattggc tttcttgttc agattttact aattgttcta aatgatacag 120

cttagtgtgc agaaaatatc ctcttgattg gaaaatagcc aaatatttac aaaacaggta 180

tactagtttg aagaggctct atatgggggg agggggtgct ggaaaacatt agtgggtgac 240

cagtaatggt ggtacagctc taactcctag caccaggagt ccgaggcagg gggatcttca 300

ggctgcatga tgtgcatagt agcatgctgg tattagggag tgggtatctg tggttcccac 360

tttgaaaata accaaaattc tccaaagtgg gcagacctaa gccaggagag ggctggccac 420

agacatttgg tactgcttgc tgagaaagca ctgattgttt tcctaaccta aagattatat 480

atggcccacc ataccatctt tgaaacaatg tgtactggcc tttggttcac ctttcttgtc 540

tttgaagttg tacttggtgg gtgcatttaa ccttgccaca gagtggggag gatagagtct 600

aatggacctt aagtggtctc tggtggccat gtcggcagtg cttaggttgt agcccagggt 660

tggagtcggc agtgacaagc aaataactat attcttgctt gctgtggcag ctatacagaa 720

atttacggta taggtaagag ggttctctag aagtactacc tgtcttagtg aaagagattg 780

cttggttaac atcctgttat gtagtggggc tacttttaaa ctgtgtgaag tccccattag 840

ccacctccat aggcaatgga gctaacattc ttgctacagt ggccgcagct cattaacacc 900

taatgatgtg tttaacatgt gtccacatgg tgtgaatgtg ggtacgcatg tgcccagtat 960

tcagttcaca gaattgtcat catcttccat catgtcttca gtgagggact ctgcagatgc 1020

ccaccccagt ccttggttgt ggtgattctg ttagcattaa atgcactgga gagcttc 1077

<210> 131

<211> 1658

<212> DNA

<213> Mus musculus

<400> 131

gacacattgg tctgcaatgt tttgtattaa ttcataaata aaatttagga acaaaaccgg 60

tggtttatcc ttgcatctct gcagtgtgga ttggacagga agttggaaat gacagggact 120

180

ctccagtagc aatggccaaa cagaagaacc aggctaggggg gctgcatctg acagagcaag 240

tagacgagag gctgggtggt gggctccggc cagcccgagt cttagagctg gtggttggtt 300

atatctggtg cctgtctcga ggagggcttg agacacagtg tggtgctcct cagaagcaga 360

caggtgattt ctttgtgtga ttttcttttt cccctgggac aatgacagtc agtaagacag 420

gtttcaggga cttttgtgtc caggtctgag cactagtcgc tcacagttgt gtgtactaac 480

cttcttcctt cctattgaaa tggcaggggt ctttgagtct cactgctgca tgttctgcct 540

tcatagggat ctgtaagtat gctgggcatc tgggctttta gggggctctc tatagggtgt 600

ctgagataga ggtcaacaag ggcttataga caactcaaac aaagcccatg gcttgagcaa 660

gtctgcaaca agctgtttgt ctagcctcca gcagagggcg agggagacag cttccagatg 720

ttcccagtag gtggagcccc tccaagccca gggctcagga ggcttacagg gtgggaactc 780

caatactggt ggagggagga gggcgtttga tgggaagata gggaagttgc tgcttcctaa 840

actgtcacaa ctgggcttgg ataggagtca tagtctggga ccacagccct gtggtagaat 900

gctagcctgg tgtgctccag gtttaatctc catcactgca gaaatgagtc caagctgtgt 960

gtacctccag ggcactggggc atggggttcc cttgccattg tgtgtgcccg gagaactggc 1020

aggcgggaaa tgtctttatc aagggttacc ttggaagagg tcccaacact gtagggtgct 1080

cctgttgtca aaacctatgc agaggcatct gcttgctctc taataacagt atgcaatgct 1140

aaagggctcg cttacagccg gtggccacac tggaggcctg cacatcaggt ggccacaagt 1200

tctgctgctg cgcctccgag gaaacacttg gtcctccgat cgattttaac ctgttgaggc 1260

tttgcaatcc ccctgtggca aaggctccag tgttttctat ttctatgcaa atttcttgaa 1320

gcagaactgt tactgtcttt ctcctctgcc ctgggaggag gcgctagcgt ttccttccaa 1380

cttcaggtgc agcccccctc gtggttagcg gtcttaagtt cgtgacttgg gtttgcagat 1440

cttttttgtt acatcgccgg accatgtggt ggtctttagc tgtaaacaac attaaccctg 1500

ggttgattag catatgcttc taaaagatgg tcccagattc tgcgacttgt aataaaatgg 1560

1620

tgtattaaag tggctgacac atggctactg tcactgtg 1658

<210> 132

<211> 1795

<212> DNA

<213> Mus musculus

<400> 132

aatatctcac caaaaaatat ttgaagaaga acaacctccg agactggctg cgtgttgtcg 60

ccaacagcaa agagagttac gagctgcgtt acttccagat taaccaggat gaagagggagg 120

aggaagacga ggattaggac acattggtct gcaatgtttt gtattaattc ataaataaaa 180

tttaggaaca aaaccggtgg tttatccttg catctctgca gtgtggattg gacaggaagt 240

tggaaatgac agggacttta actgggctgc tgctcctttg tatatagaca cttttttcct 300

gctcagaaac ttgagttctc cagtagcaat ggccaaacag aagaaccagg ctagggggct 360

gcatctgaca gagcaagtag acgagaggct gggtggtggg ctccggccag cccgagtctt 420

agagctggtg gttggttata tctggtgcct gtctcgagga gggcttgaga cacagtgtgg 480

tgctcctcag aagcagacag gtgatttctt tgtgtgattt ttcttttccc ctgggacaat 540

gacagtcagt aagacaggtt tcagggactt ttgtgtccag gtctgagcac tagtcgctca 600

cagttgtgtg tactaacctt cttccttcct attgaaatgg caggggtctt tgagtctcac 660

tgctgcatgt tctgccttca tagggatctg taagtatgct gggcatctgg gcttttaggg 720

ggctctctat agggtgtctg agatagaggt caacaagggc ttatagacaa ctcaaacaaa 780

gcccatggct tgagcaagtc tgcaacaagc tgtttgtcta gcctccagca gagggcgagg 840

gagacagctt ccagatgttc ccagtaggtg gagcccctcc aagcccaggg ctcaggaggc 900

ttacagggtg ggaactccaa tactggtgga gggaggaggg cgtttgatgg gaagataggg 960

aagttgctgc ttcctaaact gtcacaactg ggcttggata ggagtcatag tctgggacca 1020

cagccctgtg gtagaatgct agcctggtgt gctccaggtt taatctccat cactgcagaa 1080

atgagtccaa gctgtgtgta cctccagggc actgggcatg gggttccctt gccattgtgt 1140

gtgcccggag aactggcagg cgggaaatgt ctttatcaag ggttaccttg gaagaggtcc 1200

caacactgta gggtgctcct gttgtcaaaa cctatgcaga ggcatctgct tgctctctaa 1260

taacagtatg caatgctaaa gggctcgctt acagccggtg gccacactgg aggcctgcac 1320

atcaggtggc cacaagttct gctgctgcgc ctccgaggaa acacttggtc ctccgatcga 1380

ttttaacctg ttgaggcttt gcaatccccc tgtggcaaag gctccagtgt tttctatttc 1440

tatgcaaatt tcttgaagca gaactgttac tgtctttctc ctctgccctg ggaggaggcg 1500

1560

1620

aaacaacatt aaccctgggt tgattagcat atgcttctaa aagatggtcc cagattctgc 1680

gacttgtaat aaaatggaaa cttgctggtt tttatgcctt tctaactctt gtatttgaat 1740

gaatgttgat cactttttgt attaaagtgg ctgacacatg gctactgtca ctgtg 1795

<210> 133

<211> 48

<212> DNA

<213> Mus musculus

<400> 133

actgagtcca gatggctaat tctaaatata tacttttttc accataaa 48

<210> 134

<211> 140

<212> DNA

<213> Mus musculus

<400> 134

attcagcata aaataaaggc agataaagtt aaaggtcttc tggtggtctt taatgagccc 60

tgttgggagt gaggtgcttt aacatggaga agcatgttat taaacagtga aatagatggt 120

tcaaaaccac gtgaccatgt 140

<210> 135

<211> 46

<212> DNA

<213> Mus musculus

<400> 135

tgtggtagag cagagttgga aataaagctc tatctttaac tctagg 46

<210> 136

<211> 40

<212> DNA

<213> Mus musculus

<400> 136

agacatctcg tgcacggctt tcattaaaga ctgcttaagt 40

<210> 137

<211> 109

<212> DNA

<213> Mus musculus

<400> 137

gtatattttt gttttggtca ttaaaaatta aaaaaaaaaa aatacaagtg tctgcctatt 60

gcatttgtgt gggaagagac tggggaaata aaacaggtgt gctgttgtg 109

<210> 138

<211> 45

<212> DNA

<213> Mus musculus

<400> 138

acaagaaagt tttcctttaa taaaactttg ccagagctcc ttttg 45

<210> 139

<211> 640

<212> DNA

<213> Mus musculus

<400> 139

aagccacacc ggaggttaat taaatgctaa cattttccat gtggtctttg catccttcct 60

tgtctgcatg ttggaaatct gcctaacatt ctaggaagag gtgaggtgtg ggcccttgag 120

agtcagtctg tgggaataag tgtagcccaa ctatgcacag ttgtaaattc ctacatcccc 180

gtgtgtattg gtcttgatat tcaaagaatt gatgaatgcc attactttca gtccaaagtg 240

aagaaacctg gtctcaaaaa atcccgagga ccagaaatga gatgggtttt cctgaaaatc 300

taaagttctt gaaaaacctt gccatccaga ttgctagcaa ctgcctagct ttgtaagctt 360

actgtgatgg acaggtagct caggacgact ggtcacttaa tactggacag attagcatgg 420

aaaacttaag gggaggagga ggtagtaggt tccatccagc ttcgctttgt tggtggcatc 480

taggtgttgt tccaagggag catgcctacc tgcaacagga catcactggt tgggaatact 540

gtagaaccag agctgtgacc tttgaactac tagaaagatg aaattttatg taaaagagtac 600

cttggagtaa ataaataaag cccaagatcc tgattgtcta 640

<210> 140

<211> 40

<212> DNA

<213> Mus musculus

<400> 140

gccccacacg cccgaagcaa taaaagtcc actgacttcc 40

<210> 141

<211> 124

<212> DNA

<213> Mus musculus

<400> 141

aaaaggctcc tgccagtgtg aagacagacg gactgctgtg acacacctcc ccacacacta 60

tttgcagatg accagtgtcc tatgctgttc ttacaaataa actcaggcaa gatctgttag 120

cttg 124

<210> 142

<211> 575

<212> DNA

<213> Mus musculus

<400> 142

cctgctcgtg tcaaataaag ttgcagaact gccttcaggg tttggttttc ctttctgttg 60

tctgcctcat gggtggaatt tttgggtcta cagggtgttg gaaattaatc tgagaatctc 120

tgttctgggt acatgggaaa ttagaaatac gtgaaacatt cttttcacag aagtcacttt 180

attaggattg tggatttgggg ttggttttga aacagggttt cttgtggcac tgcttgttct 240

atagaatagg gtggccttga actcagaaat ccacctgcct ttttcctccct agtattggca 300

attaaatgcc cagcttgttt gtaagctctc attttcagtt ccaggtttat gtgtgagcct 360

aagattaggt aaagattgag gttataactt aaacgtactg aattaactta tgttgtgtgg 420

gtccggaa ttggacctgg gacatcaact ctgcctttcc agccatcttt gccaaccagt 480

agctcatctc tgggatgtgt ctgccctcaa aatgacattt taaaaaagtc agtacaaaag 540

aacgattttt attaaaaacc ttgaggacaa acatt 575

<210> 143

<211> 54

<212> DNA

<213> Mus musculus

<400> 143

atggcttgtg tgcatgtttt atgtttaaat aaaatcacaa aacctgccgt cgta 54

<210> 144

<211> 68

<212> DNA

<213> Mus musculus

<400> 144

actaatggag agtaaataaa taaaagtaga tttgtgctct tgtatttttt tttcacatct 60

gtcctaaa 68

<210> 145

<211> 504

<212> DNA

<213> Mus musculus

<400> 145

ggatttcaat cagtcataaa ataaatgttc tgctttcaaa aattctgtgg tgatctaagg 60

tactttaaca tcggttcaga gttcggttat atgattgctc tgggatccta cgcttcttcc 120

ttcatagttc ctgtgggtcc gaagctggga ggggctgggt ggactctcgg gaaagatact 180

ctgagcctgt ctcggtcccc atcgtgtttg cttggccctg ggcatggaag tgggtgagtg 240

atgagctgaa cgagcaggct tgctagagat gaggacagtt actggtgtgg ttatatcact 300

accatgccta cagtgtctta agacgcttac agtctgtaag ggacttaaat gatttgagct 360

cttacttatc ctgtagtttc tgatttttaa catttacttg aataaagcca agcaagataa 420

gcctttattc ccagcacttg gtgacaggtg gatctatgag ttggggatca gagctacaca 480

ttaaaactct taattcatct tact 504

<210> 146

<211> 306

<212> DNA

<213> Mus musculus

<400> 146

ggatttcaat cagtcataaa ataaatgttc tgctttcaaa aaaaaaaaaa attaatcctc 60

tgtgatggcc agcagttaac attcaacagt ttctctctag gctcttgatt ctctgactat 120

tgtagggatt cgatcagcac tcgcatacca gaagtgtgag atggtccgtc ctttttcaag 180

acaagatttc tctgtgtagc cctggctgtc ctggaaccca ctctgtagac caggctggcc 240

ttgaatttac aggatcccc ttgcctccgc ttgctgagtg ctaggattaa aggcatgcgc 300

actatg 306

<210> 147

<211> 246

<212> DNA

<213> Mus musculus

<400> 147

aagccctgct gtctgagact tgcctagcct gcaataaacg ggttatttac gtaacttttt 60

tttttttgcc ttgtttgtgg ttaattaaaa catttggtgt gtgttctatt ttttattttc 120

gaaagatgct tgttttgaga catactgtgt gaccctggct ggccttgagt gcctggttct 180

ccttacaagt gtagatacat ctggcttaag attttagtct ttcagaaata aaaatgttgc 240

tagac 246

<210> 148

<211> 41

<212> DNA

<213> Mus musculus

<400> 148

accagccctc tgcgtgtgac tattaaaaac cctgaaaagt g 41

<210> 149

<211> 181

<212> DNA

<213> Mus musculus

<400> 149

ggattcacac aatggcaaga ctgaggattt atactgaatt gtcatcaatc agtcctacca 60

gatggatttc aacatttaaa cctggagact cttcgtgtct tgaattagga tgtttgtcca 120

gtaataaaat atagaacctt tcaaaatgct tttctggttt ataaagtact gaattgccct 180

t 181

<210> 150

<211> 285

<212> DNA

<213> Mus musculus

<400> 150

tcccgccaaa gcaaccaagt cagcctgctt aatttgagaa agatggaaat aaaggcttac 60

ttctcttaaa actccggtct ggatttattt agtttgttca cttaagcagg atgaaaaagc 120

180

tttttgtggg ctttgtttgt tttttgagac agtctcttga cttggtgcca tagctagtct 240

gggacaaaga ttttccaggt gtgaattaaa ggtgtatgtc atcaa 285

<210> 151

<211> 40

<212> DNA

<213> Mus musculus

<400> 151

actgcttttg ttaagttggc taataaagag ctgaacctgt 40

<210> 152

<211> 42

<212> DNA

<213> Mus musculus

<400> 152

attcctgctc ccctgcaaat aaagtctttt tatgtatctt ga 42

<210> 153

<211> 1059

<212> DNA

<213> Mus musculus

<400> 153

cagggtcttg gcagctgcat ctggaggcat ttaataaaat aaagacattt aataaaatct 60

tgaacaaaga caaggcctga ctggattgtg tccagtattc aactgagtta tgttgtctat 120

ggagccatgc ttattctgtt ggtttaagct ggagggcatg agcagagctg accagagaag 180

240

tgctcttgat ggaacccagg tgcttggaca tagtaagcac acataagaca gagtaagact 300

360

gaatgaaagc agatggccca gcgagttagg tgcttcctat gaaagcatgt gtgctggttt 420

gtcatgcaca cagccctgca ggagagagta tggcaacaca gccgctcagc atcccaagat 480

aaaaagggag tttctactgc cattttgagc ttgggagttt gaaatgtaaa gcctgtccat 540

atgttttaag gatccatgta tttctgtttt gtttgttttt caaaacaggg tttctctgta 600

gccctggctg tcctggaacc cactctgtat gtagaccagg ctggccttga actcagaaat 660

ccacctgcct ctggcgatcc atatatttct aagtcctgta cttagacgct gttttggaaa 720

attcattttg gaagcattta ctgttggtgt gttttgtggg gaatgaatga tagcttggga 780

attcttttct gtttggtgag agtgaagctg tcagcccggt tgtagcctgg ctggtgctca 840

aaggctttct ctcattgtct tcacctacgt agctttacgt ggggtaagga cttaagttac 900

ttaagttggg tgcacactga ccatgtccac aacctgttaa ccaactctac atgatgagta 960

cagatgtacc tttttagaaa gtgttaatgt gtagccctgg ctggcctctg cctcagggta 1020

ttatgaataa agtgtgcaac cttcatctgg ttgattaaa 1059

<210> 154

<211> 49

<212> DNA

<213> Mus musculus

<400> 154

aatccagatt tttaatagtg acaaataaaa agtcctattt gtgatcgtt 49

<210> 155

<211> 87

<212> DNA

<213> Mus musculus

<400> 155

aatggtctct aggagacatg ctggaaaagt gtttgtacaa gcctttctag gcaacataca 60

tgctagatta aacagcatgg tgaaact 87

<210> 156

<211> 100

<212> DNA

<213> Mus musculus

<400> 156

agtggactct gagggacatt gcgggggaagg ggcgtttacg tttgtttata cttaaaagtt 60

ttttaagcag catgttgaat taaaaaagaa agcaagcttc 100

<210> 157

<211> 45

<212> DNA

<213> Mus musculus

<400> 157

tttcccagct gctgcctaat aaactgtgtc ctttggaaca actat 45

<210> 158

<211> 603

<212> DNA

<213> Mus musculus

<400> 158

gtctttaaga gcaacaaata aataatgacc ttgaatcttt cattggcttt cattaatagt 60

gtaactagat aaatgatggg aaagatgaga cagaagaagg aatacatcta taggactgct 120

agaatatggg gagagtgatt attttcaaat taatatgtat cgagcttcta ccccaaggta 180

aataaataac atttggagac cattaaaatg taggatggca tagaagaggc ctttactaag 240

attaataatt aaagaaacac agcctttaaa gtaaaaaaca cactgtgcct ttgaaacttg 300

ctaaaaagat taacttctgt cccaaaaggt atcagccatg cgctaccagc ctccctgccc 360

ctacagtggc agtggctgca ttcttggtga atggtagtgg aagggattaa acctaggcct 420

cagtcatgct tcccagtcac tggtactgat ttgtatgcac ccgcttaggt gtgaaggtag 480

ttttggtgtg tatcacaagt tagcctgtgt agcgaagaca ggttttctcc accgtgtttt 540

ttgttacaca tgactattca caaatgtgct gcagacagta aaatgagaaa tacccttcca 600

agg 603

<210> 159

<211> 269

<212> DNA

<213> Mus musculus

<400> 159

agaaaatgac tgaataaagt gtcattcata gtatttggtt gtagtaactt gtcaaaatct 60

cagggccatg ggtgcacgac agcagtagct tcttgaatga actgaagttt tcaagaggtg 120

cctggaaggt gaaaaacaca ctgaagccag tcatgttgat atgggggcat tctgctgctg 180

tgaaacagac tggggttcac acccaccttg cgggattaga acttcactgc cctccaactt 240

ctttctttgt aaacaactgt ccacatttt 269

<210> 160

<211> 42

<212> DNA

<213> Mus musculus

<400> 160

gcctcatgtg tagtgtaata aaggtgtctg ctgttctatc tg 42

<210> 161

<211> 69

<212> DNA

<213> Mus musculus

<400> 161

ttaatacttg gctgaactgg aggattgtct agttttccag ctgaaaaata aaaaagaatt 60

gatacttgg 69

<210> 162

<211> 36

<212> DNA

<213> Mus musculus

<400> 162

agttggagtt tatgttgtat tgaataaact ttaaag 36

<210> 163

<211> 47

<212> DNA

<213> Mus musculus

<400> 163

ggggactctg gccaatgccc tagaacaaat aaagttattt tccaacg 47

<210> 164

<211> 50

<212> DNA

<213> Mus musculus

<400> 164

ataaattttg gctgattttt ctcttgtatt tcttgtttgc tggtataaaaa 50

<210> 165

<211> 43

<212> DNA

<213> Mus musculus

<400> 165

atgctgttgt gtgcacaagc aataaaatca ctttgagtaa ctt 43

<210> 166

<211> 34

<212> DNA

<213> Mus musculus

<400> 166

ccgaggccaa taaagactgg ttttggtccc tgga 34

<210> 167

<211> 3272

<212> DNA

<213> Mus musculus

<400> 167

acgtaaagca taaataaaaa gcctttgtgg actgtgctca gggtcagtcc ttttgaatct 60

ctgcagcaga gtagctggct gtgctgactg gtgacacttc tggtgatgct cagctgtgag 120

180

atgccagtaa catcagcata aggtggttaa tgacaggatg gtcccttgag tgtgcagtga 240

gtctggttta tttgccaatg agaagcacag gcctcctgta tgggtctttg cctacagccc 300

cctttcatca ccgacttg gtagacttac attctgtcac actgttggct cttaatctca 360

gccctgaaaa atgccatttc ttgggtatca aggctagtct agattcagaa accatataaa 420

ggttgacagc tggtttaaaa aaaaaaaggc ttggagcttg agttggggttc gcaggtttatt 480

ccagggtatc tgtctgcact ttgtctccca gatttaaagg taagtgccac catgcctagc 540

atggtgactt attagctttg ttgctgtgga acatacatca aatgaaacat tggtatggct 600

660

catcccagaa ctaaggagcc attccccaga gacttgtctt cctactaagt tccatcccta 720

cagcttctag tagtagcttc agaggttgtg aattgtggac ttagtctgcc ataacattta 780

aaataggtat taaattcaag tcatttggtc actcagcacc cacgtggctc ttcagaacaa 840

cagaagcccc tcggacttgt ctgttggaaa aaccagtttg aaataatgta cctgctttag 900

ttgagaaaac gctacaactg gtgctgtgtc ctgccatgct gatgagctct gctctgcgac 960

ctgccgaact tgggggatct ctacccccag actttgctca gatctgttga tgatttgtcc 1020

atgcaggaaa gtttacaagg tctctgtgtg tctactactt actagttgct gtgacaaaaa 1080

tactgaaagt gtttactgtg tgtgaggcac aaagtttgtg ggaagctgta ccctccctca 1140

ttttggtgct gctcctgcct tgactgacag gatgagctgc cccaaccatc gttgccatct 1200

tccataagaa gcaggtggct ctattgagtt ccctggaggt gatcccaagg gaaggaggag 1260

cctgggaaag tggatctcaa gtcccctagt ctggcagttg gctgtttagg aaagtccagt 1320

gtcagtgttt gatatgttgt aaggaaacaa attcagtttt atttagctta ttggctctgg 1380

ggaaatggca gttcccatta attggtgctg tctttctctt tgaggatcaa aattagcttc 1440

ctgttcagtt gttaagcata tttcatagtc aaataatcct cttatcttta caagtgaggt 1500

tttccttcga gtggatatca gagtccctcc cacggcttct atccctccta tccttgtgta 1560

1620

tgtgtactct acacatgatt gattctaagc ccccttccct ttccatgtcc ttcaaaaatt 1680

tttttattct gagacagtgt taggattttt cctgggctag ccaaatgcaa gaagtgttgg 1740

tcccagactt gtaatctttc tgccttagcc tcccaaattc taggattata gatttatgtc 1800

1860

gtagttttgg ctttcttggg acttgctctg tagacccagg atggtcttta ctcagatctg 1920

cttgcctctg cctcccaact gctaggatta aagcatgagc ctgagccttc atgcctggct 1980

gacaggtgaa ttctcaatac ctacctagcc atagggaaag tgattgtgtc cccttcctca 2040

tagaggggca tagtcaccca ggccatacct ttagcttggg cttttggtca gtgaagaagt 2100

atgaagggac aagaacacta tcaagagcct aatgtgctct ggcctggatg gtcagcacaa 2160

2220

cctaaattta gcagactata ctgtcagcct atcagactat aggctgccgg agggcaagtc 2280

tgctacctat ttccatctca tgcctgcatt gttcatcccc ctatgtaacc cacctacctg 2340

tcactcattc ctccatccaa aaactattgt aggttcagtg gaaatttcaa gcttgcctgt 2400

ctcagcatct ttcttacctt acccctaagg atggcatctc tcttggctac atctttggtt 2460

2520

ccagtgtccc agagaccggc atttgagaga tccctgatag caaacccata gggtggcctt 2580

tttttcatcc accccattct ccctcccacc tccctctttt gaccttgagt cctcaccaga 2640

gagaaaccag gcccacttaa ttagttctac atgtgtacac tacatgggtg cagtgcccaa 2700

gcaggagagg tgttagattc ccttttaact atagttaata gacagttttt aagccccagt 2760

ggatgctggg aagcaaacct acatcctcta gaagagcagc tatttctctg agccatttct 2820

ccagcaccat tttcccctct tttaaaagca ggtcttgcag tgtggcctag tctggccccc 2880

tgaggtgttt gcattgcatg gcaggcatgt ccacaggaac accatagttc tcaccactcg 2940

tacagcacag caagtggggt gccgcagggg attatcactt gagtataaaa taagggttgc 3000

tttagattga ataggataac cacgcgttct cagaacaatc aaggaaggct ggggtgagcc 3060

agcaccgacc ttaattgttt acttagtaaa ctactaaatg tatgcacgtg taagcttttg 3120

3180

ggcagcaagc acctttgcct gctgagccta catgttgtat agaatggcaa cgttgtgtag 3240

aatggcagta cattaaatgg gtttttcatt ta 3272

<210> 168

<211> 59

<212> DNA

<213> Mus musculus

<400> 168

gcccatctca aggatcgggg tttacctttg taataaacat cctaggattt taacgttcc 59

<210> 169

<211> 35

<212> DNA

<213> Mus musculus

<400> 169

aacaaaggat gctgggttaa taaattgcct cattc 35

<210> 170

<211> 3167

<212> DNA

<213> Mus musculus

<400> 170

gacaactgaa taaatcgtct taatggtcaa attttgctgg cttttgttca ggtttttttt 60

ttttaattca tgtttatgag tgtaaatgtg tgtgtcacag gggtgtcaga tgttctttga 120

accaccatgt aggtgctgga aacccaacct gcatcctcgg agagaacagg ttccttaacc 180

actgagcaag tactgaagca ttaaactgct tttaaaaatg aaggtgtgct aacagattgg 240

tcaggtgaaa aagagacgtt aggtttcctg cagggggcgc taagccaatt taaagactaa 300

gttgggttag aaaagagcag attgcatcct tgatctttta agcctgggga ttttgttttg 360

ttttgggata gggtcccaac acagaacagg ctgacctcat taaatatcaa tcttatttga 420

ttgcctctgc tccgagta ctggaattaa aggcagggac cactgtatta gccattctga 480

gttatttgaa atggactctg caggccatac ttggtcaaaa ttctgccttc tcaattacag 540

gcatgagcca ctatgcctgg tttacttact aatagatgtc caaagactag tgtatgaaaa 600

ttttgctttt ccaggtgatt tgtgaaaggc agggtggcct ctcccatgtc acactacttg 660

ggttactcat gttgcaacat atctgcaact ttaggttgag gggatttgag cctgcatgtg 720

ccactttggc caactgaact aatctttaat tccatctaaa acttttaaat ctcagtcatg 780

tgttcaactg gaaaataacc tagagtgtgc tatgttgact tcaggtacac atcaaagcag 840

gttttagtga tgtagaagct gtgtttgagt tgaactagtg ttgaggctag gcttaggtac 900

catagaactt tggtttttca agacagggtt tctctgtgta gccctggctg tcctggaact 960

cactgtagac caggctggcc ttgaactcag aaatctgcct gcctgtgcct cccaacacac 1020

ccagctctag ctttaaattc cttgcaccaa gagatgcttt atccctccgc tgagaataca 1080

ggtgcatgtc agcatgctaa actctagata aaatttcatc ttgtttgaaa ggacaataat 1140

ataagaaaag tgtatttgca ctgtatacca tgcccttttg tgtttaaagt taaactggca 1200

acagtgtccc atagaggttc cagaagaaac tgcttctaag gggaggacca taaagggaaa 1260

tgcttaccat agaacttttt aaatgttcct acaggttgta ctctggatag gtatatgaac 1320

acctttctat tagaacagtt ttatagtagt acttagtgaa tatgtaaata atactatttg 1380

tgaaatagtt tgaggtttct ctatagtcct ggctggcttg agctctatac cagattggct 1440

tctaaatcag aaatctgcct ctatctcctg aatggttatg atagagatgc aagttactta 1500

1560

tggattagac tgtctttatt acatttactg gcttgatact ttacagtctt aagcccactt 1620

gcctgggtgt gatgcacacc tttaatccca gcactttggg atttctgagt tccaggacag 1680

ccagggctac gcagagaaac cctgtcttgg aggggaaaaa aagaaacttt ggaaatcaaa 1740

acttcttgga aagccacttt tagagacttg aatctaagga taactaacca ggtagtaacc 1800

acgagtcatt gattctgtga atcttgtatg agtgggttac aggcagaaat taacttcctc 1860

tgagagcact gctgttttag aaatgcgacc tagtaattac caaaggcatt gaagccactt 1920

gactacagtc tcaggtttct gcatctgaca ttgctgggac tgtgtggggt ttatgggtgt 1980

aaaataaaac aggcgaaagg atgttgagtg gaaagctttg gcttcagaat caaattccaa 2040

atcaattacc agatcaaatc aaatgccaga tcaaattaca agacttcctt atgactaaat 2100

2160

cccagtgaag ccaagattca catgctatgg gcatggattt cctgaggaca tgctagccta 2220

tggtatgagt tacttgaaag gactctgaga aatcttagtc cccagctgtg aggtttttaa 2280

agatcatgct caatggaaag tggggcagta atttgagagc atggcacaaa tgaggtttta 2340

tcttttgaaa ctaagtgaat ctatgtcctt ggactagcat attttaaatc acacatcaaa 2400

tgaaatttct gttcaattcc tataaacagt ttatttcata ttttgtagtt accattttca 2460

ttaacagcat acacccttca attgtgttgc taaaactgag tcacattatt ctgtaagaac 2520

ttactccagt atcacaactt ggtgctcatc caatattttt attttcattc tattttccct 2580

gtctagccac gtatggccct attctctctt cttggattga ccctagcttc aacacaagaa 2640

agcttgcagt attttttttt tgcgcctggt tcattgtttt gtcctcaagt tctatccatg 2700

tgtccagaaa ttcgaggatt ttttaaaatt tacttttctg cctaaatact ggtatgtgga 2760

tagtctgtta tttttactgt tggttgcata tctgtagcat atttctgtat ttgcaatgac 2820

tacattgaat gtcccttagt taacctcatt cttcttaact attttgtagg cgtttgctat 2880

tcaaagcaca atctcaatta aaatgttttt aatagcatct ttccacttgg atgtgtaaag 2940

aaagtatttc tagaagtctg aatttttgtt gcattgtaga tgtgtacaca atagggctgg 3000

3060

cccagaatct ataaggtaga agaaaaaaac cccagctccc acaagttatc tggccctctg 3120

cacacatata aatagtgcaa taaaaattaa ccatttaaaa atataaa 3167

<210> 171

<211> 58

<212> DNA

<213> Mus musculus

<400> 171

gcagaagaaa tcgggaattt gttacaaata aaagttttaa gtacctgtga cagttaag 58

<210> 172

<211> 49

<212> DNA

<213> Mus musculus

<400> 172

aatttgacaa tggaaacaca gtaataaatt ttcatattct gaaaaaata 49

<210> 173

<211> 264

<212> DNA

<213> Mus musculus

<400> 173

acaggttcaa tcagctgtac atttggaaaa ataaaacttt attgaatcaa atgaatgggt 60

gcatctgttt cctaaggcag ccggggagga tttggtctta ggaataatag ctggaattgg 120

tttgttggcc atgaagtcag atgcaattgc gcctgggaac cttcagcttt tccctttacg 180

tggttgcttg cttcttgttg cagcttcggt tttgaattga tgcctgaaag aaaataaaaa 240

cttagcaaga ctaatggtaa atct 264

<210> 174

<211> 71

<212> DNA

<213> Mus musculus

<400> 174

agaggccgtt ttgtaaggac ggaaggaaaa ttaccctgga aaaataaaat ggaagttgta 60

ctttacatgg c 71

<210> 175

<211> 56

<212> DNA

<213> Mus musculus

<400> 175

aagcccctga ttgaagatga gtgggaacct tcccaataaa cacgttttgg atatat 56

<210> 176

<211> 38

<212> DNA

<213> Mus musculus

<400> 176

ttgtgtatgc gttaataaaa agaaggaact cgtactta 38

<210> 177

<211> 92

<212> DNA

<213> Mus musculus

<400> 177

tcttgcagct gggttctgga tatccactac ttagcccacg gaatgatctg caactgttaa 60

ataaagcatt tatattaatt cttgtctaga aa 92

<210> 178

<211> 91

<212> DNA

<213> Mus musculus

<400> 178

gcgaccttga atggattcga ctgactacta ccaagtggaa ccgatcatgc tagtctttgt 60

acacaaagaa taaaaatgtg aagaacttta a 91

<210> 179

<211> 1187

<212> DNA

<213> Mus musculus

<400> 179

tacacgtgat atttgtaaaa ttcatatcca ataaacaatt taggacagtc atttctgctt 60

aaaggtgtta tttgttaaaa ctagtctaca gattgtcatg agtgttctgt gaaaatgtag 120

aagttaagtg caataattga aaactgcagg tgatggcata tcttgtttct gatgtacttt 180

240

taagaccctg taagaggtaa aaagtcctga aactgaccct ggatgtgttg gtgcatgaga 300

tagaatctac agctttacga tggcatcttt tggttcactg aagtggctgc ttgggagttt 360

gatgagtaca aactttatag agttggattt tgcttagaaa tgtgtaggaa gagagggttt 420

caaaacctgt tttgtgcata gaaaagtgag atcaactata acccacattt tgagaattga 480

atccagtgtt attttcagaa gaccaggtaa atttgttgga agaggtttca ctttctgttg 540

gatctagagt atggatttgg agatgaaggt tgaacattgg atgtagtagg gcttatttag 600

ggcagattat ttccattaga tttgtaaact tggttgtggt tgcaagttaa tatcaaccaa 660

gcaaatataa gtttgattaa gcttgcagac attaagcttt tctagcagct ggtgtgtgca 720

gaggcagatg gctctctggt tccaggacta cctacaatat agagaaattt tctctattcc 780

agtcaattca tcatgggtaa gaatagtcca ttttggtagt gttatttcat cgtttacaat 840

ctacctatgg gtagtggctg gtaactgcct ggatgttgac atttcacaaa ggccatactt 900

aaccacactt ttatttctat ttgtgcgatg ccttggagta gtttcccaaa gtgattttga 960

gtgtggaaga aatggtattg tccccgaaca gctggcttgg tctcaaaatc taattgatgc 1020

ttttattaaa ttggttttcc tttggagatt ttaaaaggat gatttgattt ccagaaaata 1080

ctagactcaa aatcttgata gctaaaattc ttttctattc agcaaaacaa gtcactgtat 1140

agaggttgtt caaatcaact aaagtaataa atgtcttaaa caagtgg 1187

<210> 180

<211> 43

<212> DNA

<213> Mus musculus

<400> 180

gggtttttat atgagaaaaa taaaagaatt aagtctgctg att 43

<210> 181

<211> 259

<212> DNA

<213> Mus musculus

<400> 181

ggctacacag aataataaag gttctttttg acggtggtaa atctcatgtg tggactctaa 60

gcttgtcgcc aagtgggaaa tagactggtg ggattgtaga taggatgggc tacttaaact 120

cattctaccc aggccttagt acttagcata cagccagagt caaactgatc ctttatacag 180

ggggtaccat gacagtacaa cagtgtcgtt aaccctaaca aataaatttc ccaccaacgg 240

gtggaattcc ttcattttg 259

<210> 182

<211> 48

<212> DNA

<213> Mus musculus

<400> 182

acaggacttc tcattatttt ctgttaataa attgctttgt gtaagcta 48

<210> 183

<211> 67

<212> DNA

<213> Mus musculus

<400> 183

aggcttcaat agttctccta taccctacca aatcgttcaa taataaaatc tcgcatcaag 60

ttcgctt 67

<210> 184

<211> 33

<212> DNA

<213> Mus musculus

<400> 184

gatgctccaa taaacctcac tgctgccact cag 33

<210> 185

<211> 36

<212> DNA

<213> Mus musculus

<400> 185

gatgacaacg acaataaagt gcgagactga ctggct 36

<210> 186

<211> 388

<212> DNA

<213> Mus musculus

<400> 186

acccaataaa gactgtttgc ctcatgcctg cctggcctgc ccttcctccg ccgccaacta 60

gggaagtggg gaccaaaggt tccttaggca ctgctcctgt gggtagaggg gacattagag 120

agctgacagc gcaccacctg catgagtttt tattaaagtg caaaccatgg gatgaatcag 180

ttgagcttca gtgttgaaaa tgagtagcag ggctgcccca cccacctgac caagtaccct 240

attctgcagc tatgaaaatg agatctgcac atgagctggg gttcacaagt gcacacttgg 300

360

tgctaataaa ttatttgact taagtgtg 388

<210> 187

<211> 32

<212> DNA

<213> Mus musculus

<400> 187

tgaaccctcc cccaataaaa gatggttcct ac 32

<210> 188

<211> 103

<212> DNA

<213> Mus musculus

<400> 188

60

tcgtttggga gggaatacat taaaagcttt cagaaattac ctg 103

<210> 189

<211> 277

<212> DNA

<213> Mus musculus

<400> 189

gccagcccgt gcacctacga cgcctgcagg agcagaagtg agggatgctg agggccggga 60

caagctatcg gactgtgtgc tgccatcggt aatgagtctc agtagacctg gaacgtcacc 120

tcgccgcgat cgcctggaga aatgaccgcc tttcttacaa ccaaaacagt ccctctgccc 180

tggacccccg gcactctgga ctagctctgt tctcttgtgg ccaagtgtag ctcgtgtaca 240

ataaaccctc ttgcagtcag ctgaagaatc aaactgc 277

<210> 190

<211> 44

<212> DNA

<213> Mus musculus

<400> 190

gtctctggggc ctttgctgtt aataaatagt ttatatacct atga 44

<210> 191

<211> 404

<212> DNA

<213> Mus musculus

<400> 191

aatacctagc agtgagtgga gattggatac agccaaagga gtagatctgc ggtgacttga 60

tgttttgctg tgatgtgcag atttctgaga ggacaaataa actaaaaagc tcctacacgt 120

ctgctctgct gcttattggg cattagaaga atcaggtggc tgcttgggtg ttgatgcagt 180

caagtgcact gggcttggtg aaaagcccag tgtaagaggc cggtacagat ccttcctggc 240

agagggtggt gatggagaga acataaataa ctacatgggc aaagtgtagg accaattacc 300

ctgttagcat cgtctttgct caacaccttt ctgtgtccct agactctgag tttttttcta 360

attgattttt attgaacact gagtgttttg aggttttatt tttt 404

<210> 192

<211> 39

<212> DNA

<213> Mus musculus

<400> 192

agttcaggag ctaataaagt acgtcctttg gctaatccg 39

<210> 193

<211> 48

<212> DNA

<213> Mus musculus

<400> 193

atatgcacat tttttaagta ataaaaatca agacttgatc tacgcttc 48

<210> 194

<211> 36

<212> DNA

<213> Mus musculus

<400> 194

tctgttatgc catattttca ataaacctga aaacaa 36

<210> 195

<211> 875

<212> DNA

<213> Mus musculus

<400> 195

gctgctgtgc aggtgcctga gcaaagggaa aaaagatgga aggaaaataa agttgctaaa 60

agctgtctta tggtcctcac tgcagactgt acctggattg gcatttggct atacaacaga 120

ggcatggtcc tactgacatg ttttgtgttg aatacttaag catgtgaaca catgggtttt 180

tttttttttt aatgtaaaat gtagtaacac aatgtttagg tggctttggt gttagctctg 240

gagacttcat gtgtcatcta ggtgaggtgt tctttaacac agggtctctg ttctgtcatg 300

cctcatagat ccttctacct ccagattgga gagggaaaag gcttatgtca ctgaacctgg 360

ccagattggg attttgtgtc cggaacaa agttaatgct aaaaagttaa tgccttggtg 420

agactgatag tctgatggtg tgaattcaca gtaagtggtt gggattgcca gatggaattc 480

cctgagctgc cgtgacaggt ggcattgcag aagtgaagga ttcaggaatt tgagtgttgg 540

gtggggggcct gtgaatagca cttgggctgg gaggggagac tgctgcccct gaatgtcctg 600

660

ctggcattgg cggcgcacac ctttaatccc agcacttggg agtcaggcag gtggatttct 720

ggcctggtct acagagagtt ccaggatagc cagggataca cagaaatcct gcctcaggaa 780

aaaaccaaaa agaagtttgc taaaaataag catttttgct tgatggtatt gaagattgta 840

agacattaaa ttgtgtcatt acttctccag gtact 875

<210> 196

<211> 67

<212> DNA

<213> Mus musculus

<400> 196

agctgtggtc atacctggca tgtgaccccg ggaccaaata aagtcccctt ccatccactg 60

gagcagc 67

<210> 197

<211> 50

<212> DNA

<213> Mus musculus

<400> 197

gtcctattgc caccctgcca tgctaataaa gccactgtgt ccagacttct 50

<210> 198

<211> 84

<212> DNA

<213> Mus musculus

<400> 198

ttggcctctg cttgtaatac aatgaaagta ttctaaagaa atataaaatt ggactttatg 60

agaaaataaa agtcatttca ctct 84

<210> 199

<211> 36

<212> DNA

<213> Mus musculus

<400> 199

tctgttatgc catattttca ataaacctga aaacaa 36

<210> 200

<211> 446

<212> DNA

<213> Mus musculus

<400> 200

ggaaccacac gtacttatgc tgtaacttac tgtagcgttt acagcgttac cgctgtctgg 60

acagctgagt gtgtttctta ggaatataag ttttctttct gtgctttagt gagttcattc 120

agcagttcag taataaatat gtgaaacctt ttgtttcgaa aaaaattgct tctgtgttac 180

aacttacttt gttttatggt tcaggatcat ctgcataata gacaagtatt ctatcagtga 240

gcgatatcct ggatcttgtt tgtgtagttt ggggttgaga caagtccaga ctgccctaaa 300

actcacgatc ttccttcctc ggccttctaa atgattggag gactccaaac ctaagtgatg 360

gaagtaaaaa gaaacttata tgtcaagact cattttctct atcatttcat gtgacaattg 420

aaattagatt atttcttttt tcaatc 446

<210> 201

<211> 332

<212> DNA

<213> Mus musculus

<400> 201

ggactttgct ggcagaaaag aagtacagat gaggttatag tttgaaaaac gttaattccg 60

tttattgact ttagaatgtt actttgcata gtatagacca ttacaatgga aagtacctgc 120

cttaagaaac aagaaaactg cagtttatag agaaagaatt ttcaattttg acccatgtac 180

ttaaaatttt tggtgtatac tgcagtgtag caaatgtttt gtggtgacgg tataaatggt 240

actgtttgtt atcttggatt aagagtggct agagaagttg gaagacgtgt gagaagttct 300

ttatagagaa ttaaacatga aaattacatc tc 332

<210> 202

<211> 80

<212> DNA

<213> Mus musculus

<400> 202

aacttgtgtt ctctgagagg aaaatactga agcagtagag aaatgacctg ctagagaata 60

aagttactgt taatgatacc 80

<210> 203

<211> 93

<212> DNA

<213> Mus musculus

<400> 203

gatgtctgga atggagcaca cctgggatgt agcactcttg ctatctgtcc ggtccttttt 60

gttcaataaa gtcctgaggc aactctctct gtc 93

<210> 204

<211> 152

<212> DNA

<213> Mus musculus

<400> 204

tgagctatga agttccggaa tttgtgtttt tcacagaaag ccttaccaac ttcagttact 60

ttaccaagac aatgtaatta ttgtttgatt ttataaagtc tacaacaatg atctcctatt 120

ttggtgtcag tttttcaata aagttttaat ta 152

<210> 205

<211> 1841

<212> DNA

<213> Mus musculus

<400> 205

agcaggagca ggttttccaa aagcacccct cggaagtgtt tttgtcgtcg tttaaaatta 60

tcaagtatct tcagagaaga ttatttttctg ccttcagaaa ctgaaggaag gcttgggcct 120

agagaacgac agtaaggtgc gagcaccggga gacacttaac acagctcagt ccatggaagg 180

acgagttccc tcattggctg cctgtctcga aatccacgca agctgtggag gaaagaatta 240

ccctgctcat ccctgccttct atcttggtgt ttaatgttgg gtgggcaaca agcacaaacc 300

tccctcccac cccctccaag actgttagag cagtgggcca gaccaagcgg cgcacttgaa 360

catggatcaa gagggtcccg gttttacttt ttatttttgt cagggtaggc agtcttgtgt 420

ttgctttgtt caaagcaggg tctccctgtt ggccctggct ggccttatac tccacagcag 480

tcctgcctcc tcctcctagg tgctgggatt aaaggcgtgc gccaccacgc ccggctacag 540

cctgcatttt tatgcacatt ggtctgttaa gctagttgca ttctgtgcta ccggagggga 600

aactagtgtt tgcctgggcc tggtgtgtat 660

acctttagtt gcagcgcttg ggaggcagag gcaggcagac ttcatgagtt cagggacagg 720

caagcctgct ctacagattt ccaggatacc cagggctaca catagagaaa atgttaaaga 780

taaacaaaaa gctggacagt gggggagcac acctttaatc ccagcactcg ggaggcagag 840

gcaggcggat ttctgagttc gaggccagcc tggtctacag agtgagttcc aggacagcca 900

ggactacaca gagaaaccct gtctcagaaa aaaaacaaaa caaaaaccaa tgcagtgata 960

gattgttgtt tcctaaacca catgtaccca ggaaatgccc actaaatttc acctggatca 1020

gtgttaactg atcattggga aatgaggtga ccaaaaatgc atcgcaacct tggacaaaca 1080

gcatggctat ttaacattct gggatcctgc agaatcctgc atcttcctaa gtagggaagc 1140

actgtagcat tggagagagg cctgggcgag cagagctaag gcttccattt ctggcttgct 1200

1260

tgagtacatg ccaaagtagc caggaagtgt cacttgccct ggagctagaa ttactggcaa 1320

atgaaggctc agaggtggat gctgggacca attctaggcc ctctgagaaa gcaggtgcac 1380

ttggcttgtg cctccagccc caaaggcgat ggcttattgt gagcctgagg cgccaggg 1440

tcagagac tcaagaaaca agtggggttg tccatgttgc tggagatgac ccaggtctat 1500

taggaccttg actacatgga tagacattct ggcagctagt atactgccat tggggcctat 1560

ggagaagtag ccaccgagcc tatttagaaa gaaggactgc tggcaagctt ggtgtcacta 1620

tgaaggcaga caaagatcga tgtattaacg accccgactc caaaaacact cgaggggggcc 1680

caaggtgggc tcagtggtta agagccgttc gcccaggggc tggagagttg gctcagtggt 1740

accacatggt ggctcacaac catctgtaat gagatctgac gccctcttct ggtgtatctg 1800

aagacagcta cagtgtactt acataaaata aataaatctt t 1841

<210> 206

<211> 2035

<212> DNA

<213> Artificial sequence

<220>

<223> mRNA coding sequence rpl32 - PpLuc(GC) - albumin - A64

- C30 - histoneSL

<400> 206

ggggcgctgc ctacgggaggt ggcagccatc tccttctcgg catcaagctt gaggatggag 60

gacgccaaga acatcaagaa gggcccggcg cccttctacc cgctggagga cgggaccgcc 120

ggcgagcagc tccacaaggc catgaagcgg tacgccctgg tgccggggcac gatcgccttc 180

accgacgccc acatcgaggt cgacatcacc tacgcggagt acttcgagat gagcgtgcgc 240

ctggccgagg ccatgaagcg gtacggcctg aacaccaacc accggatcgt ggtgtgctcg 300

gagaacagcc tgcagttctt catgccggtg ctgggcgccc tcttcatcgg cgtggccgtc 360

gccccggcga acgacatcta caacgagcgg gagctgctga acagcatggg gatcagccag 420

ccgaccgtgg tgttcgtgag caagaagggc ctgcagaaga tcctgaacgt gcagaagaag 480

ctgcccatca tccagaagat catcatcatg gacagcaaga ccgactacca gggcttccag 540

tcgatgtaca cgttcgtgac cagccacctc ccgccgggct tcaacgagta cgacttcgtc 600

ccggagagct tcgaccggga caagaccatc gccctgatca tgaacagcag cggcagcacc 660

ggcctgccga agggggtggc cctgccgcac cggaccgcct gcgtgcgctt ctcgcacgcc 720

cgggacccca tcttcggcaa ccagatcatc ccggacaccg ccatcctgag cgtggtgccg 780

ttccaccacg gcttcggcat gttcacgacc ctgggctacc tcatctgcgg cttccgggtg 840

gtcctgatgt accggttcga ggaggagctg ttcctgcgga gcctgcagga ctacaagatc 900

cagagcgcgc tgctcgtgcc gaccctgttc agcttcttcg ccaagagcac cctgatcgac 960

aagtacgacc tgtcgaacct gcacgagatc gccagcgggg gcgccccgct gagcaaggag 1020

1080

accgagacca cgagcgcgat cctgatcacc cccgaggggg acgacaagcc gggcgccgtg 1140

ggcaaggtgg tcccgttctt cgaggccaag gtggtggacc tggacaccgg caagaccctg 1200

ggcgtgaacc agcggggcga gctgtgcgtg cggggggccga tgatcatgag cggctacgtg 1260

aacaacccgg aggccaccaa cgccctcatc gacaaggacg gctggctgca cagcggcgac 1320

atcgcctact gggacgagga cgagcacttc ttcatcgtcg accggctgaa gtcgctgatc 1380

1440

aacatcttcg acgccggcgt ggccgggctg ccggacgacg acgccggcga gctgccggcc 1500

gcggtggtgg tgctggagca cggcaagacc atgacggaga aggagatcgt cgactacgtg 1560

gccagccagg tgaccaccgc caagaagctg cggggcggcg tggtgttcgt ggacgaggtc 1620

ccgaagggcc tgaccgggaa gctcgacgcc cggaagatcc gcgagatcct gatcaaggcc 1680

aagaagggcg gcaagatcgc cgtgtaagac tagtgcatca catttaaaag catctcagcc 1740

taccatgaga ataagagaaa gaaaatgaag atcaatagct tattcatctc tttttctttt 1800

tcgttggtgt aaagccaaca ccctgtctaa aaaacataaa tttctttaat cattttgcct 1860

cttttctctg tgcttcaatt aataaaaaat ggaaagaacc tagatctaaa aaaaaaaaaa 1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa atgcatcccc 1980

cccccccccc cccccccccc cccccccaaa ggctcttttc agagccacca gaatt 2035

<---

Claims (37)

1. An artificial nucleic acid molecule for increasing protein yield, comprising: a. at least one open reading frame (ORF); and b. at least one element of the 3'-untranslated region (3'-UTR element), which contains a nucleotide sequence derived from the 3'-UTR of a ribosomal protein gene or from a variant of the 3'-UTR of a ribosomal protein gene selected from the group consisting of from ribosomal protein S9 (RPS9), ribosomal protein S19 (RPS19), ribosomal protein S21 (RPS21), ribosomal protein S27 (RPS27), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29) and ribosomal protein L35a (RPL35A). 2. An artificial nucleic acid molecule according to claim 1, wherein the 3'-UTR is heterologous to ORF. 3. An artificial nucleic acid molecule according to claim 1 or 2, in which 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 globin proteins (primarily beta- globin), luciferase protein, beta-glucuronidase (GUS) proteins and GFP or variants thereof, preferably not from EGFP, or variants whose sequences are at least 70% identical to the sequence of the globin protein, luciferase protein or GFP protein. 4. Artificial nucleic acid molecule according to one of paragraphs. 1-3, in which the open reading frame (ORF) does not encode a ribosomal protein, preferably does not encode a eukaryotic ribosomal protein. 5. Artificial nucleic acid molecule according to one of paragraphs. 1-4, in which the open reading frame (ORF) does not encode a human or plant ribosomal protein, in particular human ribosomal protein S6 (RPS6), human ribosomal protein L36a-like (RPL36AL), or Arabidopsis ribosomal protein S16 (RPS16). 6. Artificial nucleic acid molecule according to one of paragraphs. 1-5, in which the open reading frame (ORF) does not encode ribosomal protein S6 (RPS6), ribosomal protein L36a-like (RPL36AL), or ribosomal protein S16 (RPS16). 7. Artificial nucleic acid molecule according to one of paragraphs. 1-6, in which the 3'-UTR, preferably an artificial nucleic acid molecule, does not contain a poly(A)-sequence or polyadenylation signal, or in which the 3'-UTR, preferably an artificial nucleic acid molecule, additionally contains a poly(A)- polyadenylation sequence and/or signal. 8. An artificial nucleic acid molecule according to claim 7, wherein the poly(A) sequence or polyadenylation signal is/is localized at the 3'-end of the 3'-UTR element. 9. An artificial nucleic acid molecule according to claim 7 or 8, wherein the polyadenylation signal contains the consensus sequence NN(U/T)ANA, where N is A or U, preferably AA(U/T)AAA or A(U/T) (U/T)AAA. 10. Artificial nucleic acid molecule according to one of paragraphs. 7-9 in which the polyadenylation signal, preferably the NNUANA consensus sequence, is located less than about 50 nucleotides downstream of the 3' end of the 3' UTR element. 11. Artificial nucleic acid molecule according to one of paragraphs. 7-10, wherein the poly(A) sequence is from about 20 to about 300 adenine nucleotides in length, preferably from about 40 to about 200 adenine nucleotides, more preferably from about 50 to about 100 adenine nucleotides, even more preferably from about 60 up to about 70 adenine nucleotides. 12. An artificial nucleic acid molecule according to one of paragraphs. 1-11, in which the nucleic acid contains an additional 5'-terminal element, preferably a 5'-UTR, a promoter, or a sequence containing a 5'-UTR and a promoter. 13. An artificial nucleic acid molecule according to claim 12, wherein the 5'-UTR is a 5'-UTR of 5'-TOP. 14. An artificial nucleic acid molecule according to claim 12 or 13, in which the 5'-UTR does not contain a 5'-TOP motif. 15. Artificial nucleic acid molecule according to one of paragraphs. 1-14, in which the artificial nucleic acid molecule also contains a 5'-UTR and in which all 3'-UTRs, ORFs, and 5'-UTRs are heterologous to each other. 16. An artificial nucleic acid molecule according to one of paragraphs. 1-15, in which at least one 3'-UTR element contains or consists of a nucleotide sequence identical to at least about 1, 2, 3, 4, 5, 10, 15, 20, 30, or 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 40, 58, 66, 69, 73, 75, 76, 144, 161, 169 , 171, 175, 177 and 178, or in which at least one 3'-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 an example but 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 NOs: 40, 58, 66, 69, 73, 75, 76, 144, 161, 169, 171, 175, 177 and 178. 17. The artificial nucleic acid molecule of claim 16, wherein the fragment 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. 18. Artificial nucleic acid molecule according to one of paragraphs. 1-17, in which at least one 3'-UTR element has a length of 3 to about 500 nucleotides, preferably from 5 to about 150 nucleotides, more preferably from 10 to 100 nucleotides, even more preferably from 15 to 90, most preferably 20 to 70 nucleotides. 19. Artificial nucleic acid molecule according to one of paragraphs. 1-18, additionally containing a 5' cap structure, a poly(C) sequence, a histone stem-loop structure, and/or an IRES motif. 20. An artificial nucleic acid molecule according to one of paragraphs. 1-19, in which the histone stem-loop structure contains the sequence shown in SEQ ID NO: 5. 21. An artificial nucleic acid molecule according to one of paragraphs. 1-20, wherein the artificial nucleic acid molecule, preferably an open reading frame, has at least partially modified G/C content, wherein preferably the G/C content of the open reading frame is increased compared to the wild-type open reading frame. 22. An artificial nucleic acid molecule according to one of paragraphs. 1-21, wherein the open reading frame contains a codon-optimized region, wherein the open reading frame preferably has optimized codons. 23. An artificial nucleic acid molecule according to one of paragraphs. 1-22, which is an RNA, preferably an mRNA molecule. 24. An expression vector containing an artificial nucleic acid molecule according to one of paragraphs. 1-23. 25. The vector according to claim 24, which is a DNA vector. 26. Vector according to one of paragraphs. 24-25, which is a plasmid vector or a viral vector, preferably a plasmid vector. 27. Vector according to one of paragraphs. 24-26, which is a circular molecule. 28. The vector of claim 27, wherein the poly(A) sequence, poly(C) sequence, stem-loop histone structure, or 3'-UTR element in the coding strand is located 5'→3' direction of a restriction site intended to linearize a circular vector molecule. 29. Cell for obtaining the target protein containing an artificial nucleic acid molecule according to one of paragraphs. 1-23 or a vector according to one of paragraphs. 24-28. 30. A cell according to claim 29, which is a mammalian cell. 31. A cell according to claim 29 or 30, which is a mammalian cell, preferably an isolated mammalian cell, preferably a human. 32. A method for increasing protein yield from an artificial nucleic acid molecule, preferably from an mRNA molecule or a vector, comprising the step of associating the nucleic acid molecule, preferably an mRNA molecule or vector, with a 3'-UTR element, where the 3'-UTR element contains or consists from a nucleotide sequence derived from the 3'-UTR of a ribosomal protein gene or from a 3'-UTR variant of a ribosomal protein gene selected from the group consisting of ribosomal protein S9 (RPS9), ribosomal protein S1 (RPS19), ribosomal protein S21 (RPS21 ), ribosomal protein S27 (RPS27), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), and ribosomal protein L35a (RPL35A). 33. The method according to p. 32, in which the 3'-UTR element contains or consists of a nucleotide sequence identical to 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% a nucleotide sequence selected from the group consisting of SEQ ID NOs: 40, 58, 66, 69, 73, 75, 76, 144, 161, 169, 171, 175, 177 and 178, or in which the 3'-UTR 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% identical, more preferably at least about on the 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: 40, 58 , 66, 69, 73, 75, 76, 144, 161, 169, 171, 175, 177 and 178. 34. The method of claim 33, wherein the fragment is 3 to about 500 nucleotides in length, preferably 5 to about 150 nucleotides, more preferably 10 to about 100 nucleotides, even more preferably 15 to 90, most preferably 20 to 70 nucleotides. 35. The method according to one of paragraphs. 32-34, wherein the 3'UTR element 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.

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