Synthetic transfer RNA with extended anticodon loop

Abstract

To provide a frameshift mutation suppressor for treating genetic disorders associated with frameshift mutations, such as neurofibromatosis.SOLUTION: A synthetic transfer RNA comprises an extended anticodon loop with a four-nucleotide anticodon or a five-nucleotide anticodon, which base-pairs with the codon on an mRNA having a mutation in the coding sequence and with the remaining of the following codon (in the case of a -1 frameshift) or with the remaining of the preceding codon (in the case of a +1 frameshift), so that the amino acid carried is incorporated into the growing amino acid chain and the reading frame is corrected, being a frameshift mutation suppressor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to synthetic transfer RNAs with extended anticodon loops. [Background technology]

[0002] Transfer ribonucleic acid (tRNA) is an essential part of the protein synthesis machinery of living cells, as it is a necessary component for translating the nucleotide sequence of messenger RNA (mRNA) into the amino acid sequence of a protein. Natural tRNAs contain an amino acid binding stem that can covalently bind to an amino acid, and an anticodon loop containing a base triplet, called an "anticodon," that can noncovalently bind to a corresponding base triplet, called a "codon," on mRNA. Proteins are synthesized by assembling the amino acids carried by the tRNA using the codon sequence on mRNA as a template, with the assistance of a multicomponent system that includes, inter alia, ribosomes and several accessory enzymes.

[0003] Some diseases within the group of genetic disorders are based on alterations in genetic information, such as mutations in the DNA of coding genes. This includes the exchange, deletion, or insertion of single or multiple nucleotides. In this case, the mRNA transcribed from the mutated gene also carries altered genetic information, resulting in the formation of abnormal and potentially nonfunctional proteins. The deletion or insertion of one or more nucleotides (collectively referred to as indels), for example, alters the reading frame within the coding region (i.e., the three-nucleotide region through which ribosomes read the information in the mRNA), resulting in the creation of an entirely new amino acid sequence downstream of the mutation and the production of a nonfunctional protein. As an example, mutations in the genes encoding neurofibromin 1 (NF1) and neurofibromin 2 (NF2), respectively, can cause the diseases neurofibromatosis type 1 (NF1) and neurofibromatosis type 2 (NF2), respectively. The NF1 and NF2 genes are thought to function as tumor suppressors. Mutations in NF1 and NF2 occur not only at birth (inherited or de novo) but also somatically. The most common mutations are frameshift mutations accompanied by deletions or insertions of 1 to 12 nucleotides, with deletions occurring more frequently than insertions (Non-Patent Documents 1 and 2).

[0004] Although some antibiotics, such as macrolides, promote frameshifting, no attempts to use macrolides to correct frameshifting have been reported (Non-Patent Documents 3 and 4).

[0005] Although still in its infancy, gene therapy, which involves the introduction of corrective genetic material into a patient's cells, is becoming increasingly important for the treatment of genetic diseases. Approaches based on modifying downstream entities of genes, primarily mRNA, are preferable because they do not fall under classical gene therapy approaches, as the gene sequence (or DNA) remains unchanged. However, mRNA is inherently short-lived, and the length of mRNA sequences poses challenges for therapeutic applications. Certain mRNAs may be longer than the payload capacity of currently available vectors for gene delivery and therapy, for example.

[0006] Compared to mRNA, tRNA molecules are much more stable and on average about 10 times shorter, which reduces the problem of their introduction into target tissues. For this reason, attempts have been made to use tRNA in gene therapy to prevent the formation of truncated proteins from mRNAs with premature stop codons and instead introduce the correct amino acid (see, for example, Non-Patent Document 5, Patent Document 1, and Patent Document 2).

[0007] Natural tRNAs with extra nucleotides or short codon doublets in the anticodon have been found in bacteria. These unusual codons are naturally used to suppress specific frameshift positions (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8). Sako et al. (2006) (Patent Document 9) reported an approach to read through PTC-containing mRNA using suppressor tRNAs introduced into cells by transfection. Non-sense triplet codons and four-base codons were read by the corresponding suppressor tRNAs derived from human tRNA (Ser).

[0008] To incorporate unnatural amino acids into proteins and address the molecular mechanisms of frameshift suppression for 2-, 3-, 4-, 5-, and 6-base codons with tRNAs containing 6- to 10-nt segments in the anticodon loop, tRNAs with extended anticodon loops containing 4- or 5-base anticodons have also been introduced into bacterial in vitro translation systems (Patent Document 3, Patent Document 4, Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13). However, the incorporation yield of unnatural amino acids is extremely low and is affected by the mRNA environment. Furthermore, approaches to incorporate unnatural (nonstandard or nonproteinogenic) amino acids into proteins using tRNAs modified in the anticodon loop commonly use so-called orthogonal translation systems, i.e., translation systems that employ engineered aminoacyl-tRNA synthetases to attach nonstandard amino acids to modified tRNAs (Non-Patent Document 14, Non-Patent Document 15, Patent Document 5). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 2003 / 0224479 [Patent Document 2] U.S. Patent No. 6964859 [Patent Document 3] U.S. Patent Application Publication No. 2006 / 0177900 [Patent Document 4] International Publication No. 2005 / 007870 [Patent Document 5] International Publication No. 2005 / 019415

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[0010]

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[0011] There remains a need to block the effects of frameshift mutations and / or to suppress frameshift mutations. It is therefore an object of the present invention to provide such means, in particular frameshift mutation suppressors for treating genetic diseases associated with frameshift mutations, such as neurofibromatosis. [Means for solving the problem]

[0012] In one aspect, the invention provides synthetic transfer ribonucleic acids (tRNAs) that contain an extended anticodon loop with a four- or five-base anticodon that is configured to base-pair with five consecutive nucleotide bases on a messenger RNA, correcting insertions or deletions in consecutive codon base triplets on an mRNA that change the reading frame between the -1 and +1 frames.

[0013] The present invention provides novel suppressor tRNAs that can be used to suppress -1 or +1 frameshift mutations with high specificity and in an environment-dependent manner. This restores the ability of cells to synthesize functional proteins, for example, from mRNAs with mutations in the coding sequence. Without this mutation, the protein sequence would be different, resulting in reduced or non-functional proteins. The synthetic tRNAs of the present invention contain anticodon loops extended by one or two nucleotides. The synthetic tRNAs of the present invention are complementary to four or five bases of two adjacent codons on an mRNA, where the first codon is a codon with an indel (insertion or deletion) and the second codon is an intact adjacent codon. The synthetic tRNAs of the present invention, which have four or five codons, base pair with the codon on the mRNA and the remainder of the subsequent codon (in the case of a -1 frameshift) or the preceding codon (in the case of a +1 frameshift). As a result, the amino acid carried by the tRNA is incorporated into the growing amino acid chain, correcting the reading frame. In the case of a -1 frameshift, if the synthetic tRNA of the present invention is not (pre)aminoacylated with a dipeptide, the resulting protein will have one less amino acid than the wild-type protein, i.e., the protein synthesized from the wild-type mRNA, but there is still a high probability that a functional protein will be obtained.

[0014] Advantageously, the present invention provides synthetic transfer RNAs designed with anticodon loops that confer higher binding affinity compared to prior art suppressor tRNAs used for the incorporation of unnatural amino acids. Binding to two consecutive codons, a codon with an indel (insertion or deletion) and its adjacent unmodified codon, is highly specific compared to natural dinucleotide-anticodon suppressor tRNAs. As a result, the synthetic tRNAs of the present invention can be designed to bind efficiently to specific mutation sites, significantly reducing the risk of nonspecific pairing to other partially homologous regions of the mRNA.

[0015] The term "transfer ribonucleic acid" or "tRNA" refers to an RNA molecule, typically 73–90 nucleotides long, that mediates the translation of messenger RNA nucleotide sequences into protein amino acid sequences. tRNAs covalently bind to specific amino acids through the 3' CCA tail at the end of the acceptor stem and can base-pair with messenger RNA trinucleotide sequences (codons) via a trinucleotide anticodon in the anticodon loop of the anticodon arm. Some anticodons can pair with multiple codons through a phenomenon known as unstable base pairing. The secondary "cloverleaf" structure of tRNAs includes an acceptor stem that binds to the amino acid, and three arms (the "D arm," "T arm," and "anticodon arm") that terminate in loops (the D loop, the TΨC loop, and the anticodon loop)—i.e., sites with unpaired nucleotides. Aminoacyl-tRNA synthetases load (aminoacylate) specific amino acids onto tRNAs. Each tRNA contains a distinct anticodon triplet sequence capable of base-pairing with one or more codons for amino acids. Conventionally, the nucleotides of a tRNA are often numbered 1–76, starting from the 5' phosphate end, based on a 76-nucleotide "consensus" tRNA molecule, regardless of the actual number of nucleotides in the tRNA. tRNAs are not necessarily 76 nucleotides long due to variable segments within the tRNA, such as the D-loop (see Figure 3). According to this convention, nucleotides 34–36 of a natural tRNA refer to the three nucleotides of the anticodon, and positions 74–76 refer to the terminating CCA tail. Any "supernumerary" nucleotides can be numbered either by adding an alphabetic character to the number of existing nucleotides that are part of the consensus tRNA, e.g., 20a, 20b, etc., or, in the case of variable loops, by numbering each nucleotide independently and adding a prefix, e.g., e11, e12, etc. (see, e.g., Non-Patent Document 16). Hereinafter, the tRNA-specific numbering system will also be referred to as the "tRNA numbering system" or "transfer RNA numbering system."

[0016] The term "synthetic transfer ribonucleic acid" or "synthetic tRNA" refers to a non-natural tRNA. This term also encompasses analogs of natural tRNAs, i.e., tRNAs that are structurally similar to natural tRNAs but in which the base, sugar, and / or phosphate moieties of one or more of the nucleotides from which the tRNA is composed have been modified. Modified tRNAs may have a modified phosphodiester backbone, e.g., where the phosphodiester bridge is replaced with a phosphorothioate, phosphoramidate, or methylphosphonate bridge. The sugar moiety may be modified at the 2'OH group, e.g., by dehydroxylation to deoxyribonucleotides or by substitution with a methoxy, methoxyethoxy, or aminoethoxy group. Synthetic transfer ribonucleic acids can be synthesized, e.g., chemically and / or enzymatically in vitro, or in cell-based systems, e.g., bacterial cells in vivo. The term "codon" refers to a nucleotide triplet, i.e., a sequence of three DNA or RNA nucleotides, that corresponds to a specific amino acid or stop signal during protein synthesis. A list of codons (at the mRNA level) and the amino acids they encode is provided below.

[0017] Amino acid single letter code codon Ala A GCU, GCC, GCA, GCG Arg R CGU, CGC, CGA, CGG, AGA, AGG Asn N AAU, AAC Asp D GAU, GAC Cys C UGU, UGC Gln Q CAA, CAG Glu E GAA, GAG Gly G GGU, GGC, GGA, GGG His H CAU, CAC Ile I AUU, AUC, AUA Leu L UUA, UUG, CUU, CUC, CUA, CUG Lys K AAA, AAG Met M AUG Phe F UUU, UUC Pro P CCU, CCC, CCA, CCG Ser S UCU, UCC, UCA, UCG, AGU, AGC Thr T ACU, ACC, ACA, ACG Trp W UGG Tyr Y UAU, UAC Val V GUU, GUC, GUA, GUG Start:AUG Stop: UAA, UGA, UAG, abbreviation "X"

[0018] As used herein, the term "sense codon" refers to a codon that encodes an amino acid. The term "stop codon" or "non-sense codon" refers to a codon, i.e., a nucleotide triplet, of the genetic code that does not encode one of the 20 amino acids normally found in proteins and that signals the termination of translation of messenger RNA.

[0019] The term "frameshift mutation" refers to an out-of-frame insertion or deletion of a number of nucleotides not evenly divisible by three (collectively referred to as "indels"). This prevents decoding of the nucleotide sequence, which proceeds in steps of three nucleotide bases. The term "-1 frameshift mutation" means that a single nucleotide deletion shifts the reading frame by one nucleotide, and the first nucleotide of the subsequent codon is read as part of the codon from which the nucleotide was deleted. Deletion of a single nucleotide from an upstream codon along with several triplets (i.e., deletion of 4, 7, 10, etc. nucleotides) is also considered a -1 frameshift. The term "+1 frameshift mutation" refers to the insertion of a single nucleotide into a triplet or the deletion of two nucleotides. Either event results in a one-nucleotide shift in the reading frame, and the nucleotide from the upstream codon is read as part of the downstream codon. An insertion of a single nucleotide along with several triplets (3n+1 nucleotides, where n is an integer, i.e., insertion of 4, 7, 10, etc. nucleotides) or a deletion of two nucleotides from the upstream codon along with several triplets (i.e., deletion of 5, 8, 11, etc. nucleotides) is also considered a +1 frameshift. Frameshift mutations are involved in various genetic disorders, including Duchenne muscular dystrophy (DMD), Crohn's disease (CD), Tay-Sachs disease (TSD), cystic fibrosis (CF), neuronal ceroid lipofuscinosis (NCL), and neurofibromatosis type 1 (NF1).

[0020] The term "anticodon" refers to a sequence of typically three nucleotides that base-pairs (non-covalently binds) with three bases (nucleotides) of a codon on an mRNA. Anticodons may also contain nucleotides with modified bases. The term "four-nucleotide anticodon" or "four-base anticodon" refers to an anticodon with four consecutive nucleotides (bases) that pair with four consecutive bases on an mRNA. The term "quadruplet nucleotide anticodon" or "quadruplet anticodon" may also be used to refer to a "four-nucleotide anticodon." The term "five-nucleotide anticodon" or "five-base anticodon" refers to an anticodon with five consecutive nucleotides (bases) that bind (base-pair) with five consecutive bases on an mRNA. The term "quintuplet nucleotide anticodon" or "quintuplet anticodon" may also be used to refer to a "five-nucleotide anticodon."

[0021] The term "anticodon loop" refers to the unpaired nucleotides of the anticodon arm that contains the anticodon. Natural tRNAs typically have seven nucleotides in the anticodon loop, three of which are paired with the codon in the mRNA.

[0022] The term "extended anticodon loop" refers to an anticodon loop in which the number of nucleotides in the loop is greater than that of natural tRNA. The extended anticodon loop can, for example, contain more than seven nucleotides, for example, 8, 9, or 10 nucleotides. In particular, this term refers to an anticodon loop that contains an anticodon consisting of more than three consecutive nucleotides, for example, 4, 5, or 6 nucleotides, that can base pair with the corresponding number of consecutive nucleotides in mRNA.

[0023] The term "anticodon stem" refers to the paired nucleotides of the anticodon arm that carry the anticodon loop.

[0024] The term "T-stem" refers to the paired nucleotides of the T-arm, i.e., the unpaired nucleotides of the T-arm, which carry the T-loop.

[0025] The term "variable loop" refers to the tRNA loop located between the anticodon arm and the T arm. The number of nucleotides that make up the variable loop can vary greatly from tRNA to tRNA. Therefore, the variable loop can be relatively short or even absent, or it can be relatively large, for example, forming a helix. The term "variable arm" may be used synonymously with the term "variable loop."

[0026] As used herein, the term "codon base triplet" or "anticodon base triplet" refers to the sequence of three consecutive nucleotides that form a codon or anticodon. Synonymously, the terms "three-nucleotide codon" (also referred to as "three-base codon") or "three-nucleotide anticodon" (also referred to as "three-base anticodon"), or abbreviations thereof, such as "3nt codon" or "3nt anticodon," may be used.

[0027] The term "base pair" refers to a pair of bases joined by hydrogen bonds, or the formation of such a base pair. One base of a base pair is usually a purine, and the other base is usually a pyrimidine. In RNA, the bases adenine and uracil can form a base pair, and the bases guanine and cytosine can form a base pair. However, other base pairs ("unstable base pairs") are also possible, such as guanine-uracil (GU), hypoxanthine-uracil (I-U), hypoxanthine-adenine (IA), and hypoxanthine-cytosine (IC). The term "base-pairable" refers to the ability of a nucleotide or sequence of nucleotides to form a hydrogen-bond-stabilized structure with a corresponding nucleotide or sequence of nucleotides.

[0028] As used herein, the term "base," e.g., "base A, C, G, or U," encompasses or is used synonymously with the term "nucleotide," unless the context clearly indicates otherwise.

[0029] "PTC" refers to a premature termination codon, which is a stop codon introduced into a coding nucleic acid sequence by a nonsense mutation, i.e., a mutation that changes a sense codon encoding one of the 20 proteinogenic amino acids specified in the standard genetic code into a chain-terminating codon. Thus, this term refers to a premature stop signal in the translation of the genetic code contained in mRNA.

[0030] The term "Crohn's disease" or "Crohn's disease" refers to a gastrointestinal inflammatory disease associated with the NOD2 gene. The common mutation associated with this disease is the insertion of a cytosine at position 3020.

[0031] The term "Tay-Sachs disease" refers to a genetic disorder that causes destruction of nerve cells, which becomes evident in infancy, around 2-3 months of age. It can result in severe motor disabilities, hearing loss, and epileptic seizures. In many cases, it leads to death during infancy.

[0032] The term "Duchenne muscular dystrophy" (DMD), also known as "Becker muscular dystrophy" (BMD), refers to an X-linked recessive genetic disorder characterized by progressive muscle degeneration and weakness resulting from the lack of functional dystrophin protein. The lack of dystrophin can be caused by nonsense mutations in the dystrophin gene.

[0033] Neurofibromatosis type 1 (NF1 or NF-1), also known as Recklinghausen's disease, is an autosomal dominant disorder caused by mutations in the NF1 gene, which encodes neurofibromin, located on chromosome 17. NF1 causes tumors along the nervous system.

[0034] The term "cystic fibrosis" refers to a genetic disease that is inherited in an autosomal recessive manner. Cystic fibrosis is caused by mutations in both copies of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Cystic fibrosis can be caused, for example, by a frameshift mutation that results in the introduction of a premature stop codon (see, for example, Non-Patent Document 17).

[0035] The term "neuronal ceroid lipofuscinosis" refers to a neurodegenerative lysosomal storage disease associated with alterations of tripeptides in lysosomes. Neuronal ceroid lipofuscinosis can be caused by, for example, frameshift mutations in the tripeptidyl peptidase I (TPP1) or CLN2 genes, among other mutations (see, for example, Non-Patent Document 18).

[0036] The term "frameshift suppression" refers to a mechanism that counteracts the effects of a frameshift mutation and at least partially restores the wild-type phenotype.

[0037] The term "suppressor tRNA" refers to a tRNA that alters the reading of messenger RNA in some translation systems. Examples of suppressor tRNAs include tRNAs that carry amino acids and can base pair with mutant codons covering two consecutive codons, one of which is intact and the other of which has an insertion or deletion. Thus, the reading frame can be corrected by the translation system.

[0038] The term "homology" in relation to nucleic acids refers to the degree of similarity or identity between the nucleotide sequence of a nucleic acid and the nucleotide sequence of another nucleic acid. Homology is determined by comparing a position in a first sequence with the corresponding position in a second sequence to determine whether an identical nucleotide exists at that position. Sequence gaps may need to be taken into account to allow for optimal alignment. To determine the degree of similarity or identity between two nucleic acids, it is preferable to consider a minimum length of the nucleic acids being compared, for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% of the nucleotides in each sequence. Preferably, the full length of each nucleic acid(s) is used for comparison. The degree of similarity or identity between two sequences can be determined using computer programs such as MUSCLE (Non-Patent Document 19) or MAFFT (Non-Patent Document 20). When the terms "x% homologous" or "x% homology" are used herein, this means that two nucleic acids have sequence identity or similarity, preferably x%, for example 50% sequence identity.

[0039] The term "aminoacylation" refers to the enzymatic reaction that loads a tRNA with an amino acid. Aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of a specific cognate amino acid or its precursor to a compatible cognate tRNA, forming an aminoacyl-tRNA. Therefore, the term "aminoacyl-tRNA" refers to a tRNA with an amino acid attached. Each aminoacyl-tRNA synthetase is highly specific for a given amino acid. While multiple tRNAs may exist for the same amino acid, there is only one aminoacyl-tRNA synthetase for each of the 20 proteinogenic amino acids. The terms "loading" or "addition" may also be used as synonyms for "aminoacylation." The term "aminoacylation," in relation to the synthetic tRNAs of the present invention, refers to a synthetic tRNA that is already loaded (preloaded) with an amino acid or dipeptide so that the tRNA is already acylated when it enters the target cell. In this context, the term "preaminoacylated" may also be used synonymously.

[0040] The term "modified nucleotide" (or "unusual nucleotide") with respect to a tRNA refers to a modified or unusual nucleotide base, i.e., a nucleotide having a base other than the usual bases adenine (A), uracil (U), guanine (G), and cytosine (C). Examples of modified nucleotides include 4-acetylcytidine (ac4c), 5-(carboxyhydroxymethyl)uridine (chm5u), 2'-O-methylcytidine (cm), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2u), 5-carboxymethylaminomethyluridine (cmnm5u), dihydrouridine (d), 2'-O-methylpseudouridine (fm), beta, D-galactosylqueosine (gal q), 2'-O-methylguanosine (gm), inosine (i), N6-isopentenyladenosine (i6a), 1-methyladenosine (m1a), 1-methylpseudouridine (m1f), 1-methylguanosine (m1g), 1-methylinosine (m1i), 2,2-dimethylguanosine (m22g), 2'-O-methyladenosine (am), 2-methyladenosine (m2a), 2-methylguanosine (m2g), 3-methylcytidine (m3c), 5-methylcytidine (m5c), N6-methyladenosine (m6a), 7-methylguanosine (m7g), 5-methylaminomethyluridine (mam5u), 5-methoxyaminomethyl-2-thiouridine (mam5s2u), beta, D-mannosylqueosine (man q), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2u), 5-methoxycarbonylmethyluridine (mcm5u), 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-methoxyuridine (mo5u), 2-methylthio-N6-isopentenyladenosine (ms2i6a), N-((9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine (ms2t6a), N-((9-beta-D-ribofuranosylpurine-6-yl)N-methylcarbamoyl)threonine (mt6a), uridine-5-hydroxyacetone Acid-methyl ester (mv), uridine-5-oxyacetic acid (o5u), wybutoxosin (osyw), pseudouridine (p, Ψ), queosin (q), 2-thiocytidine (s2c), 5-methyl-2-thiouridine (s2t), 2-thiouridine (s2u), 4-thiouridine (s4u), 5-methyluridine (t), N-((9-beta-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine (t6a), 2'-O-methyl-5-methyluridine (tm), 2'-O-methyluridine (um), wybutoxosin (yw), 3-(3-amino-3-carboxypropyl)uridine, (acp3)u(x).

[0041] The term "corresponding modified nucleotide" refers to a modified nucleotide at a given position in a sequence, the base of which is modified relative to the normal, i.e., unmodified, base of the nucleotide at the same position in the original sequence to be compared with the sequence containing the modified nucleotide. Therefore, the corresponding modified nucleotide can be any nucleotide that is normally produced in cells from a normal nucleotide by modifying the normal nucleotide. A modified nucleotide corresponding to uridine can be, for example, any nucleotide derived from a modified uridine. As an example, 5-(carboxyhydroxymethyl)uridine (chm5u) at a particular position in a sequence can be the modified nucleotide corresponding to the uridine at the same position in the original sequence. Additional modified nucleotides corresponding to uridine include, for example, 5-methyluridine (t), 2'-O-methyl-5-methyluridine (tm), 2'-O-methyluridine (um), or 5-methoxyuridine (mo5u). As another example, inosine is a modified nucleotide corresponding to adenosine, since it is produced from adenosine.

[0042] The synthetic transfer RNAs of the present invention may be synthesized based on natural tRNAs. However, the tRNAs of the present invention are preferably designed computationally ("in silico") and synthesized chemically and / or enzymatically in vitro. By computationally designing the synthetic tRNAs of the present invention, tRNAs that do not interfere with other tRNAs present in cells can be designed and synthesized. The synthetic tRNAs of the present invention are selected or designed so that they can carry a specific amino acid encoded by the codon adjacent to the mutant codon or the wild-type codon that has undergone an indel, using an aminoacyl-tRNA synthetase naturally present in living cells, preferably mammalian cells, such as human cells.

[0043] To counteract the potential for destabilization due to the expansion of the anticodon loop, the synthetic tRNAs of the present invention may be further structurally modified, either inside or outside the anticodon loop, to enhance their stability. Preferably, the tRNAs of the present invention are designed to have at least the same or greater stability as natural tRNAs in which the natural three-nucleotide anticodon is replaced with a four- or five-nucleotide anticodon, or at least the same or greater stability as natural tRNAs for the cognate amino acid. The term "stability" refers to the stable, correctly folded functional conformation of the tRNA in the absence of translation factors, which can be predicted by estimating the free energy change upon folding, and / or the stable binding of the tRNA to an elongation factor, allowing the tRNA to interact with the elongation factor and elongate. "High stability" refers, for example, to a large desired free energy change upon folding of the tRNA into a functional state. It also refers to a high proportion of tRNA molecules in the desired configuration, i.e., fully or completely folded. "Higher stability" can mean a higher stability of binding to an elongation factor, e.g., a higher binding affinity, but also means enabling favorable and uninterrupted elongation of the translation site. The stability of a synthetic transfer RNA of the present invention having a four-nucleotide anticodon is comparable to that of a natural transfer RNA in which a three-nucleotide anticodon is replaced with a four-nucleotide anticodon, or comparable to that of a natural tRNA for the cognate amino acid. Furthermore, the stability of a synthetic transfer RNA of the present invention having a five-nucleotide anticodon is comparable to that of a natural transfer RNA in which a three-nucleotide anticodon is replaced with a five-nucleotide anticodon, or comparable to that of a natural tRNA for the cognate amino acid. The increased stability of the synthetic tRNA of the present invention, compared to less stable tRNAs, increases the concentration of folded tRNA in cells. More preferably, the synthetic tRNA of the present invention is configured to be less stable in complex with an elongation factor (e.g., eEF1A), thereby causing promiscuity of the tRNA during translation and resulting in active repression.

[0044] The tRNA may be modified, for example, according to the nucleotide composition of the anticodon arm or components other than the anticodon arm, such as the D arm, T arm, or variable arm. For example, the synthetic tRNA of the present invention preferably has a C, i.e., nucleotide C, at position 32 and an A, i.e., nucleotide A, at position 37 in the anticodon loop. This numbering follows the tRNA numbering convention described above. Furthermore, it is preferred that the anticodon loop be flanked by a G-C or C-G pair, i.e., a G-C or C-G pair be present at the end of the anticodon stem toward the anticodon loop. The terms "G-C pair" or "C-G pair" refer to the bases (nucleotides) C and G that are paired via hydrogen bonds and separated in the 5'-3' direction by the anticodon loop. According to the tRNA numbering convention, the G-C or C-G pair occupies positions 31 and 39. That is, for a "G-C" pair, there is a G at position 31 and a C at position 39, or for a "C-G" pair, there is a C at position 31 and a G at position 39. In another preferred embodiment, the U-A pair (5'-3' direction) flanks the anticodon loop, i.e., is located at the end of the anticodon stem towards the anticodon loop, i.e., at positions 31 and 39 according to the numbering convention for tRNA.

[0045] An example of an anticodon arm suitable for use as a suppressor of a +1 frameshift mutation or a +1 frameshift mutation and having an anticodon loop with a U at position 31 and an A at position 39 (U31-A39 anticodon arm) is as follows: +1 Frameshift: AUGGUCU NNNN AAACCAU (SEQ ID NO: 17) -1 Frameshift: AUGGUCU NNNNN AAACCAU (SEQ ID NO: 18) The underlined N represents a 4-nt or 5-nt anticodon.

[0046] In a preferred embodiment of the invention, a synthetic tRNA of the invention comprises a T stem having the following structure: N1CGGG‐T‐loop‐CCCGN2, in sequence N1=A, G, or C N2=C, U, or G The first five nucleotides (N1CGGG) and the last five nucleotides (CCCGN2) are paired to form a T-stem with a T-loop between them. Preferred combinations of N1 and N2 are as follows: N1=G, N2=C N1=A, N2=U N1=G, N2=U N1=C, N2=G

[0047] In a preferred embodiment, the T arm has the following general sequence (SEQ ID NO: 19): N1CGGGNNNNNNNCCCGN2 N1 and N2 are as defined above, The internal consecutive Ns form a T-loop, and N is any nucleotide or any corresponding modified nucleotide. The T loop may have the following sequence: UUCGAAU

[0048] Thus, in a preferred embodiment, the T arm may have the sequence of SEQ ID NO: 20 below. N1CGGGUUCGAAUCCCGN2 In the sequence, N1 and N2 are as defined above. A particularly preferred embodiment of the T-arm is as follows. GCGGG‐T‐loop‐CCCGC, ACGGG‐T‐loop‐CCCGU, GCGGG-T-loop-CCCGU, or CCGGG-T-loop-CCCGG The T loop preferably has the sequence given above (UUCGAAU) and the T arm preferably has the sequence: GCGGGUUCGAAUCCCGC (SEQ ID NO: 21), ACGGGUUCGAAUCCCGU (SEQ ID NO: 22), GCGGGUUCGAAUCCCGU (SEQ ID NO: 23), or CCGGGUUCGAAUCCCGG (SEQ ID NO: 24) The synthetic tRNA of the present invention containing the T arm as described above is highly stable and is particularly useful as a suppressor tRNA for suppressing not only frameshift mutations but also nonsense mutations (mutations that convert a sense codon into a stop codon, PTCs).

[0049] In a preferred embodiment of the invention, the synthetic tRNA comprises a variable loop having the following sequence (SEQ ID NO:25): UGGGGNNNNNNCCCCGC An example of a preferred embodiment of a variable loop is shown in the following sequence (SEQ ID NO: 26): UGGGGUCCACUCCCCGC

[0050] Similar to the above-mentioned tRNA containing a T arm, the above-mentioned synthetic tRNA containing a variable loop has been confirmed to have excellent stability and can be advantageously used not only to suppress frameshift mutations but also, for example, to suppress nonsense mutations.

[0051] The synthetic tRNAs of the present invention may comprise the above-described T-arms or variable loops, either alone or in combination. More preferably, the tRNAs of the present invention comprise a U31-A39 anticodon arm, i.e., an anticodon arm having a U at position 31 and an A at position 39. The anticodon arm may have the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0052] Those skilled in the art understand that tRNAs are aminoacylated with specific amino acids by specific aminoacyl-tRNA synthetases (aaRSs), and that aaRSs can recognize their cognate tRNAs via unique recognition elements in the acceptor stem and / or anticodon loop of the tRNA. To provide tRNAs carrying their cognate amino acids in vivo, those skilled in the art may design synthetic tRNAs of the present invention with suitable unique recognition elements. Alternatively, the synthetic tRNAs of the present invention can be aminoacylated with flexizyme and transfected into eukaryotic cells as aminoacyl-tRNAs (Non-Patent Document 21).

[0053] The tRNAs of the present invention preferably have low sequence identity to any naturally occurring tRNA, preferably less than 70%, less than 65%, less than 60%, less than 65%, or less than 50%, and particularly preferably less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 44%, or less than 43%, preferably over the entire length of the tRNA.

[0054] In the synthetic transfer RNAs of the present invention, the anticodon loop is extended by a sufficient number of nucleotides to accommodate an anticodon of 4 or 5 nucleotides and base pair with mRNA. The anticodon loop of the synthetic transfer RNAs of the present invention may be composed of, for example, 7 to 12 nucleotides, preferably 7 to 10 nucleotides, or 8 to 10 nucleotides, and more preferably 8 or 9 nucleotides.

[0055] In one embodiment, the extended anticodon loop of a synthetic tRNA of the present invention comprises a four-base anticodon that can base-pair with a four-base codon, i.e., a codon base triplet, on a target mRNA to which a nucleotide has been added. In another embodiment, a synthetic tRNA of the present invention comprises a five-base anticodon that contains a base doublet that can base-pair with a codon doublet remaining from a codon triplet on an mRNA after a nucleotide in the codon has been deleted. Meanwhile, the adjacent anticodon base triplet preferably base-pairs with a sense codon, i.e., 5' or 3', preceding or following the deleted codon on the mRNA. The terms "preceding" and "following" refer to the direction of translation, i.e., the 5'-3' direction of the mRNA.

[0056] An example of a five-nucleotide anticodon in the extended anticodon loop of a synthetic tRNA of the invention is GAUUC (5'-3' orientation, or CUUAG in the 3'-5' orientation), which matches GAAAUC (5'-3') in the unmutated mRNA, where UC can base pair with the base doublet GA remaining after deletion of the A from the codon GAA, and GAU can base pair with the codon AUC encoding isoleucine.

[0057] The synthetic transfer RNAs of the present invention may be aminoacylated, i.e., may carry an amino acid or dipeptide at the end of their acceptor stem. Preferably, the tRNA is aminoacylated with an amino acid encoded by a sense codon that base-pairs with the 4- or 5-nucleotide anticodon. The synthetic tRNAs of the present invention can be chemically and / or enzymatically aminoacylated with a single amino acid or dipeptide. Adding a dipeptide to a tRNA can be achieved by methods known to those skilled in the art (see, for example, Non-Patent Document 22). To aminoacylate a tRNA with a dipeptide, for example, engineered bacterial tRNA synthetases or RNA-based catalysts may be used. In the case of a deletion, the dipeptide is preferably composed of the amino acids encoded by the consecutive unmutated codons. The use of such dipeptide-aminoacylated synthetic tRNAs suppresses -1 frameshifting as intended, resulting in the production of not only a non-truncated protein but also a protein with the amino acid sequence of the wild-type protein.

[0058] In a preferred embodiment, the synthetic transfer RNA of the invention has or comprises: a) a sequence consisting of, in the 5' to 3' direction, consecutive sequences A, B, and C portions, wherein portion A has or comprises one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; portion B has or comprises one of the sequences set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:25, SEQ ID NO:31, and SEQ ID NO:32; and portion C has or comprises one of the sequences set forth in SEQ ID NO:5, SEQ ID NO:33, and SEQ ID NO:34; or b) a sequence having at least 90%, preferably at least 95%, 96%, 97%, 98%, or 99%, sequence identity with one of the sequences according to a) above; or c) a sequence according to one of the sequences according to a) or b) above, wherein at least one of the nucleotides is replaced by a corresponding modified nucleotide. The tRNA of the present invention may be composed of any combination of the A, B, and C portions described above, provided that the order of the portions is ABC in the 5'-3' direction.

[0059] In a preferred embodiment, a synthetic tRNA of the invention consists of three sequences, A, B, and C, which are covalently linked in a 5' to 3' direction, eg, via phosphodiester bonds.

[0060] In a 4 nt anticodon tRNA, the A portion comprises a 4 nt-anticodon (hereinafter underlined "N") and preferably has the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:27, or SEQ ID NO:28. SEQ ID NO: 1 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNN AUGCNNNUN SEQ ID NO: 2 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNN AGCNNNUN SEQ ID NO: 27 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCUNNNN AAACNNNUN SEQ ID NO: 28 NNCAGNNUGNNCGAGNNGUCUAAGNNNAUGGUCU NNNN AAACCAUUN N=any nucleotide, or any corresponding modified nucleotide It should be noted that the tRNA sequence of the present invention can contain a combination of unknown nucleotides (N), i.e., any nucleotide or corresponding modified nucleotide can be present at any position in the sequence, except for nucleotides that are not involved in base pairing, such as nucleotides in one of the loops, e.g., the T-loop or anticodon loop, which are limited to nucleotides that can form a functional tRNA molecule through base pairing.

[0061] The B portion may be of variable length and comprises or consists of a variable loop, preferably having the sequence of SEQ ID NO: 3, 4, 6, 25, 31 or 32 (N=any nucleotide, or any corresponding modified nucleotide). SEQ ID NO: 3 NNNNNNNNNNN SEQ ID NO:4 NNNNNNNNNNNNNNN SEQ ID NO:6 UGGGGUCACUCCCCG SEQ ID NO: 25 UGGGGNNNNNNCCCCGC SEQ ID NO: 31 NNNNNNNNNNNNNNNNN SEQ ID NO: 32 UGGGGUCCACUCCCCGC

[0062] Preferably, the C portion has the sequence set forth in SEQ ID NO: 5, 33, or 34. SEQ ID NO:5 CNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 33 CNCGGGNNNNNNNCCCGNNNCUGNNACCA SEQ ID NO: 34 CNCGGGUUCGAAUCCCGNNNCUGNNACCA

[0063] A preferred 4 nt-anticodon tRNA of the present invention consisting of the above-mentioned A to C portions has, for example, the following sequence (SEQ ID NOs: 9 to 12, 35 to 38; N = any nucleotide, underlined is the anticodon). SEQ ID NO:9 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNN AUGCNNNUNNNNNNNNNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 10 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNN AGCNNNUNNNNNNNNNNNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 11 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNN AUGCNNNUNUGGGGUCACUCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 12 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNN AGCNNNUNUGGGGUCACUCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 35 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCU NNNN AAACNNNUNNNNNNNNNNNNNNNNNNNCNCGGGNNNNNNNCCCGNNNCUGNNACCA SEQ ID NO: 36 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUC UNNNN AAACNNNUNUGGGGUCCACUCCCCCGCCNCGGGNNNNNNNCCCGNNNCUGNNACCA SEQ ID NO: 37 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCU NNNN AAACNNNUNUGGGGUCCACUCCCCCGCCNCGGGUUCGAAUCCCGNNNCUGNNACCA SEQ ID NO: 38 NNCAGNNUGNNCGAGNNGUCUAAGNNNAUGGUCU NNNN AAACNNNUNUGGGGUCCACUCCCCCGCCNCGGGUUCGAAUCCCGNNNCUGNNACCA

[0064] In the case of a 5 nt-anticodon, the A portion of the tRNA comprises the 5 nt-anticodon (underlined "N" below) and preferably has the sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:29, or SEQ ID NO:30 (N=any nucleotide). Part A: SEQ ID NO:7 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNNN AUGCNNNUN SEQ ID NO:8 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNNN AGCNNNUN SEQ ID NO: 29 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCU NNNNN AAACNNNUN SEQ ID NO: 30 NNCAGNNUGNNCGAGNNGUCUAAGNNNAUGGUCU NNNNN AAACCAUUN

[0065] The B portion may be of variable length and may comprise or consist of a variable loop. In a preferred embodiment, the B portion has one of the following sequences: SEQ ID NO: 3 NNNNNNNNNNN SEQ ID NO:4 NNNNNNNNNNNNNNN SEQ ID NO:6 UGGGGUCACUCCCCG SEQ ID NO: 25 UGGGGNNNNNNCCCCGC SEQ ID NO: 31 NNNNNNNNNNNNNNNNN SEQ ID NO: 32 UGGGGUCCACUCCCCGC

[0066] The C portion preferably has the following sequence: SEQ ID NO:5 CNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 33 CNCGGGNNNNNNNCCCGNNNCUGNNACCA SEQ ID NO: 34 CNCGGGUUCGAAUCCCGNNNCUGNNACCA

[0067] The 5 nt-anticodon tRNA consisting of portions A to C of the present invention preferably has the following sequence (N=any nucleotide; the underlined portion is the anticodon): SEQ ID NO: 13 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNNN AUGCNNNUNNNNNNNNNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 14 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNNN AGCNNNUNNNNNNNNNNNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 15 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNNN AUGCNNNUNUGGGGUCACUCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 16 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCU NNNNN AGCNNNUNUGGGGUCACUCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 39 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCU NNNNN AAACNNNUNNNNNNNNNNNNNNNNNNNCNCGGGNNNNNNNCCCGNNNCUGNNACCA SEQ ID NO: 40 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCU NNNNN AAACNNNUNUGGGGUCCACUCCCCCGCCNCGGGNNNNNNNCCCGNNNCUGNNACCA SEQ ID NO: 41 NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGUCU NNNNN AAACNNNUNUGGGGUCCACUCCCCCGCCNCGGGUUCGAAUCCCGNNNCUGNNACCA SEQ ID NO: 42 NNCAGNNUGNNCGAGNNGUCUAAGNNNAUGGUCU NNNNN AAACNNNUNUGGGGUCCACUCCCCCGCCNCGGGUUCGAAUCCCGNNNCUGNNACCA

[0068] In the above sequences, N represents any of the bases A, C, G, or U, or any modified base. Preferably, the base does not violate base pairing as shown in Figure 3. Allowed base pairs are, for example, G-C, C-G, A-U, U-A, and unstable base pairs such as G-U, U-G, I-U, U-I, I-A, A-I, and I-C, C-I. As used herein, the symbols G, C, A, or U may represent an unmodified base or any of the corresponding modified bases (see below). Thus, the tRNAs of the invention may contain one or more modified nucleotides.

[0069] For clarity, it should be noted that the synthetic transfer RNAs of the present invention may or may not be synthesized to contain any modified nucleotides. Thus, the synthetic transfer RNAs of the present invention may not contain any modified nucleotides. However, after entering a cell, one or more nucleotides of the synthetic tRNA may nevertheless be modified within the cell by cellular enzymatic machinery. Consequently, a synthetic tRNA of the present invention that is designed, synthesized, and administered without modified nucleotides may contain one or more modified nucleotides in a living cell due to modifications made to the nucleotides by the cell. Indeed, it is preferred that the synthetic tRNAs of the present invention be synthesized and administered so as to be completely free of modified nucleotides, leaving any modifications in the cell. When the synthetic tRNA of the invention is synthesized using modified nucleotides so that it already contains the modified nucleotides prior to administration, the tRNA of the invention preferably contains one or more of the following modified nucleotides (Table 1).

[0070] Table 1. Possible modified nucleotides and their positions within tRNAs (position numbering according to the specific tRNA numbering convention for popular "consensus" tRNAs; see also Figure 3)

[0071] [Table 1]

[0072] m1g, 1-methylguanosine; am, 2'-O-methyladenosine; cm, 2'-O-methylcytidine; gm, 2'-O-methylguanosine; Ψ, pseudouridine; m2g, N2-methylguanosine; ac4c, N4-acetylcytidine; d, dihydrouridine; m22g, N2,N2-dimethylguanosine; m2g, N2-methylguanosine; I, inosine; m5c, 5-methylcytidine; mcm5u, 5-methoxycarbonylmethyluridine; mcm5s2u, 5-methoxycarbonyl-methyl-2-thiouridine; ncm5u, 5-carbamoylmethyluridine; ncm5um, 5-carbamoylmethyl-2'-O-methyluridine; q, queosine; m5c, 5-methylcytidine.

[0073] As described above, unmodified nucleotides in the sequence of a synthetic tRNA of the present invention may be replaced with the corresponding modified nucleotide. Thus, the symbols A, C, G, or U in the above sequences of a tRNA of the present invention may represent an unmodified base or any corresponding modified base. A in the sequence may represent, for example, an adenine nucleotide (A) or a corresponding modified nucleotide, such as 1-methyladenosine (m1a). When synthesizing a tRNA, the base used may be an unmodified base. However, the base of the synthesized tRNA may be chemically and / or enzymatically modified in vitro. Once introduced or incorporated into a cell, the tRNA, whether synthesized in vitro using unmodified or modified nucleotides, may be modified by the cell.

[0074] In a further aspect, the present invention relates to a synthetic transfer RNA according to the first aspect of the present invention for use as a pharmaceutical. The transfer RNA of the present invention is particularly useful for treating patients suffering from diseases associated with frameshifts that result in the loss of functional proteins or reduced protein function. Examples of diseases for which the tRNA of the present invention can be advantageously used include neurofibromatosis type 1, Duchenne muscular dystrophy, Crohn's disease, cystic fibrosis, neuronal ceroid lipofuscinosis, and Tay-Sachs disease. Suitable compositions or means for delivering tRNA to cells are known. These means include viral vectors, such as those based on adeno-associated virus (AAV), and encapsulation or linkage to nanoparticles.

[0075] In a further aspect, the present invention relates to a method for treating a patient suffering from a disease associated with a frameshift mutation, comprising administering to the patient an effective amount of a synthetic transfer RNA of the invention or a composition comprising the synthetic transfer RNA of the invention. In a preferred embodiment, the method is for treating neurofibromatosis type 1, Duchenne muscular dystrophy, Crohn's disease, cystic fibrosis, neuronal ceroid lipofuscinosis, and / or Tay-Sachs disease.

[0076] In yet another aspect, the present invention relates to a synthetic transfer RNA having an extended anticodon loop, comprising: a) a variable arm having or comprising the sequence of SEQ ID NO: 32, and / or b) a T-arm having or comprising one of the sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; and / or c) U at position 31 and A at position 39 according to the tRNA numbering convention.

[0077] The tRNAs of this aspect of the invention, particularly those comprising a variable arm having or comprising the sequence of SEQ ID NO: 32, or a T-arm having or comprising one of the sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, or those comprising a variable arm having or comprising the sequence of SEQ ID NO: 32 in combination with a T-arm having or comprising one of the sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, are stable in a cellular environment, e.g., in mammalian cells, and are useful for suppressing nonsense or frameshift mutations. The extended anticodon loop may comprise a 4nt-, 5nt-, or 6nt-anticodon to suppress frameshift or nonsense mutations. Therefore, this tRNA can be advantageously used as a suppressor molecule to treat patients suffering from diseases associated with such mutations, such as neurofibromatosis type 1, Duchenne muscular dystrophy, Crohn's disease, cystic fibrosis, neuronal ceroid lipofuscinosis, or Tay-Sachs disease.

[0078] The tRNA of this embodiment of the invention preferably has an anticodon arm with a U at position 31 and an A at position 39. The anticodon arm may have, for example, the sequence of SEQ ID NO: 17 or SEQ ID NO: 18. The tRNA of this embodiment of the invention may comprise such an anticodon arm alone, or may comprise such an anticodon arm in combination with the variable loop described above, in combination with the T arm described above, or in combination with the variable loop and T arm described above. The invention will now be described by way of example only with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0079]

Figure 1

Figure 2

Figure 3

[0080] Figure 1 shows a schematic example of a synthetic tRNA1 of the invention useful as a +1 frameshift suppressor and a target mRNA 15 bearing a mutant codon 17 with nucleotide 13 inserted. Figure 1A shows synthetic tRNA1 bound to mRNA 15 with mutant codon 17 with nucleotide 13 inserted, Figure 1B shows mRNA 15 with mutant codon 17, and Figure 1C shows the original, non-mutated (wild-type) mRNA 15 with the addition of nucleotide 13. Synthetic tRNA1 of the invention consists of tRNA nucleotide 11 and has the consensus cloverleaf structure of natural tRNAs, including acceptor stem 2 with CCA tail 10, T-arm 3 with TΨC loop 6, D-arm 4 with D-loop 7, 5-nucleotide stem portion 8, and anticodon arm 5 with anticodon loop 9. Amino acid 14 is attached to CCA tail 10 of acceptor stem 2. The extended anticodon loop 9 consists of nine nucleotides 11 and includes a four-nucleotide (quadruplet) anticodon 12, i.e., a "codon" consisting of four nucleotides (filled circle) instead of the usual three nucleotides. The four-nucleotide anticodon 12 can base pair with a mutant codon 17 (also filled) on the target mRNA 15, consisting of mRNA nucleotide 16. The mutant codon 17 contains an insertion such that the original base triplet codon is extended by the inserted nucleotide 13. The four-nucleotide anticodon 12 (filled circle) can base pair with the mutant codon 17 (also filled) on the mRNA 15. The tRNA preferably carries the amino acid encoded by the unmutated codon. The variable loop between the T arm and the anticodon arm is not shown.

[0081] Figure 2 shows a schematic example of a synthetic tRNA1 of the invention useful as a -1 frameshift suppressor. As shown in Figure 2A, this embodiment of a synthetic tRNA1 of the invention carries a five-nucleotide anticodon 19, i.e., a five-nucleotide anticodon capable of base-pairing with the complementary five nucleotides on mRNA 15. Figures 2B and 2C show the mutated mRNA 15 (B) and the original, unmutated mRNA 15 (C). Mutant mRNA 15 carries a mutated codon 22 (hatched) in which nucleotide 20 has been deleted. The preceding unmutated codon 21, i.e., the original nucleotide triplet codon, is shown in black. In the embodiment shown herein, the five-nucleotide anticodon 19 consists of three nucleotides (filled circles) that can base-pair with the complementary codon 21 preceding the mutated (truncated) codon 22, and the remaining two nucleotides (hatched circles) can base-pair with the mutated codon 22 (also hatched), i.e., the nucleotide doublet remaining after deletion of nucleotide 20 from the original codon 22. tRNA1 may carry either the amino acid 14 encoded by the codon 21 preceding the mutated codon 22 or the amino acid 14 encoded by the original, unmutated codon 22. It is also possible to add a dipeptide consisting of the codon 21 preceding the mutated codon and the amino acid encoded by the mutated codon to the tRNA. Of course, the tRNA can also be designed so that the mutated codon will base-pair with the two codons preceding the unmutated codon.

[0082] Figure 3 shows an example of a tRNA numbered according to conventional numbering, which applies to popular "consensus" tRNAs and begins with 1 at the 5' end and ends with 76 at the 3' end. In such "consensus" tRNAs, the nucleotides of the natural anticodon triplet 25 are always at positions 34, 35, and 36, regardless of the actual number of preceding nucleotides. The tRNA may also contain additional nucleotides, e.g., between positions 1 and 34, e.g., in the D-loop, or in the variable loop 24 between positions 45 and 46. The additional nucleotides may be numbered with an alphabetical prefix, e.g., 20a, 20b, etc. In the variable loop 24, the additional nucleotides are numbered with an "e" prefix and a number suffix, depending on the nucleotide's position in the loop. Modified nucleotides, e.g., those listed in Table 1 above, may also be present in the sequence. [Example]

[0083] To test the effects of various nucleotides and nucleotide pairs on tRNA stability and its ability to read non-canonical codons (nucleotide triplets that do not encode proteinogenic amino acids), various constructs were tested in Hep3B cells. To do this, tRNAs were first transcribed in vitro and then transfected into Hep3B cells along with a reporter plasmid. The reporter plasmid contained a luciferase gene in which a non-canonical codon was incorporated downstream of the start codon, to which all of the tested tRNAs paired. The tRNA variations occurred within the T-stem and anticodon stem, while the rest of the tRNA structure was kept constant.

[0084] The following T-arms and anticodon arms were tested alone or in combination (see Table 2 below): Only the stem portion was varied, with the loop of the T-arm (UUCGAAU) and the anticodon arm (CU UCA The loop (AA, anticodon underlined) remained intact.

[0085] [Table 2]

[0086] [Table 3]

[0087] In combination experiments (modified T stem + modified anticodon stem), the A-stem variant 1 (U31-A39) described above was used as the anticodon stem.

[0088] [Table 4]

[0089] In vitro tRNA transcription Templates for T7 promoter-driven transcription of the designed tRNAs were generated by annealing and primer extension of two overlapping DNA oligonucleotides (commercially available) covering the entire length of each tRNA, including the T7 promoter sequence (5'-TAATACGACTCACTATA-3'). Both oligonucleotides were denatured at 95°C for 2 min and then annealed at their overlapping regions by incubation in 0.2 M Tris-HCl (pH 7.5) at room temperature for 3 min. To complement the DNA template and generate fully double-stranded DNA, 0.4 mM dNTPs, 4 U / μL RevertAid Reverse Transcriptase (Thermo Fisher Scientific, USA), and 1x RT buffer were added to the annealed oligonucleotides, and the reaction was incubated at 37°C for 40 min. The DNA templates were purified with phenol / chloroform.

[0090] To perform in vitro T7-driven transcription of tRNA, 30 μg of dsDNA template was mixed with 2 mM NTPs, 5 mM GMP, and 0.6 U / μL T7 RNA polymerase (Thermo Fisher Scientific, USA) in 1× transcription buffer and incubated overnight at 37°C. tRNA was precipitated with ethanol, separated on a 10% denaturing polyacrylamide gel, and eluted overnight at 4°C with 50 mM KOAc and 200 mM KCl pH 7.0 under constant shaking (1000 rpm). tRNA was filtered, recovered by ethanol precipitation, and resuspended in DEPC-H2O. tRNA integrity was analyzed by 10% denaturing polyacrylamide gel electrophoresis, and the tRNA was stored at -80°C for future use.

[0091] tRNA efficiency test in Hep3B cells Hep3B cells were maintained at 37°C under 5% CO2 in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% glutamine (GIBCO). Approximately 10,000 cells were seeded into a 96-well plate one day before co-transfection. The transfection mixture contained reporter plasmid (pGL4.51 [luc2 / CMV / Neo] backbone (Promega, USA)) (25 ng / well), repressor tRNA (100 ng / well), Lipofectamine 3000 (0.3 μl / well), and P3000 reagent (0.2 μl / well) (Thermo Fisher Scientific, USA) in Opti-MEM (Thermo Fisher Scientific, USA). Cells were incubated with the transfection mixture for 5 hours. The medium was then replaced with fresh medium. After 24 hours, the cells were washed with 1x PBS and lysed in 1x Passive Lysis Buffer (Promega, USA) for 15 minutes at room temperature with stirring. Firefly luciferase reagent (Promega, USA) was added, and luciferase activity was measured using a Tecan Spark microplate reader (Tecan, Switzerland).

[0092] In the designed tRNAs, nucleotide pairs in the T-stem and anticodon stem were changed, while all other portions of the tRNA remained constant (see above). 100 ng of in vitro transcribed tRNA was cotransfected with pGL4.51[luc2 / CMV / Neo], which expresses a luciferase reporter with a non-canonical codon, and expression was allowed for 24 hours. Pairing to the non-canonical codon resulted in luciferase activity, which was monitored by measuring Firefly luciferase expression using a microplate reader. The results are shown in Table 2 below.

[0093] Table 2: Activity of various tRNA designs with optimized T-stem or anticodon (A) stem. In the designed tRNAs, nucleotide pairs in the T-stem and anticodon stem were varied, thereby keeping all other portions of the tRNA constant. 100 ng of in vitro transcribed tRNA was cotransfected with pGL4.51[luc2 / CMV / Neo], which expresses a luciferase reporter with a non-canonical codon, and expression was allowed for 24 hours. Efficient pairing to the non-canonical codon resulted in luciferase activity, which was monitored by firefly luciferase enzyme activity using a microplate reader. Data are means ± SD (n = 3).

[0094] [Table 5] TIFF2025131687000007.tif172166

[0095] a The stabilization energy of the T-stem mutant is calculated as the difference (ΔΔG°) between the stabilization energy through binding of the natural human Ser-tRNA mutant to the elongation factor, which has an energy of ΔG° = 0.5 kcal / mol. Because the elongation factor binds specifically to the T-stem, this energy is contributed only to the T-stem mutant and not to the compound mutant. bNote that multiple sets of experiments were performed using different luciferase substrates, so each experiment had to be compared to its own set of controls. The two sets are separated by a thick lane. c The percentage of Fluc activity was calculated by subtracting wild-type luciferase activity or mock values. d In A / T variants 3 and 4, the T-stem is further mutated to stabilize a Although these mutants increased the T-stem activity, they were not tested as single T-stem mutants.

Claims

[Claim 1] Contains an extended anticodon loop with a four-nucleotide anticodon or a five-nucleotide anticodon A synthetic transfer RNA characterized by:

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