LU101182B1

LU101182B1 - Synthetic transfer RNA with extended anticodon loop

Info

Publication number: LU101182B1
Application number: LU101182A
Authority: LU
Inventors: Andrew Torda; Zoya Ignatova
Original assignee: Univ Hamburg
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2020-10-12

Abstract

The invention relates to a synthetic transfer RNA with an extended anticodon loop. The invention provides a synthetic suppressor transfer RNA useful for the treatment of a genetic disease like neurofibromatosis associated with a frameshift mutation. The synthetic transfer RNA comprises an extended anticodon loop having a four-nucleotide anticodon or a five- nucleotide anticodon.

Description

PAT 1690 LU -1- LU101182

SYNTHETIC TRANSFER RNA WITH EXTENDED ANTICODON LOOP

DESCRIPTION The invention relates to a synthetic transfer RNA with an extended anticodon loop. Transfer ribonucleic acids (tRNAs) are an essential part of the protein synthesis machinery of living cells as necessary components for translating the nucleotide sequence of a messenger RNA (mRNA) into the amino acid sequence of a protein. Naturally occurring tRNAs comprise an amino acid binding stem being able to covalently bind an amino acid and an anticodon loop containing a base triplet called “anticodon”, which can bind non-covalently to a corresponding base triplet called “codon” on an mRNA. A protein is synthesized by assembling the amino acids carried by tRNAs using the codon sequence on the mRNA as a template with the aid of a multi component system comprising, inter alia, the ribosome and several auxiliary enzymes. Some diseases belonging to the group of genetic or monogenic diseases are based on a change in the genetic information, e.g. a mutation in the DNA of the encoding genes. This includes single- or multiple nucleotide exchanges, deletions or insertions. In this case the mRNA transcribed from the mutated gene will also carry the altered genetic information and an aberrant, possibly non-functional protein is formed. A deletion or insertion (collectively termed indel) of one or multiple nucleotides may, for example, change the reading-frame within a coding region (i.e. the trinucleotide register in which the ribosome reads the information in mRNA) resulting in an entirely new amino acid sequence down-stream of the mutation and the production of a non-functional protein. As an example, the diseases neurofibromatosis type I (NF1) and neurofibromatosis type 2 (NF2) can be caused by a mutation of the gene coding for neurofibromin 1 (NF) and neurofibromin 2 (NF2), respectively. The genes NF1 and NF2 are believed to function as tumor suppressors. Mutations in NF1 and NF2 occur at birth (inherited or de novo) but also in the somatic cell state. The most common mutations are frameshift mutations with deletion or insertion, ranging from 1 to 12 nucleotides, with deletions being more frequent than insertions (Pros, E., Gomez, C., Martin, T., Fabregas, P., Serra, E. and Lazaro, C. (2008), Nature and mRNA effect of 282 different NF1 point mutations: focus on splicing alterations, Hum. Mutat., 29: E173-E193. doi:10.1002/humu.20826; Ars E, Kruyer H,

VW

PAT 1690 LU

-2- LU101182 Morell M, et al., Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients, J.

Med.

Genet. 2003;40:e82, doi:10.1136/jmg.40.6.e82). Some antibiotics such as macrolides promote frameshifting, but no attempts to use them to correct frameshifting so far have been reported (Brierley, I. 2013, Macrolide-Induced Ribosomal Frameshifting: A New Route to Antibiotic Resistance, Mol.

Cell 52, 613-615, doi:10.1016/j.molcel.2013.11.017; Atkins, J.

F., & Baranov, P.

V. (2013). Molecular biology: Antibiotic re-frames decoding.

Nature, 503(7477), 478-479. Do1:10.1038/503478a). Although still in its beginnings, gene therapy involving the introduction of corrective genetic material into the cells of a patient is becoming important for treating genetic diseases.

Approaches based on correcting entities down-stream of the gene, mostly mRNA, are preferred as they do not belong to the classic gene therapy approaches, since the gene sequence (or DNA) remains unchanged.

However, mRNA is intrinsically short-lived and the length of the mRNA sequences presents problems for therapeutic application.

A particular mRNA may, for example, be longer than the cargo capacity of currently available vectors for gene delivery and therapy.

Compared to mRNA, tRNA molecules offer significantly higher stability and are on average 10-fold shorter, alleviating the problem of introduction into the target tissue.

This has led to attempts to use tRNA in gene therapy in order to prevent the formation of a truncated protein from an mRNA with a premature stop codon and to introduce the correct amino acid instead (see, e.g., Koukuntla, R 2009, Suppressor tRNA mediated gene therapy, Graduate Theses and Dissertations, 10920, Iowa State University, http:/lib.dr.iastate.edu/etd/10920; US 2003/0224479 A1; US 6964859). Natural tRNAs with an extra nucleotide in the anticodon or with a shortened codon doublet have been found in bacteria.

These unusual codons are used naturally to suppress a specific frameshift position (Qian, Q., Li J-N., Zhao H., Hagervall T.G., Farabaugh P.J., Bjork G.R., A New Model for Phenotypic Suppression of Frameshift Mutations by Mutant tRNAs, Mol cell 1, 471-482, 1998; O'Mahony DJ, Hughes D, Thompson S, Atkins JF, Suppression of a -1 Frameshift Mutation by a Recessive tRNA Suppressor Which Causes Doublet Decoding, J Bact, 171 (7), 1989). Sako et al. 2006 (Sako Y, Usuki F, Suga H.

A novel therapeutic approach ig

PAT 1690 LU -3- LU101182 for genetic diseases by introduction of suppressor tRNA.

Nucleic Acids Symp Ser (Oxf). 2006;(50):239-240) describe an approach to read through PTC-containing mRNAs using suppressor tRNA that is introduced to cells by transfection.

Nonsense triplet codons and four- base codons were read by the corresponding suppressor tRNAs derived from human tRNA(Ser). tRNAs with an extended anticodon loop comprising a four-base or five-base anticodon have also been introduced to in vitro translation systems of bacteria to incorporate unnatural amino acids into proteins and address the molecular mechanism of frameshift suppression of two-, three-, four-, five-, and six-base codons with tRNAs containing 6-10 nt in their anticodon loops (US 2006/0177900 A1; WO 2005/007870; Hohsaka T, Ashizuka Y, Murakami H, Sisido M.

Five-base codons for incorporation of nonnatural amino acids into proteins.

Nucleic Acids Research. 2001;29(17):3646-3651; Hohsaka T, Sisido M.

Incorporation of non-natural amino acids into proteins.

Curr Opin Chem Biol. 2002 Dec;6(6):809-15; Anderson JC, Magliery TJ, Schultz PG.

Exploring the limits of codon and anticodon size.

Chem Biol. 2002, 9(2):237-44. DOI: 10.1016/S1074-5521(02)00094-7). The yields of incorporation of unnatural amino acids are, however, extremely low, and are influenced by the mRNA context.

There is still a need to counteract the effects of and/or suppressing a frameshift mutation.

It is therefore an object of the invention to provide such means, in particular a frameshift mutation suppressor for the treatment of a genetic disease like neurofibromatosis associated with a frameshift mutation.

In one aspect the invention provides a synthetic transfer ribonucleic acid (tRNA), the synthetic transfer RNA comprising an extended anticodon loop with a four- or five-base anticodon.

The four-base anticodon is configured to base-pair to four, the five-base anticodon is configured to base-pair to five consecutive nucleotide bases on a messenger RNA in order to correct insertions and deletions in consecutive codon base triplets on an mRNA, which change the reading-frame register into a —1 and +1 frame.

The invention provides novel suppressor tRNAs that can be used to suppress a —1 or +1 frameshift mutation with high specificity, in a context-dependent manner, for example, to

{4

PAT 1690 LU

—4- LU101182 restore the ability of a cell to synthesize a functional protein from an mRNA having a mutation in its coding sequence, which would otherwise lead to a different protein sequence and a less functional or non-functional protein.

The synthetic tRNA of the invention comprises an anticodon loop being extended by one or two nucleotides.

The synthetic tRNA of the invention is complementary to four or five bases of two adjacent codons on the mRNA, the first being the codon with an indel (insertion or deletion) and the second being an intact adjacent codon.

The synthetic tRNA of the invention with four- or five-base codons base-pairs with the codon and the remaining of the following (in case of a —1 frameshifting) or of the preceding (in case of +1 frameshifting) codon on the mRNA resulting in the incorporation of an amino acid carried by the tRNA into the growing amino acid chain and the correction of the reading frame.

In case of a —1 frameshift, unless the synthetic tRNA of the invention is (pre)aminoacylated with a dipeptide, the resulting protein will have one amino acid less than the wild-type protein, i.e. a protein synthesized from the wild-type mRNA, but there is still a good chance that this will lead to a functional protein.

Advantageously, the invention provides synthetic transfer RNA being designed to have an anticodon loop leading to higher binding affinity when compared to prior art suppressor tRNAs used for non-natural amino acid incorporation.

The binding to two consecutive codons, the one with the indel (insertion or deletion) and the adjacent unaltered codon, is associated with higher specificity compared to natural two-nucleotide-anticodon suppressor tRNAs.

Consequently, the synthetic tRNA of the invention can be designed to effectively bind to a specific mutation site, considerably reducing the risk of unspecific pairing to other partially homologous regions of the mRNA.

The terms “transfer ribonucleic acid” or “tRNA” refer to RNA molecules with a length of typically 73 to 90 nucleotides, which mediate the translation of a nucleotide sequence in a messenger RNA into the amino acid sequence of a protein. tRNAs are able to covalently bind a specific amino acid at their 3' CCA tail at the end of the acceptor stem, and to base-pair via a three-nucleotide anticodon in the anticodon loop of the anticodon arm with a three-nucleotide sequence (codon) in the messenger RNA.

Some anticodons can pair with more than one codon due to a phenomenon known as wobble base pairing.

The secondary “cloverleaf” structure of tRNA comprises the acceptor stem binding the amino acid and three arms (“D arm”, “T arm”

of

PAT 1690 LU 75 LU101182 and “anticodon arm”) ending in loops (D loop, TyC loop, anticodon loop), i.e. sections with unpaired nucleotides. Aminoacyl tRNA synthetases charge (aminoacylate) tRNAs with a specific amino acid. Each tRNA contains a distinct anticodon triplet sequence that can base-pair to one or more codons for an amino acid. By convention, the nucleotides of tRNAs are often numbered 1 to 76, starting from the 5’-phosphate terminus, based on a “consensus” tRNA molecule consisting of 76 nucleotides, and regardless of the actual number of nucleotides in the tRNA, which are not always of length 76 due to variable portions, such as the D loop in the tRNA (see Fig. 3). Following this convention, nucleotide positions 34-36 of naturally occurring tRNA refer to the three nucleotides of the anticodon, and positions 74-76 refer to the terminating CCA tail. Any “supernumerary” nucleotide can be numbered by adding alphabetic characters to the number of the previous nucleotide being part of the consensus tRNA and numbered according to the convention, for example 20a, 20b etc, or by independently numbering the nucleotides and adding a leading letter, as in case of the variable loop such as ell, e12 etc. (see, for example, Sprinzl M, Horn C, Brown M, Ioudovitch A, Steinberg S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1998;26(1):148-53). In the following, the tRNA-specific numbering will also be referred to as “tRNA numbering convention”. The terms “synthetic transfer ribonucleic acid” or “synthetic tRNA” refer to a non-naturally occurring tRNA. The term also encompasses analogues to naturally occurring tRNAs, i.e. tRNAs being structurally similar to naturally occurring tRNAs, but being modified in the base component, the sugar component and/or the phosphate component of one or more of the nucleotides, of which the tRNA is composed. The modified tRNA may, for example, have the phosphodiester backbone modified in that the phosphodiester bridge is replaced by a phosphorothioate, phosphoramidate or methyl phosphonate bridge. The sugar component may, for example, be modified at the 2' OH group, e.g. by dehydroxylating it to a deoxy ribonucleotide, or by replacing it with a methoxy-, methoxyethoxy- or aminoethoxy group. A synthetic transfer ribonucleic acid can, for example, be synthesized chemically and/or enzymatically in vitro, or in a cell based system, e.g. in a bacterial cell in vivo.

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PAT 1690 LU -6- LU101182 The term “codon” refers to a sequence of nucleotide triplets, i.e. three DNA or RNA nucleotides, corresponding to a specific amino acid or stop signal during protein synthesis. A list of codons (on mRNA level) and the encoded amino acids are given in the following: Amino acid One Letter Code Codons 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, abbreviated “X” The term “sense codon” as used herein refers to a codon coding for an amino acid. The term “stop codon” or “nonsense codon” refers to a codon, i.e. a nucleotide triplet, of the genetic code

V

PAT 1690 LU -7- LU101182 not coding for one of the 20 amino acids normally found in proteins and signalling the termination of translation of a messenger RNA. The term “frameshift mutation” refers to an out-of-frame insertion or deletion (collectively called “indels”) of nucleotides with a number not evenly divisible by three. This perturbs the nucleotide sequence decoding which proceeds in steps of three nucleotide bases. The term ‘1 frameshift mutation” relates to the deletion of a single nucleotide causing a shift in the reading frame by one nucleotide leading to the first nucleotide of the following codon being read as part of the codon from which the nucleotide has been deleted. Deletion of one nucleotide from the upstream codon along with several triplets (i.e. deletion of 4, 7, 10 etc. nucleotides) is also considered as —1 frameshifting. The term “+1 frameshift mutation” relates to the insertion of a single nucleotide into a triplet, or the deletion of two nucleotides. The result of either event is to shift the reading frame by one nucleotide, such that a nucleotide of an upstream codon is being read as part of a downstream codon. Insertion of one nucleotide along with several triplets (3n+1 nucleotides, n being an integer, i.e. insertion of 4, 7, 10 etc. nucleotides), or deletion of two nucleotides from the upstream codon along with several triplets (i.e. deletion of 5, 8, 11 etc nucleotides) is also considered as +1 frameshifting. Frameshift mutations are implicated in a variety of genetic disorders such as Duchenne muscular dystrophy (DMD), Crohn disease (CD), Tay-Sachs disease (TSD) or neurofibromatosis type 1 (NF1). The term “anticodon” refers to a sequence of usually three nucleotides that base-pair (non- covalently bind) to the three bases of the codon on the mRNA. An anticodon may also contain nucleotides with modified bases. The terms “four nucleotide anticodon” or “four base anticodon” relate to an anticodon having four consecutive nucleotides (bases) which pair with four consecutive bases on an mRNA. The terms “quadruplet nucleotide anticodon” or “quadruplet anticodon” may also be used to denote a “four nucleotide anticodon”. The terms “five nucleotide anticodon” or “five base anticodon” relate to an anticodon having five consecutive nucleotides (bases) which bind (base-pair) to five consecutive bases on an mRNA. The terms “quintuplet nucleotide anticodon” or “quintuplet anticodon” may also be used to denote a “five nucleotide anticodon”.

Ww

PAT 1690 LU

-8- LU101182 The term “anticodon loop” refers to the unpaired nucleotides of the anticodon arm containing the anticodon.

Naturally occurring tRNAs usually have seven nucleotides in their anticodon loop, three of which pair to the codon in the mRNA.

The term “extended anticodon loop” refers to an anticodon loop with a higher number of nucleotides in the loop than in naturally occurring tRNAs.

An extended anticodon loop may, for example, contain more than seven nucleotides, e.g. eight, nine, or ten nucleotides.

The terms “codon base triplet” or “anticodon base triplet”, when used herein, refer to sequences of three consecutive nucleotides which form a codon or anticodon.

Synonymously, the terms “three nucleotide codon” (also “three base codon”) or “three nucleotide anticodon” (also “three base anticodon”), or abbreviations thereof, e.g. “3nt codon”, “3nt anticodon” etc, may be used.

The term "base pair" refers to a pair of bases, or the formation of such a pair of bases, joined by hydrogen bonds.

One of the bases of the 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, the formation of other base pairs (“wobble base pairs”) is also possible, e.g. base pairs of guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). The term “being able to base- pair” refers to the ability of nucleotides or sequences of nucleotides to form hydrogen-bond- stabilised structures with a corresponding nucleotide or nucleotide sequence. “PTC” refers to a premature termination codon.

This is a stop codon introduced into a coding nucleic acid sequence by a nonsense mutation, i.e. a mutation in which a sense codon coding for one of the twenty proteinogenic amino acids specified by the standard genetic code is changed to a chain-terminating codon.

The term thus refers to a premature stop signal in the translation of the genetic code contained in mRNA.

The terms “Crohn disease” or “Crohn’s disease” relate to a gastro-intestinal inflammatory disease associated with the NOD2 gene.

A common mutation associated with this disease is an insertion of cytosine at position 3020.

PAT 1690 LU

—9- LU101182 The term “Tay-Sachs disease” relates to a genetic disorder resulting in nerve cells destruction and is apparent at early childhood at around 2-3 months after birth.

It leads to severe movement disability, hearing loss, seizures etc.

Death often occurs in early childhood.

The term “Duchenne muscular dystrophy” (DMD) (also “Becker muscular dystrophy”, BMD) refers to an X-linked recessive genetic disorder characterized by progressive muscle degeneration and weakness caused by an absence of a functional dystrophin protein.

The absence of dystrophin can be caused by a nonsense mutation in the dystrophin gene. “Neurofibromatosis type 1” (NF1 or NF-1), also called “Recklinghausen disease”, is an autosomal dominant inherited disorder caused by the mutation of the NF1 gene on chromosome 17 coding for neurofibromin.

NF1 causes tumours along the nervous system.

The term “frameshift suppression” refers to mechanisms masking the effects of a frameshift mutation and at least partly restoring the wild-type phenotype.

The term “suppressor tRNA” relates to a tRNA altering the reading of a messenger RNA in some translation systems.

An example of a suppressor tRNA is a tRNA carrying an amino acid and being able to base-pair to mutated codons covering two consecutive codons, of which one is intact and one has an insertion or deletion.

The translation system can thus correct the reading frame.

The term “homology” in relation to a nucleic acid refers to the degree of similarity or identity between the nucleotide sequence of the nucleic acid and the nucleotide sequence of another nucleic acid.

Homology is determined by comparing a position in the first sequence with a corresponding position in the second sequence in order to determine whether identical nucleotides are present at that position.

It may be necessary to take sequence gaps into account in order to produce the best possible alignment.

For determining the degree of similarity or identity between two nucleic acids it is preferable to take a minimum length of the nucleic acids to be compared into account, 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 the respective sequences.

Preferably the full length of the respective nucleic acid(s) is used for

PAT 1690 LU -10- LU101182 comparison. The degree of similarity or identity of two sequences can be determined by using a computer program such as muscle (Edgar, R.C. (2004), Muscle: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res., 32, 1792-1797, doi:

10.1093 /nar/gkh340) or mafft (Katoh, K. and Standley, D.M. (2013) MAFFT Multiple Sequence Alignment Software Version 7, Mol. Biol. Evol., 30, 772-780, doi.org/10.1093/molbev/mst010). Where such terms like “x % homologous to” or “homology of x %” are used herein, this means that two nucleic acids have a sequence identity or similarity of x %, e.g. 50%. The term “aminoacylation” relates to the enzymatic reaction in which a tRNA is charged with an amino acid. An aminoacyl tRNA synthetase (aaRS) catalyses the esterification of a specific cognate amino acid or its precursor to a compatible cognate tRNA to form an aminoacyl-tRNA. The term “aminoacyl-tRNA” thus relates to a tRNA with an amino acid attached to it. Each aminoacyl-tRNA synthetase is highly specific for a given amino acid, and, although more than one tRNA may be present for the same amino acid, there is only one aminoacyl tRNA synthetase for each of the 20 proteinogenic amino acids. The terms “charge” or “load” may also be used synonymously for “aminoacylate”. The term “aminoacylated” in relation to the synthetic tRNA of the invention relates to a synthetic tRNA already charged (precharged) with an amino acid or a dipeptide, such that the tRNA is already acylated when entering the target cell. The term “preaminoacylated” may synonymously be used in this context. The term “modified nucleotides” (or “unusual nucleotides”) in reference to tRNA relates to nucleotides having modified or unusual nucleotide bases, i.e. other than the usual bases adenine (A), uracil (U), guanine (G) and cytosine (C). Examples of modified nucleotides include 4- acetylcytidine (acdc), 5-(carboxyhydroxymethyl)uridine (chmSu), 2’-O-methylcytidine (cm), 5- carboxymethylaminomethyl-2-thiouridine (cmnm5s2u), 5-carboxymethylaminomethyluridine (cmnm5u), dihydrouridine (d), 2’-O-methylpseudouridine (fm), beta, D-galactosylqueuosine (gal q), 2’-O-methylguanosine (gm), inosine (i), N6-isopentenyladenosine (i6a), 1- methyladenosine (m1a), 1-methylpseudouridine (m1f), 1-methylguanosine (m1g), 1- methylinosine (m11i), 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

PAT 1690 LU

-11- LU101182 (mamSu), 5-methoxyaminomethyl-2-thiouridine (mam5s2u), beta, D-mannosylqueuosine (man q), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2u), S-methoxycarbonylmethyluridine (memS5u), 5-carbamoylmethyluridine (nem5U), 5-carbamoylmethyl-2’-O-methyluridine (nemSUm), 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-oxyacetic acid- methylester (mv), uridine-5-oxyacetic acid (o5u), wybutoxosine (osyw), pseudouridine (p, P), queuosine (q), 2-thiocytidine (s2c), 5-methyl-2-thiouridine (s2t), 2-thiouridine (s2u), 4- thiouridine (s4u), 5-methyluridine (t), N-((9-beta-D-ribofuranosylpurine-6-yl)- carbamoyl)threonine (t6a), 2’-O-methyl-5-methyluridine (tm), 2’-O-methyluridine (um), wybutosine (yw), 3-(3-amino-3-carboxy-propyl)uridine, (acp3)u (x), The term “corresponding modified nucleotide” relates to a modified nucleotide at a given position in a sequence, which base has been modified based on the usual, 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.

À corresponding modified nucleotide is thus any nucleotide that, in a cell, is usually produced from a usual nucleotide by modifying the usual nucleotide.

A modified nucleotide corresponding to uridine, for example, is thus any nucleotide derived by the modification of uridine.

As an example, 5-(carboxyhydroxymethyl)uridine (chmS5u) at a particular position in a sequence may be a modified nucleotide corresponding to uridine at the same position in the original sequence.

Further modified nucleotides corresponding to uridine are, for example, 5-methyluridine (t), 2-O-methyl-5-methyluridine (tm), 2’-O-methyluridine (um), or 5-methoxyuridine (mo5u). Inosine, as another example, is produced from adenosine and thus is a modified nucleotide corresponding to adenosine.

The synthetic transfer RNA of the invention may be synthesized based on a naturally occurring tRNA.

However, the tRNA of the invention is preferably designed computationally (“in silico”) and synthesized in vitro chemically and/or enzymatically.

The computational design of a synthetic tRNA according to the invention allows the design and synthesis of a tRNA that does not interfere with other tRNAs present in the cell.

The synthetic tRNA of the invention is selected or designed in such a manner that an aminoacyl tRNA synthetase that naturally occurs in a living cell, preferably a mammalian cell, e.g. a human cell, is able to charge the tRNA with

4

PAT 1690 LU -12- LU101182 a specific amino acid that is encoded by the codon adjacent to the mutated codon or by the wild-type codon receiving indels In order to counteract a possible destabilization due to the anticodon loop expansion, the synthetic tRNA of the invention may be further structurally modified, within or outside its anticodon loop, to have a high stability. Preferably, the tRNA of the invention is designed to have at least the same or a higher stability than a naturally occurring tRNA in which the natural three-nucleotide anticodon is replaced with a four- or five-nucleotide anticodon. The term “stability” relates to the adoption of a stable and correctly folded and functional conformation of the tRNA in the absence of a translation factor, and can be predicted based on the calculation of the free energy change upon folding. “Higher stability” means that the favourable free energy change upon the tRNA folding to a functional state is greater. This includes a higher fraction of tRNA molecules in the desired configuration, i.e. adequately or completely folded. The stability of a synthetic transfer RNA of the invention with a four-nucleotide anticodon is compared with a naturally occurring transfer RNA whose three-nucleotide anticodon has been replaced with a four-nucleotide anticodon, and the stability of a synthetic transfer RNA of the invention with a five-nucleotide anticodon is compared with a naturally occurring transfer RNA whose three-nucleotide anticodon has been replaced with a five-nucleotide anticodon. A high stability of the synthetic tRNA of the invention alone, i.e. in the absence of translation factors, improves the longevity of the tRNA within the cell. Further preferred, the synthetic tRNA of the invention is configured to have less stability in a complex with the elongation factor (e.g. eEF1A), thus increasing the functional promiscuity of the tRNA in translation and consequently the suppression activity. The tRNA may, for example, be modified regarding the nucleotide composition of its anticodon loop or components outside the anticodon arm, for example of its D-arm, T-arm or variable arm. It is preferred, for example, that the synthetic tRNA of the invention has a C at position 32 and/or an A at position 37 in the anticodon loop, the numbering following the above-mentioned tRNA numbering convention. It is further preferred to have a G-C or C-G pair at the end of the anticodon stem in direction of the anticodon loop, that is to have a G-C or C-G pair flanking and stabilizing the anticodon loop. According to tRNA numbering convention, the G-C or C-G pair would take the positions 31 and 39.

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PAT 1690 LU -13- LU101182 The skilled person is aware of the fact that a tRNA is aminoacylated with a specific amino acid by a specific aminoacyl tRNA synthetase (aaRS), and that the aaRS is able to recognize its cognate tRNA through unique identity elements at the acceptor stem and/or anticodon loop of the tRNA. In order to provide a tRNA which is loaded with its cognate amino acid in vivo, the skilled person will design the synthetic tRNA of the invention with suitable unique identity elements. It is also possible to aminoacylate a synthetic tRNA of the invention with a flexizyme and transfect it into an eukaryotic cell as aminoacyl-tRNA (Goto, Y., Kato, T. and Suga, H., Flexizymes for genetic code reprogramming, Nat Protoc., 6(6), 779, 2011, doi:10.1038/nprot.2011.331). The tRNA of the invention preferably has a low sequence identity to any naturally occurring tRNA, and has preferably a sequence identity of less than 50%, especially preferred of less than 49%, 48%, 47%, 46%, 45%, 44% or 43%. In the synthetic transfer RNA according to the invention the anticodon loop has been extended by a large enough number of nucleotides to accommodate the four or five nucleotide anticodon and to allow base-pairing with an mRNA. The anticodon loop of a synthetic transfer RNA of the invention may, for example, consist of 7-12, preferably 7 to 10 or 8-10, further especially preferred 8 or 9 nucleotides. The extended anticodon loop of the synthetic tRNA of the invention comprises, in one embodiment, a four-base anticodon, which is able to base-pair to a four-base codon, i.e. a codon base triplet on a targeted mRNA with an additional nucleotide. In another embodiment, the synthetic tRNA of the invention comprises a five-base anticodon, including a base doublet being able to base-pair to a codon doublet left from a codon triplet on the mRNA after deletion of a nucleotide in that codon, whereas the neighbouring anticodon base triplet preferably base- pairs to a sense codon preceding or following, i.e. 5' or 3' to the codon with the deletion on the mRNA. The terms “preceding” or “following” relate to the direction of translation, i.e. the 5'-3' direction of the mRNA. An example of a five-nucleotide anticodon in the extended anticodon loop of the synthetic tRNA of the invention is GAUUC (in 5'-3' direction, or CUUAG in 3'-5' direction), matching

Ÿ

PAT 1690 LU -14- LU101182 with GAAAUC (5'-3") in the unmutated mRNA, where UC is able to base-pair with the base doublet GA left after deletion of an A in the codon GAA, and GAU is able to base-pair with the codon AUC coding for isoleucine. The synthetic transfer RNA according to the invention may be aminoacylated, i.e. carrying an amino acid or a dipeptide at the end of its acceptor stem. Preferably, the tRNA is aminoacylated with an amino acid being encoded by a sense codon base-pairing with the four or five nucleotide anticodon. The synthetic tRNA of the invention can be chemically and/or enzymatically aminoacylated with a single amino acid or dipeptide. The loading of a tRNA with a dipeptide can be accomplished with methods known to those skilled in the art (see, for example, Maini R, Dedkova LM, Paul R, Madathil MM, Chowdhury SR, Chen S, Hecht SM, 2015, Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro, J. Am. Chem. Soc, 137, 11206-11209, doi 10.1021/jacs.5b03135). Engineered bacterial tRNA synthetases or RNA-based catalysts may, for example, be used to aminoacylate the tRNA with a dipeptide. A dipeptide is preferably composed of the amino acids encoded by the consecutive unmutated codons in case of a deletion. The use of a synthetic tRNA aminoacylated with such a dipeptide would not only result in the intended suppression of a -1 frameshift and the production of a non-truncated protein, but also in the production of a protein having the amino acid sequence of the wild-type protein. In preferred embodiments, the synthetic transfer RNA of the invention has or comprises a) a sequence being composed of, in 5° to 3° direction, consecutive sequence parts A, B and C, part A having or comprising one of the sequences according to SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 07 and SEQ ID NO: 08, part B having one of the sequences according to SEQ ID NO: 03, SEQ ID NO: 04 and SEQ ID NO: 06, and part C having or comprising the sequence according SEQ ID NO: 5, 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 sequences of a) or b) above, where at least one of the nucleotides is replaced with a corresponding modified nucleotide.

nr

PAT 1690 LU -15- LU101182 In a preferred embodiment the synthetic tRNA of the invention is composed of three sequence parts A, B and C, which are covalently bonded in the 5° to 3° direction, e.g. via phosphodiester bonds. In 4-nt-anticodon tRNAs the part A comprises the 4-nt-anticodon (underlined “N”’s below) and preferably has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNAUGCNNNUN SEQ ID NO: 2

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNAGCNNNUN N= any nucleotide Part B comprises the variable loop and preferably has the sequences of SEQ ID NO: 3, 4 or 6. SEQ ID NO: 3

NNNNNNNNNNN SEQ ID NO: 4

NNNNNNNNNNNNNNN SEQ ID NO: 6

UGGGGUCACUCCCCG Part C preferably has the sequence with SEQ ID NO: 5. SEQ ID NO: 5

CNUGGNUUCGAAUNCCANNNCUGNNACCA

PAT 1690 LU —16- LU101182 A 4-nt-anticodon tRNA composed of parts A to C of the invention thus preferably has the following sequences (N = any nucleotide): SEQ ID NO: 9

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNAUGCNNNUNNNNNNN

NNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 10

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNAGCNNNUNNNNNNNN

NNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 11

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNAUGCNNNUNUGGGGU

CACUCCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 12

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNAGCNNNUNUGGGGUC

ACUCCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA In the case of a 5-nt-anticodon, tRNAs part A comprises the 5-nt-anticodon (underlined “Ns below) and preferably has the sequence of SEQ ID NO: 7 or SEQ ID NO: 8 (N = any nucleotide). Part A: SEQ ID NO: 7

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNNAUGCNNNUN SEQ ID NO: 8

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNNAGCNNNUN Part B preferably has the following sequences:

PAT 1690 LU —17- LU101182 SEQ ID NO: 3

NNNNNNNNNNN SEQ ID NO: 4

NNNNNNNNNNNNNNN SEQ ID NO: 6

UGGGGUCACUCCCCG Part C preferably has the following sequence: : SEQ ID NO: 5

CNUGGNUUCGAAUNCCANNNCUGNNACCA A 5-nt-anticodon tRNA composed of parts A to C of the invention thus preferably has the following sequences (N = any nucleotide): SEQ ID NO: 13

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNNAUGCNNNUNNNNNN

NNNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 14

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNNAGCNNNUNNNNNNN

NNNNNCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 15

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNNAUGCNNNUNUGGGG

UCACUCCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA SEQ ID NO: 16

PAT 1690 LU -18- LU101182

NNCAGNNUGNNCGAGNNGUCUAAGNNNNNNGCCUNNNNNAGCNNNUNUGGGGU

CACUCCCCGCNUGGNUUCGAAUNCCANNNCUGNNACCA In the above sequences, N stands for any of the bases A, C, G or U, or any modified base. Preferably, the base doesn’t violate the base pairing as given in Fig. 3. Allowed base pairs are, for example, G-C, C-G, A-U, U-A, and wobble base pairs like G-U, U-G, I-U, U-I, I-A, A-I and I-C, C-I.

The symbols G, C, A or U may represent the unmodified or any corresponding modified base (see below). The tRNA of the invention may thus contain one or more modified nucleotides. For clarification, it is noted that the synthetic transfer RNA of the invention may or may not be synthesized to contain any modified nucleotides. The synthetic transfer RNA of the invention may thus not contain any modified nucleotide. However, after entering a cell, one or more nucleotides of that synthetic tRNA may nevertheless be modified within the cell by the cellular enzymatic machinery. Consequently, a synthetic tRNA of the invention, which has been designed, synthesized and administered without any modified nucleotide, may, in a living cell, contain one or more modified nucleotides due to modifications the cell has made to them. In fact, it is preferred that the synthetic tRNA of the invention is synthesized and also administered without containing any modified nucleotides and to leave any modifications to the cell.

If a synthetic tRNA of the invention is synthesized with modified nucleotides, such that the tRNA already contains modified nucleotides prior to administration, it is preferred that the tRNA of the invention contains one or more of the following modified nucleotides (Table 1): Table 1. Possible modified nucleotides and positions within the tRNA (position numbering according to the specific tRNA numbering convention for a generalized “consensus” tRNA, see also Fig. 3) Position Modification 1 P 4 cm, am

PAT 1690 LU -19- LU101182 9 mlg 12 acdc 16 d 17 d 18 m2g 20, 20a-b d 26 m22g 28 F 29 F

F 32 ¥, 2'O-methylribose, cm 34 I, ¥, m5c, cm, gm, 2'O-methylribose, q, mecm5u, ncm5u, ncmSum, mem5s2u,

F mlg, 1-methylguanosine; am, 2'-O-methyladenosine; cm, 2'-O-methylcytidine; gm, 2'-O- methylguanosine; ¥, pseudouridine; m2g, N2-methylguanosine; acdc, N4-acetylcytidine; d, dihydrouridine; m22g, N2,N2-dimethylguanosine; m2g, N2-methylguanosine; I, inosine; m5c, 5-methylcytidine; mem5u, 5-methoxycarbonylmethyluridine; memS5s2u, 5-methoxycarbonyl- methyl-2-thiouridine; ncm5u, 5-carbamoylmethyluridine; nemSum, 5-carbamoylmethyl-2'-O- methyluridine; q, queuosine; m5c, 5-methylcytidine. As mentioned above, an unmodified nucleotide in a sequence for a synthetic tRNA of the invention may be replaced with a corresponding modified nucleotide. The symbols A, C, G or U in the above sequences for tRNAs of the invention may therefore represent an unmodified or any corresponding modified base. An A in a sequence may, for example, represent an adenine nucleotide (A) or a corresponding modified nucleotide, e.g. 1-methyladenosine (m1la). During synthesis of the tRNAs, the bases used can be unmodified bases. The bases of the synthesized tRNA may, however, be modified chemically and/or enzymatically in vitro. Once introduced or incorporated in a cell, the tRNAs, whether in vitro synthesized with unmodified or modified nucleotides, may be modified by the cell. y

PAT 1690 LU

-20- LU101182 In a further aspect the invention relates to the synthetic transfer RNA according to the first aspect of the invention for use as a medicament.

The transfer RNA of the invention is especially useful for treating patients with a disease associated with a frameshifting causing the absence of a functional protein or the dysfunction of a protein.

Examples for diseases, in which the tRNA of the invention may advantageously be employed are neurofibromatosis type 1, Duchenne muscular dystrophy, Crohn disease and Tay-Sachs disease.

Suitable compositions or means for delivering tRNAs to a cell are known.

These include viral vectors such as adeno-associated virus (AAV)-based viral vectors, encapsulation in or coupling to nanoparticles.

The invention will be described in the following by way of examples and the appended figures for illustrative purposes only.

Figure 1. Schematic example of a synthetic tRNA of the invention and a targeted mRNA for supressing a +1 frameshift mutation.

A.

Synthetic tRNA bound to targeted mRNA.

B. mRNA with codon carrying insertion.

C.

Original (wild-type) mRNA without insertion.

Figure 2. Schematic example of a synthetic tRNA of the invention and a targeted mRNA for supressing a —1 frameshift mutation.

Figure 3. Schematic drawing of a generalized “consensus” tRNA structure and its numbering according to tRNA numbering convention.

Figure 1 shows a schematic example of a synthetic tRNA 1 of the invention, useful as a +1 frameshift suppressor, and a targeted mRNA 15 carrying a mutated codon 17 with an inserted nucleotide 13. Fig. 1A shows the synthetic tRNA 1 bound to an mRNA 15 having a mutated codon 17 with an inserted nucleotide 13, Fig. 1B shows the mRNA 15 with the mutated codon 17, and Fig.

C depicts the original unmutated (wild-type) mRNA 15 receiving the additional nucleotide 13. The synthetic tRNA 1 of the invention is composed of tRNA nucleotides 11 and has the common cloverleaf structure of natural tRNA comprising an acceptor stem 2 with the CCA tail 10, a T arm 3 with the TyC loop 6, a D arm 4 with the D loop 7 and an anticodon arm with a five nucleotide stem portion 8 and the anticodon loop 9. An amino acid 14 is bound to the CCA tail 10 of the acceptor stem 2. The extended anticodon loop 9 consists of nine

PAT 1690 LU —21- LU101182 nucleotides 11 and contains a four-nucleotide (quadruplet) anticodon 12, i.e. a “codon” composed of four nucleotides (solid black circles) instead of the usual three nucleotides. The four-nucleotide anticodon 12 is able to base-pair to a mutated codon 17 (also solid black) on a targeted mRNA 15 composed of mRNA nucleotides 16. The mutated codon 17 carries an insertion, such that the original base triplet codon is extended by the inserted nucleotide 13. The four-nucleotide anticodon 12 (solid black circles) is able to base-pair with the mutated codon 17 (also solid black) on the mRNA 15. The tRNA preferably carries an amino acid encoded by the unmutated codon. A variable loop between the T arm and the anticodon arm is not shown here. Figure 2 shows a schematic example of a synthetic tRNA 1 of the invention, useful as a —1 frameshift suppressor. As shown in Fig. 2A, this embodiment of a synthetic tRNA 1 of the invention carries a five-nucleotide anticodon 19, i.e. an anticodon composed of five nucleotides being able to base-pair with complementary five nucleotides on an mRNA 15. Fig. 2B and 2C show the mutated (B) and the original (C) unmutated mRNA 15. The mutated mRNA 15 carries a mutated codon 22 (hatched) from which a nucleotide 20 has been deleted. The preceding unmutated, i.e. original nucleotide triplet codon, 21 is shown in solid black. In the embodiment shown here, the five-nucleotide anticodon 19 is composed of three nucleotides (solid black circles) being able to base pair to the complementary codon 21 preceding the mutated (truncated) codon 22, and the remaining two nucleotides (hatched circles) are able to base-pair with the mutated codon 22 (also hatched), i.e. with the nucleotide doublet left after the deletion of the nucleotide 20 from the original codon 22. The tRNA 1 may carry an amino acid 14 encoded by the codon 21 preceding the mutated codon 22 or an amino acid 14 encoded by the original unmutated codon 22. It is also possible to load the tRNA with a dipeptide being composed of the amino acids encoded by the codon 21 preceding the mutated codon and the mutated codon. The tRNA could, of course, also be designed to base-pair with two codons where the mutated codon precedes the unmutated codon. Figure 3 depicts an example of a tRNA numbered according to the conventional numbering applied to a generalized “consensus” tRNA, beginning with 1 at the 5° end and ending with 76 at the 3° end. In such a “consensus” tRNA the nucleotides of the natural anticodon triplet 25 is always at positions 34, 35 and 36, regardless of the actual number of previous nucleotides. A tRNA may, for example, contain additional nucleotides between positions 1 and 34, e.g. in the

VW

PAT 1690 LU

-22- LU101182 D loop, and in the variable loop 24 between positions 45 and 46. Additional nucleotides may be numbered with added alphabetic characters, e.g. 20a, 20b etc.

In the variable loop 24 the additional nucleotides are numbered with a preceding “e” and a following numeral depending on the position of the nucleotide in the loop.

Modified nucleotides, as e.g. listed in Table 1 above, may be present in the sequence.

PAT 1690 LU —23 — LU101182

SEQUENCE LISTING <110> Universitaet Hamburg <120> Synthetic transfer RNA with extended anticodon loop <130> PAT 1690 LU <160> 16 <170> BiSSAP 1.3.6 <210> 1 <211> 47 <212> RNA <213> Artificial Sequence <220> <223> Part A 4nt anticodon tRNA <220> <221> misc feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 16..17

PAT 1690 LU

-24- LU101182 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 35..38 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..38 <223> /note="extended (4nt) anticodon" <220> <221> misc feature <222> 43..45 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 47 <223> /note="n = A, C, G or U, or any modified base" <400> 1 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnau geNNNUN 47 <210> 2 <211> 46 <212> RNA <213> Artificial Sequence <220> <223> Part A 4nt anticodon tRNA

Ie

PAT 1690 LU —25- LU101182 <220> <221> misc_feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..38 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..38 <223> /note="extended (4nt) anticodon" <220> <221> misc_feature <222> 42..44

Y

PAT 1690 LU -26- LU101182 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 46 <223> /note="n = A, C, G or U, or any modified base" <400> 2 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnag cnnnun 46 <210> 3 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> Part B variable loop <220> <221> misc feature <222> 1..11 <223> /note="n = A, C, G or U, or any modified base" <400> 3 nnnnnnnnnn n 11 <210> 4 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> Part B variable loop <220>

PAT 1690 LU -27- LU101182 <221> misc feature <222> 1..15 <223> /note="n = A, C, G or U, or any modified base" <400> 4 nnnnnnnnnn nnnnn 15 <210> 5 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> Part C <220> <221> misc feature <222> 2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 6 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 14 <223> /note="n = A, C, G or U, or any modified base" <220> <221> mat peptide <222> 18..20 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature

PAT 1690 LU 728 — LU101182 <222> 24..25 <223> /note="n = A, C, G or U, or any modified base" <400> 5 cnuggnuucg aaunccannn cugnnacca 29 <210> 6 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> Part B variable loop <400> 6 uggggucacu ccccg 15 <210> 7 <211> 48 <212> RNA <213> Artificial Sequence <220> <223> Part A 5 nt anticodon tRNA <220> <221> misc feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base"

T

PAT 1690 LU -29- LU101182 <220> <221> misc_ feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 35..39 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..39 <223> /note="5nt anticodon" <220> <221> misc feature <222> 44..46 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc binding <222> 48 <223> /note="n = A, C, G or U, or any modified base" <400> 7 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnna ugennnun 48 : T

PAT 1690 LU — 30 — LU101182 <210> 8 <211> 47 <212> RNA <213> Artificial Sequence <220> <223> Part A 5 nt anticodon tRNA <220> <221> misc_feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..39 <223> /note="n = A, C, G or U, or any modified base" y

PAT 1690 LU —31- LU101182 <220> <221> misc feature <222> 35..39 <223> /note="5nt anticodon" <220> <221> misc feature <222> 43..45 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc binding <222> 47 <223> /note="n = A, C, G or U, or any modified base" <400> 8 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnna gonnnun 47 <210> 9 <211> 87 <212> RNA <213> Artificial Sequence <220> <223> 4nt anticodon tRNA <220> <221> misc_feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220>

PAT 1690 LU —32— 3 LU101182 <221> misc feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_ feature <222> 35..38 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..38 <223> /note="4nt anticodon" <220> <221> misc feature <222> 43..45 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 47..58 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 60 <223> /note="n = A, C, G or U, or any modified base"

Ÿ

PAT 1690 LU -33- LU101182 <220> <221> misc_feature <222> 64 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> "72 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 76..78 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 82..83 <223> /note="n = A, C, G or U, or any modified base" <400> 9 nncagnnugn ncgagnnguc uaagnnnnnn gceunnnnau gonnnunnnn nnnnnnnnen 60 uggnuucgaa unccannncu gnnacca 87 <210> 10 <211> 86 <212> RNA <213> Artificial Sequence <220> <223> 4nt anticodon tRNA <220> <221> misc feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base"

PAT 1690 LU —34- LU101182 <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" 4 <220> <221> misc_feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..38 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 35..38 <223> /note="4nt anticodon" <220> <221> misc feature <222> 42..44 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 46..57

PAT 1690 LU —35— LU101182

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 59

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 63

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 71

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 75..77

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_ feature

<222> 81..82

<223> /note="n = A, C, G or U, or any modified base"

<400> 10 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnag cnnnunnnnn nnnnnnncnu 60 ggnuucgaau nccannncug nnacca 86 | <210> 11

<211> 91

<212> RNA

<213> Artificial Sequence

Wr PE ee

PAT 1690 LU — 36 — LU101182

<220>

<223> Ant anticodon tRNA

<220>

<221> misc feature

<222> 1..2

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 6..7

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 10..11

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

| <222> 16..17

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 25..30

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature | <222> 35..38

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 35..38

<223> /note="4nt anticodon"

<220> y ee r—————

PAT 1690 LU —37— LU101182

<221> misc_feature <222> 43..45 <223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_ feature

<222> 47

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_ feature

<222> 64

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 68

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 76

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 80..82

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 86..87

<223> /note="n = A, C, G or U, or any modified base"

<400> 11 nncagnnugn ncgagnnguc uaagnnnnnn gecunnnnau gennnunugg ggucacucce 60 cgcnugqnuu cgaaunccan nncugnnacc a 91 ete

PAT 1690 LU —38— LU101182 <210> 12 <211> 90 <212> RNA <213> Artificial Sequence <220> <223> 4nt anticodon tRNA <220> <221> misc_ feature <222> 1..2 | <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature | <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..38 <223> /note="n = A, C, G or U, or any modified base" EL m tt ES

PAT 1690 LU —39— LU101182

<220>

<221> misc_feature

<222> 35..38

<223> /note="4nt anticodon"

<220>

<221> misc_feature

<222> 42..44

<223> /note="n = A, C, G or U, or any modified base" <220>

<221> misc_feature

<222> 46

<223> /note="n = A, C, G or U, or any modified base" <220>

<221> misc_feature

<222> 63

<223> /note="n = A, C, G or U, or any modified base" <220>

<221> misc feature

<222> 67

<223> /note="n = A, C, G or U, or any modified base" <220>

<221> misc feature

<222> 75

<223> /note="n = A, C, G or U, or any modified base" <220>

<221> misc_feature

<222> 79..81

<223> /note="n = A, C, G or U, or any modified base" <220>

<221> misc_feature

<222> 85..86

Ed

PAT 1690 LU — 40 — LU101182

<223> /note="n = A, C, G or U, or any modified base"

<400> 12 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnag cnnnunuggg gucacucecc 60 gonuggnuue gaaunccann ncugnnacca 90

<210> 13

<211> 88

<212> RNA

<213> Artificial Sequence

<220>

<223> 5nt anticodon tRNA

| <220>

<221> misc_feature

<222> 1..2

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 6..7

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 10..11 | <223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc_feature

<222> 16..17

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

. y er —————

PAT 1690 LU

— 41 -

LU101182

<222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..39 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..39 <223> /note="5nt anticodon" <220> <221> misc feature <222> 44..46 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 48..59 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_ feature <222> 61 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 65 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 73 <223> /note="n = A, C, G or U, or any modified base" <220>

PAT 1690 LU

—42—

LU101182

<221> misc feature <222> 77..79 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 83..84 <223> /note="n = A, C, G or U, or any modified base" <400> 13 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnna ugcnnnunnn nnnnnnnnnc 60 nuggnuucga aunccannnc ugnnacca 88 <210> 14 <211> 87 <212> RNA <213> Artificial Sequence <220> <223> 5nt anticodon tRNA <220> <221> misc feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base"

PAT 1690 LU — 43 — LU101182

<220>

<221> misc_feature

<222> 16..17

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 25..30

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 35..39

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 35..39

<223> /note="5nt anticodon"

<220>

<221> misc feature

<222> 43..45

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc feature

<222> 47..58

<223> /note="n = A, C, G or U, or any modified base"

<220>

<221> misc _ feature

<222> 60

<223> /note="n = A, C, G or U, or any modified base" | <220>

<221> misc feature

<222> 64

<223> /note="n = A, C, G or U, or any modified base"

PAT 1690 LU —44- LU101182 <220> <221> misc feature <222> 72 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 76..78 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 82..83 <223> /note="n = A, C, G or U, or any modified base" <400> 14 nncagnnugn ncgagnnguc uaagnnnnnn gcocunnnnna gennnunnnn nnnnnnnnen 60 uggnuucgaa unccannncu gnnacca 87 <210> 15 <211> 92 <212> RNA <213> Artificial Sequence <220> <223> 5nt anticodon tRNA <220> <221> misc feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 6..7

PAT 1690 LU —45—

LU101182 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_ feature <222> 35..39 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 35..39 <223> /note="5nt anticodon" <220> <221> misc feature <222> 44..46 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 48 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature

PAT 1690 LU

— 46 —

LU101182

<222> 64 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 69 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 77 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 81..83 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 87..88 <223> /note="n = A, C, G or U, or any modified base" <400> 15 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnna ugennnunug gggucacucc 60 ccgenuggnu ucgaauncca nnncugnnac ca 92 <210> 16 <211> 91 <212> RNA <213> Artificial Sequence <220> <223> 5nt anticodon tRNA <220>

PAT 1690 LU

—47—

LU101182

<221> misc feature <222> 1..2 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 6..7 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 10..11 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 16..17 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 25..30 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 35..39 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 35..39 <223> /note="5nt anticodon" <220> <221> misc feature <222> 43..45 <223> /note="n = A, C, G or U, or any modified base"

PAT 1690 LU

— 48 —

LU101182

<220> <221> misc feature <222> 47 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 64 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 68 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc _ feature <222> 76 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc feature <222> 80..82 <223> /note="n = A, C, G or U, or any modified base" <220> <221> misc_feature <222> 86..87 <223> /note="n = A, C, G or U, or any modified base" <400> 16 nncagnnugn ncgagnnguc uaagnnnnnn gccunnnnna gennnunugg ggucacucec 60 cgcnuggnuu cgaaunccan nncugnnacc a 91

Claims

PAT 1690 LU 195 LU101182 CLAIMS

1. A synthetic transfer RNA comprising an extended anticodon loop having a four- nucleotide anticodon or a five-nucleotide anticodon.

2. The synthetic transfer RNA according to claim 1, wherein the anticodon loop consists of 7 to 12, preferably 7 to 10 or 8 to 10 nucleotides, especially preferred 8 or 9 nucleotides.

3. The synthetic transfer RNA according to one of claims 1 or 2, wherein the transfer RNA is aminoacylated.

4. The synthetic transfer RNA according to claim 3, wherein the transfer RNA is aminoacylated with a dipeptide.

5. The synthetic transfer RNA according to one of the preceding claims, wherein the synthetic tRNA has a sequence identity of less than 50%, preferably of less than 49%, 48%, 47%, 46%, 45%, 44% or 43% to any naturally occurring tRNA.

6. The synthetic transfer RNA according to one of the preceding claims, wherein the synthetic transfer RNA has the same or a higher stability compared to a naturally occurring transfer RNA, whose three-nucleotide anticodon has been replaced with a four-nucleotide anticodon or a five-nucleotide anticodon, wherein the stability of a synthetic transfer RNA with a four-nucleotide anticodon is compared with a naturally occurring transfer RNA whose three- nucleotide anticodon has been replaced with a four-nucleotide anticodon, and the stability of a synthetic transfer RNA with a five-nucleotide anticodon is compared with a naturally occurring transfer RNA whose three-nucleotide anticodon has been replaced with a five-nucleotide anticodon.

7. The synthetic transfer RNA according to one of the preceding claims, wherein the synthetic tRNA has a C at position 32 and/or an A at position 37 in the anticodon loop, the numbering following tRNA numbering convention.

nt

PAT 1690 LU —>0- LU101182

8. The synthetic transfer RNA according to one of the preceding claims, wherein the extended anticodon loop is flanked by a G-C or C-G pair.

9. The synthetic transfer RNA according to one of the preceding claims, the synthetic transfer RNA having or comprising a) a sequence being composed of, in 5° to 3° direction, consecutive sequence parts A, B and C, part A having or comprising one of the sequences according to SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 07 and SEQ ID NO: 08, part B having one of the sequences according to SEQ ID NO: 03, SEQ ID NO: 04 and SEQ ID NO: 06, and part C having or comprising the sequence according SEQ ID NO: 5, 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 sequences of a) or b) above, where at least one of the nucleotides is replaced with a corresponding modified nucleotide.

10. The synthetic transfer RNA according to one of the preceding claims for use as a medicament.

11. The synthetic transfer RNA according to one of claims 1 to 9 for use as a medicament in a disease, which is at least partly caused by a frameshift mutation leading to the production of a protein being dysfunctional or non-functional compared to the wild-type protein.

12. The synthetic transfer RNA according to one of claims 1 to 9 for use as a medicament for treating Crohn disease, Tay-Sachs disease, Duchenne muscular dystrophy or neurofibromatosis type 1.