Universal inhibitor tRNA and uses thereof

Abstract

The present invention relates, at least in part, to tRNA therapeutic compositions having specificity for more than one termination codon.

Description

[0001] Related Applications

[0002] This application, filed on January 25, 2022, pursuant to 35 U.S.C. § 119(e), is entitled “ UNIVERSAL SUPPRESSOR tRNAS ”, the entire contents of which are incorporated herein by reference.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (H101570007WO00-SEQ-ZJG.xml; size: 19,216 bytes; and creation date: January 24, 2023) are incorporated herein by reference in its entirety. Background Art

[0005] DNA molecules carry genetic information in the form of nucleotide base sequences that make up DNA polymers. Only four nucleotide bases are used in DNA: adenine, guanine, cytosine and thymine. This information is transcribed into messenger RNA (mRNA) in the form of codons of three consecutive bases, and then translated by transfer RNA (tRNA) and ribosomes to form proteins. Four nucleotide bases are used in RNA: adenine, guanine, cytosine and uracil. The genetic code is the relationship between triplet codons and specific amino acids. Sixty-four possible codon triplets form the genetic code, of which three stop (also called "terminating" or "nonsense") codons provide signals to the translation machinery (cellular ribosomes) to terminate protein production at a specific codon. The other sixty-one codon triplets (also called "sense codons") correspond to one of the 20 standard amino acids. Summary of the invention

[0006] Premature termination codons (PTCs) caused by mutations such as nonsense mutations can lead to premature termination of protein translation, and thus can lead to protein dysfunction and / or disease. Such methods are provided herein, which provide tRNAs that can restore (at least partially restore) protein function. Such tRNAs can reverse or treat diseases or conditions that can be caused by one or more PTCs or are associated with one or more PTCs.

[0007] The present invention relates (at least in part) to tRNA-mediated therapeutic agents targeting nonsense mutations, wherein the replaced amino acid may or may not be a natural amino acid that has been encoded or would be encoded in the absence of a nonsense mutation (e.g., an amino acid of a wild-type protein). That is, tRNA-mediated therapeutic agents can provide "read-through" of PTCs and / or at least partial (if not complete) functional restoration of gene products. Therefore, the resulting gene product may or may not have amino acid sequence identity with the original, wild-type or non-disease-related gene product. It is suggested that a preferred embodiment of the present invention is a tRNA composition that targets more than one possible premature stop codon and replaces it with an amino acid (e.g., serine). In one embodiment of any one of the compositions or methods provided herein, the tRNA composition targets all three possible stop codons and replaces them with an amino acid (e.g., serine).

[0008] Although there are only 3 stop codons, there are 23 different nucleotide substitutions that can lead to stop codons. For example, the UGA stop codon differs by one nucleotide from the following codons: arginine (CGA, AGA), glycine (GGA), leucine (UUA), serine (UCA), cysteine ​​(UGU, UGC) and tryptophan (UGG). The most common nonsense mutations are CGA (arginine) -> UGA and CAG (glutamine) -> UGA. The disease state can be caused by any protein truncation induced by premature stop codons. The disease-free or disease-mitigated state can be induced by reading through PTC with suppressor tRNA, which inserts amino acids at the position wrongly encoded by PTC.

[0009] Although the specific rare genetic diseases caused by PTC may have known specific nonsense mutations and therefore have specific restorative therapeutic suppressor tRNAs, there are also other disease states, including cancer, for which specific PTCs or groups of PTCs may be unknown. In these cases, the treatment of PTCs carried out by suppressor tRNAs that provide read-through or functionally similar amino acid encodings may have a therapeutic effect. It is worth noting that serine, as a medium-sized hydrophilic amino acid, can provide structural values ​​similar to, for example, arginine, leucine, glutamine, cysteine ​​or glycine in many cases. It is also worth noting that some aminoacyl tRNA synthetases do not engage with the anticodon region of tRNA substantially, while others engage with it. The tRNAs of alanine, serine and leucine are mainly recognized by the synthetase of the tRNA through the non-anticodon elements of tRNA.

[0010] Thus, use of tRNA compositions in which at least two, if not all three, stop codons are targeted to replace an amino acid (eg, serine) can be used to treat disease without requiring knowledge of the specific PTC that causes or contributes to the disease.

[0011] Some aspects of the present disclosure provide compositions comprising more than one tRNA, each of the tRNAs having an anticodon specific for a stop codon, wherein the composition can target at least two stop codons, and wherein each of the tRNAs can provide an amino acid for replacement of its specific stop codon.

[0012] In some embodiments, the composition comprises more than two tRNAs, each of which has an anticodon specific for a stop codon, and wherein the composition can target all three stop codons. In some embodiments, each tRNA can provide the same amino acid for replacing its specific stop codon. In some embodiments, the amino acid for replacing the stop codon is serine. In some embodiments, the amino acid for replacing the stop codon is arginine. In some embodiments, the amino acid for replacing the stop codon is glutamine. In some embodiments, the amino acid for replacing the stop codon is glutamic acid.

[0013] In some embodiments, tRNA can provide two different amino acids for replacing at least two of its specific stop codons. In some embodiments, the two amino acids for replacing the stop codon are selected from arginine, glutamine and glutamic acid. In some embodiments, the two amino acids for replacing the stop codon are arginine and glutamine.

[0014] In some embodiments, the tRNA can provide three different amino acids for replacing three of its specific stop codons. In some embodiments, the amino acids for replacing the stop codon are arginine, glutamine and glutamic acid.

[0015] In some embodiments, the stop codon is selected from TGA, TAA, and TAG.

[0016] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0017] Also provided herein are oligonucleotides encoding tRNA described herein. In some embodiments, the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 1 to 14. Also provided are groups of such oligonucleotides, which together encode the tRNA described herein. In some embodiments, each oligonucleotide in the group comprises the nucleotide sequence of any one of SEQ ID NO: 1 to 14. In some embodiments, the oligonucleotide is DNA. In some embodiments, the oligonucleotide is RNA.

[0018] Also provided herein is an expression cassette, which comprises a promoter and a nucleic acid encoding a tRNA, an oligonucleotide or a group of oligonucleotides as described herein. In some embodiments, a promoter is operably connected to a group of such nucleic acid, an oligonucleotide or an oligonucleotide. In some embodiments, the group of expression cassettes co-encodes all of tRNA as described herein. In some embodiments, one or more expression cassettes also comprise a terminator. In some embodiments, each expression cassette also comprises a terminator.

[0019] In some aspects, a vector comprising an oligonucleotide or expression cassette described herein is provided. In some embodiments, the vector is a viral vector or a plasmid vector.

[0020] Also provided herein are compositions comprising tRNA, oligonucleotides, expression cassettes, or vectors as described herein, and a pharmaceutically acceptable carrier. In some embodiments, the composition is contained in a particle, such as a nanoparticle. In some embodiments, the particle is a liposome or lipid nanoparticle. In some embodiments, the composition is contained in an exosome.

[0021] Also provided herein are cells comprising a tRNA, oligonucleotide, expression cassette, vector or composition described herein.

[0022] Other aspects of the present disclosure provide methods of reading through one or more stop codons, comprising delivering to a cell a tRNA, oligonucleotide, expression cassette, vector or composition described herein in an amount effective to read through one or more stop codons.

[0023] Other aspects of the present disclosure provide methods of restoring at least partial protein function, comprising delivering to a cell a tRNA, oligonucleotide, expression cassette, vector or composition described herein in an amount effective to restore at least partial protein function.

[0024] Other aspects of the present disclosure provide methods of reducing or inhibiting cell survival or proliferation, comprising delivering to a cell a tRNA, oligonucleotide, expression cassette, vector or composition described herein in an amount effective to reduce or inhibit cell survival or proliferation.

[0025] In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.

[0026] Other aspects of the present disclosure provide methods for treating a subject, comprising delivering tRNA, oligonucleotides, expression cassettes, vectors or compositions described herein to cells in an amount effective to treat the subject. In some embodiments, the subject suffers from a disease caused by one or more nonsense mutations or one or more premature termination codons or due to one or more nonsense mutations or one or more premature termination codons. In some embodiments, the subject suffers from a genetic disease or illness. In some embodiments, the subject suffers from a hyperproliferative disease or illness. In some embodiments, the hyperproliferative disease or illness is cancer. In some embodiments, administration is systemic or local administration. In some embodiments, the subject is a human.

[0027] Each limitation of the present invention can cover multiple embodiments of the present invention. Therefore, it is expected that each limitation of the present invention involving any element or combination of elements can be included in each aspect of the present invention. The present invention is not limited to the details of the arrangement and construction of the components set forth in the following description or illustrated in the accompanying drawings in terms of its application. The present invention can have other embodiments and can be practiced or implemented in a variety of ways. In addition, the wording and terminology used in this disclosure are for the purpose of description and should not be considered as restrictive. The "include", "comprising", or "having", "containing", "involving" and its variations used in this disclosure are intended to cover the items listed thereafter and their equivalents and additional items. Unless the content is clearly indicated otherwise, as used in this specification and the appended claims, the nouns not defined by quantifiers mean "one / kind or more / kind".

[0028] In some embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of. The phrase "consisting essentially of" is used herein to claim the specified integers or steps and those that do not materially affect the characteristics or functions of the claimed invention. As used herein, the term "consisting of" is used to indicate that only the enumerated integers (e.g., features, elements, properties, attributes, methods / processing steps, or limitations) or groups of integers (e.g., features, elements, properties, attributes, methods / processing steps, or limitations) are present. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A table of the genetic code is provided.

[0030] Figure 2The general four-arm structure of a tRNA is shown, comprising a T arm, a D arm, an anticodon arm and an acceptor stem (or arm) (SEQ ID NO: 15). These regions may also be referred to as 'loops' throughout this application.

[0031] Figure 3 A map of a plasmid encoding a single tRNA is provided.

[0032] Figure 4 Maps of plasmids encoding both tRNAs are provided.

[0033] Figure 5 Maps of plasmids encoding the three tRNAs are provided.

[0034] Figure 6 A map of a plasmid encoding three tRNAs (anti-codon edited (ACE) or native tRNA) and additionally encoding a transfection reporter (superfolded GFP) is provided.

[0035] Figure 7 A graph is provided showing that a plasmid encoding a universal suppressor tRNA transfected into HEK293T PTC NLuc cells induced expression of Nanoluciferase, whereas a control plasmid lacking the suppressor tRNA coding sequence (hCR000.p014) did not.

[0036] Figure 8 Provided is an image of a Western blot gel showing that the expression of full-length p53 is increased in HT1299 cancer cells containing an integrated CMV-promoted p53 gene with a premature TGA stop codon, which results in a truncated p53 protein without a universal suppressor tRNA. A control plasmid (hCR000.p014) lacking a suppressor tRNA coding sequence did not induce increased expression of full-length p53.

[0037] Fig.9A and 9B Provided is a graph showing the expression level of full-length p53 in HT1299 cancer cells containing an integrated CMV-promoted p53 gene with a premature TGA stop codon and treated with a plasmid encoding a universal tRNA ( Fig.9A ) or truncated p53 expression levels ( Fig. 9B) relative to the corresponding expression levels in untreated cells. Cells treated with a plasmid encoding a universal suppressor tRNA showed enhanced expression of full-length p53 and reduced expression of truncated p53, whereas cells treated with a control plasmid lacking the suppressor tRNA coding sequence (hCR000.p014) showed full-length p53 and truncated p53 levels comparable to untreated cells. DETAILED DESCRIPTION

[0038] Provided herein is a therapeutic agent for treatment (e.g., for the treatment of cancer), wherein the therapeutic agent provides more than one nonsense suppressor tRNA, and the tRNA is specific to more than one (if not all) stop codons. These nonsense suppressor tRNAs can provide one or more amino acids, such as serine, arginine, glutamine and / or glutamic acid, and read through PTC. Nonsense suppressor tRNA may not provide complete and / or accurate recovery of natural or wild-type proteins, but may still have a therapeutic effect even if the function is partially restored or similar. In some embodiments, tRNA can target PTC and replace with serine, which is effectively replaced in many cases due to the nature of amino acids and synthetase recognition. In some embodiments, tRNA can target PTC and replace with arginine. In some embodiments, tRNA can target PTC and replace with glutamine. In some embodiments, tRNA can target PTC and replace with glutamic acid. In some embodiments, tRNA can target PTC and replace with more than one (e.g., two, three or more) amino acids. In some embodiments, more than one (eg, two, three, or more) amino acids are selected from serine, arginine, glutamine, and glutamate.

[0039] Transfer RNA (tRNA) is a decoder of the DNA and RNA "blueprints". DNA transcription produces messenger RNA (mRNA) that encodes the primary amino acid structure, which can be modified after transcription and ultimately become a folded or unfolded protein after interacting with the ribosome and tRNA. Once the mRNA binds to the ribosome, the tRNA delivers the amino acid to the ribosome and forms an amino acid chain based on the mRNA code ( Figure 1 ).

[0040] tRNA has a general four-arm structure, which includes a T arm, a D arm, an anticodon arm, and an acceptor stem or arm ( Figure 2 ). The T arm is composed of a "T stem" and a "TΨC loop". Any tRNA provided herein may comprise this four-arm structure. In some embodiments, the tRNA is about 100 nucleotides in length and can be easily introduced into cells.

[0041] RNA molecules can be modified or engineered to enable them to systematically "recode" the genetic code. It has been discovered that tRNAs can be altered by molecular editing of the anticodon sequence within the tRNA. This approach allows reprogramming of stop codons for undesirable amino acid substitutions.

[0042] Provided herein are compositions and methods related to the technology for introducing one or more suppressor tRNAs into diseased cells or tissues that can read through one or more PTCs. In particular, the one or more suppressor tRNAs can act extensively on a variety of premature termination codons (PTCs). The tRNA compositions provided herein can be modified to target any one or more undesirable termination codons, such as any one or more termination codons provided herein. In some embodiments, replaceable one or more amino acids are any amino acids applicable to the present disclosure. Without wishing to be bound by any theory, serine, as a medium-sized hydrophilic amino acid, is expected to provide structural values ​​similar to other amino acids (such as arginine, leucine, glutamine, cysteine ​​or glycine). Therefore, in many cases, serine is expected to become an effective amino acid replacement for common PTCs. In some embodiments, the one or more tRNAs can be used to suppress miscoded PTCs during polypeptide or protein production and replace them with amino acids (such as serine, arginine, glutamine or glutamic acid). In some embodiments, the one or more tRNAs can be used to suppress more than one type of mis-encoded PTC during polypeptide or protein production and replace them all with an amino acid (e.g., serine, arginine, glutamine, or glutamate) or with a different amino acid (e.g., an amino acid selected from serine, arginine, glutamine, glutamate, or a combination thereof).

[0043] As used herein, "early termination codon" or PTC refers to a single nucleotide mutation that converts a canonical triplet nucleotide codon into one of three termination codons (e.g., TAG, TGA, or TAA). Although only 3 termination codons are currently known in the art, 23 different nucleotide replacements can result in termination codons. For example, the UGA termination codon can be derived from codons for: arginine (CGA, AGA), glycine (GGA), leucine (UUA), serine (UCA), cysteine ​​(UGU, UGC), and tryptophan (UGG). The presence of a stop codon (stop codon or termination codon) indicates that protein synthesis is terminated (i.e., the translation process of the current protein is terminated). PTC can result in protein truncation, thereby causing abnormalities and diseases.

[0044] In some embodiments, the disclosure provides compositions comprising one or more tRNAs as described herein. In some embodiments, the compositions described herein comprise more than one tRNA, each of the tRNAs having an anticodon specific for a stop codon, wherein the composition can target at least two stop codons (e.g., 2 or 3 stop codons selected from TAA, TGA, and TAG), and wherein each tRNA can provide an amino acid (e.g., the same amino acid or a plurality of different amino acids) for replacing its specific stop codon.

[0045] In some embodiments, the composition comprises more than two tRNAs, each of which has an anticodon specific for a stop codon, and wherein the composition can target all three stop codons (ie, all of TAA, TGA, and TAG).

[0046] In some embodiments, the compositions described herein comprise more than one tRNA, wherein each of the tRNAs can provide the same amino acid for replacing its specific stop codon. In some embodiments, the amino acid for replacing the stop codon is serine. In some embodiments, the amino acid for replacing the stop codon is arginine. In some embodiments, the amino acid for replacing the stop codon is glutamine. In some embodiments, the amino acid for replacing the stop codon is glutamic acid.

[0047] In some embodiments, the compositions described herein comprise more than one tRNA, wherein the tRNA can provide two different amino acids for replacing at least two of its specific stop codons. In some embodiments, the two amino acids for replacing the stop codon are selected from arginine, glutamine and glutamic acid. In some embodiments, the two amino acids for replacing the stop codon are arginine and glutamine.

[0048] In some embodiments, the compositions described herein comprise more than one tRNA, wherein the tRNA can provide three different amino acids for replacing three of its specific stop codons. In some embodiments, the amino acids for replacing the stop codon are arginine, glutamine and glutamic acid.

[0049] Also provided herein are oligonucleotides or groups of oligonucleotides encoding any one or more tRNAs as provided herein. In some embodiments, the oligonucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 14. In some embodiments, the compositions described herein comprise an oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 1 to 14; or comprise a tRNA encoded by the oligonucleotide.

[0050] In some embodiments, the disclosure provides such a composition, which comprises one or more nucleic acids or expression constructs encoding one or more tRNA as described herein. In some embodiments, the composition also comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier can be any carrier known in the art and applicable to the disclosure. In some embodiments, the disclosure provides such a cell, which comprises one or more tRNA as described herein or one or more nucleic acids encoded by them. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0051] The present disclosure provides methods for treating one or more PTCs or achieving read-through of one or more PTCs by using one or more suppressor tRNAs that can provide functionally similar amino acid coding (e.g., serine, arginine, glutamine, or glutamate). Inserting a functionally similar amino acid into an amino acid codon that is incorrectly encoded by a PTC can restore partial, if not complete, function of the gene product. In some embodiments, the restored gene product may be identical or different from a wild-type or PTC-free gene product.

[0052] The present disclosure provides methods for treating an object suffering from a disease or condition. In some embodiments, the method comprises administering to the object one or more tRNAs or compositions as disclosed herein. In some embodiments, the disease or condition may include a genetic disease or condition or a hyperproliferative disease or condition. In some embodiments, the disease or condition may be cancer. In some embodiments, the disease or condition may be any disease or condition that may benefit from a tRNA-based treatment as provided herein.

[0053] In some embodiments, protein expression or function is at least partially restored compared to a control. An example of a "control" as used herein can be a tRNA that is not an inhibitor tRNA as provided herein, or that is not expected to read through the PTC. As another example, the control can be the same tRNA, but it cannot replace the target amino acid (e.g., serine) in a position that is miscoded by the PTC. The improvement, recovery, or comparison of any other features of a disease or condition can be evaluated in vitro, as known to those of ordinary skill in the art, or can be evaluated in vivo using a test subject.

[0054] In some embodiments, a disease or condition is improved (e.g., reduced in severity) by at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, including all values ​​therebetween.

[0055] As used herein, a "genetic disease or condition" is a disease or condition caused by a mutation that is inherited or arises within the genetic code of a subject, or that predisposes a subject to the disease or condition. Any of the compositions or methods provided herein can be used to treat or prevent a genetic disease or condition.

[0056] As used herein, "hyperproliferative disease or condition" refers to any disease or condition in which the cell proliferation rate is abnormally high by rapid division, excessive proliferation, etc. Certain embodiments of the present disclosure provide methods for treating a disease or condition, such as a hyperproliferative disease or condition, in a subject (e.g., a mammal). In certain embodiments, the mammal is a human. Certain embodiments of the present disclosure provide the use of one or more tRNA or compositions as described herein for preparing a drug that can be used for treating a disease or condition (e.g., a hyperproliferative disease or condition) in a subject (e.g., a mammal, such as a human). In certain embodiments, the treatment has the potential use of treating / managing a disease or condition (e.g., a hyperproliferative disease or condition, including tumors, cancers, and neoplastic tissues, and non-neoplastic or non-malignant hyperproliferative conditions). In certain embodiments, a hyperproliferative disease or condition is cancer.

[0057] tRNA and the nucleotide sequence encoding tRNA can be produced by synthesis. In addition, the nucleotide sequence encoding hundreds of human tRNAs is known, and those skilled in the art can generally obtain it through sources such as GenBank. In one embodiment of any one of the compositions or methods provided herein, tRNA is selected from those provided in PCT / US2018 / 059065, and the disclosure of tRNA and its sequence is incorporated herein by reference in its entirety. In one embodiment of any one of the compositions or methods provided herein, tRNA is selected from those replaceable with serine provided in PCT / US2018 / 059065. The structure of tRNA is usually highly conservative, and tRNA can function across species. Therefore, the tRNA sequence of bacteria or other eukaryotic organisms is also a potential source of tRNA provided herein. Determining whether a specific tRNA or tRNA combination functions as desired (e.g., in a desired mammalian cell) can be as described herein or confirmed by other experiments that are obvious to those of ordinary skill in the art thanks to the teachings provided herein.

[0058] The tRNA may be in any form suitable for being delivered to a target cell or object in vitro or in vivo, such as a recombinant plasmid comprising a heterologous nucleic acid sequence. The heterologous nucleic acid sequence encodes a target gene product (e.g., tRNA) for any of the uses provided herein, including disease treatment, and may optionally be in the form of an expression cassette. The term "recombinant" refers to a polynucleotide that does not exist in nature or is connected to another polynucleotide in an arrangement not found in nature. The term "heterologous" as used herein refers to a nucleic acid sequence obtained from or derived from an entity that is genetically different from the rest of the entity being compared.

[0059] The present disclosure provides a method for delivering one or more tRNA or compositions provided herein to an object or a cell of an object. As described herein, tRNA or compositions can be delivered to cells in vivo or in vitro. Administration to cells can be accomplished by any means, including simply contacting cells. Contact with cells can be sustained for any desired length of time. Cells can include any desired cell in humans and other large (non-rodent) mammals (e.g., primates, horses, sheep, goats, pigs, and dogs). Any of the objects provided herein can be humans or other mammals. The term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cattle, pigs, and sheep.

[0060] In some embodiments, administration can be systemic. In some embodiments, administration can be local. In some embodiments, administration can be delivered directly to selected organs, oral, inhaled, intraocular, intravenous (including facial vein injection) and retro-orbital injection, intraventricular (intracerebroventricular, ICV), intracerebellomedullary cisterna (intracisterna magna, ICM) injection, intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral and other parenteral administration routes. If desired, administration routes can be combined. In some embodiments, tRNA as disclosed herein can be administered by any route applicable to the present disclosure.

[0061] Suitable methods for delivery and introduction into a subject are also provided or otherwise known in the art. In one embodiment, the pharmaceutical composition will include enough genetic material to produce a therapeutically effective amount of the target tRNA. The tRNA or genetic material encoding the tRNA can be delivered in an effective amount and delivered to a cell, such as using an endogenous tRNA synthetase. If the tRNA synthetase is present in a cell into which the tRNA is introduced according to the present invention, the tRNA synthetase is considered to be "endogenous" to the cell. It will be apparent to one of ordinary skill in the art that whether the tRNA synthetase is naturally present in a cell of the relevant type, or the particular cell in question has been modified or otherwise artificially manipulated to contain or express the tRNA synthetase, for these purposes, the tRNA synthetase can be considered to be endogenous.

[0062] In some embodiments, tRNA can be formulated in a pharmaceutical composition. The pharmaceutical composition may also contain a pharmaceutically acceptable excipient. Such excipients include any agent that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts such as inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc. may be included therein; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may be present in such carriers. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0063] It is obvious to those skilled in the art in view of the teachings of this specification that the effective amount of tRNA provided can be determined empirically. Administration can be achieved continuously or intermittently in one dose throughout the treatment process. The method of determining the most effective administration mode and dosage can vary depending on the therapeutic component, target cell and subject, etc. The treating physician can select the dosage level and mode for single and multiple administrations.

[0064] The present invention can use carriers, including water, saline, artificial CSF or other known substances. In order to prepare the preparation, the purified composition can be isolated. The composition can then be adjusted to an appropriate concentration and packaged for use.

[0065] The term "treatment" and its variations as used herein refer to both therapeutic treatments and measures that can provide some benefit or alleviate symptoms to the subject, wherein the purpose is to prevent or slow down (reduce) undesirable physiological changes or conditions, such as the growth, development or spread of cancer. For purposes of the present invention, favorable or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonged survival compared to the expected survival without treatment. Those requiring treatment include those already suffering from illness, disease or disorder.

[0066] The phrase "therapeutically effective amount" means an amount of one or more compounds or compositions of the invention that (i) treats a specific disease, condition, or disorder described herein, (ii) attenuates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder described herein, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that a drug can prevent growth and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and / or determining response rate (RR).

[0067] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells. Some examples of cancer include, but are not limited to, carcinomas, malignancies, etc.

[0068] The tRNA or compositions described herein can be administered so as to cause at least one symptom associated with a disease or condition, such as a genetic disease or condition or a hyperproliferative disease or condition (e.g., cancer) to be alleviated. The amount administered will vary according to a variety of factors, including but not limited to the selected components, the specific disease, the weight, physical condition, and age of the mammal. Such factors can be readily determined by a clinician using animal models or other test systems known in the art.

[0069] Administration of tRNA or compositions according to the invention can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic, and other factors known to the skilled person. Administration can be substantially continuous over a preselected period of time, or can be carried out in a series of spaced doses.

[0070] One or more suitable unit dosage forms having one or more tRNAs or compositions of the present invention can be formulated and can be administered by a variety of routes. When preparing the medicament of the present invention for administration, the medicament of the present invention can be combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical preparation or unit dosage form. The total active ingredients in such a preparation account for 0.1% to 99.9% of the preparation by weight. "Pharmaceutically acceptable" refers to carriers, diluents, excipients and / or salts that are compatible with the other ingredients of the preparation and harmless to the recipient thereof. Pharmaceutical preparations can be prepared using well-known and readily available ingredients by procedures known in the art. The medicament of the present invention can also be formulated into solutions suitable for administration. The pharmaceutical preparations of the medicament of the present invention can also take the form of an aqueous solution or an anhydrous solution or dispersion, or alternatively an emulsion or suspension.

[0071] Therefore, the medicament can be formulated for administration and can be present in an ampoule containing an added preservative, a prefilled syringe, a small volume infusion container or a multi-dose container in a unit dose form. The active ingredient can be in such a form as a suspension, solution or emulsion in an oily or aqueous vehicle, and can include a formulation (formulatoryagent), such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, before use, the active ingredient can be in a suitable carrier, such as sterile, pyrogen-free water. It should be understood that the unit content of the active ingredient contained in the individual dose of each dosage form does not need to constitute an effective amount for treating a specific indication or disease, because the required effective amount can be achieved by administering multiple dosage units. In addition, the effective amount can be achieved using a dose less than that in the dosage form, either alone or in a series of administrations.

[0072] The pharmaceutical preparation of the present invention may comprise a pharmaceutically acceptable carrier, a diluent, a solubilizing agent or an emulsifying agent and a salt of a type well known in the art as an optional ingredient. Some specific non-limiting examples of carriers and / or diluents that can be used in the pharmaceutical preparation of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions of pH 7.0 to 8.0, and water.

[0073] Any composition provided herein can be placed in contact with cells, applied to cells or introduced into cells using genetic transfer methods (e.g., transfection). Therefore, any composition provided herein can be included in a gene delivery vehicle or included with a gene delivery vehicle. The gene delivery vehicle can be any delivery vehicle known in the art, and can include naked nucleic acids promoted by receptor and / or lipid-mediated transfection.

[0074] Compositions provided herein can be contacted with cells, or delivered to or applied to objects in particles (e.g., nanoparticles). Particles, such as nanoparticles, can be but are not limited to lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., such nanoparticles whose major materials constituting their structure are lipids) and / or particles with nanomaterial combinations. Particles can be a variety of different shapes, including but not limited to spherical, cubic, pyramidal, rectangular, cylindrical, annular, etc.

[0075] In some embodiments, the particle, such as a nanoparticle, may comprise one or more lipids. In some embodiments, the particle, such as a nanoparticle, may comprise a liposome. In some embodiments, the particle, such as a nanoparticle, may comprise a lipid bilayer. In some embodiments, the particle, such as a nanoparticle, may comprise a lipid monolayer. In some embodiments, the particle, such as a nanoparticle, may comprise a micelle.

[0076] The tRNA or composition provided herein can be contacted with a cell, or delivered to or applied to a subject using an extracellular vesicle or exosome. Generally speaking, an exosome is an extracellular vesicle (EV) (30 to 150 nm in diameter) of nanometer size that can be formed and released by many mammalian cells. EV or exosomes can be loaded with a target agent, for example, by pretreating cells with an agent, and then separating the loaded EV or exosomes to load.

[0077] EVs or exosomes can be derived from human embryonic kidney cells, bone marrow stem cells, immature dendritic cells, erythrocytes, and from milk. EVs or exosomes can be isolated and purified using a variety of different techniques. Such methods include, but are not limited to, ultracentrifugation, ultrafiltration, size exclusion chromatography (SEC), precipitation with polymers, and separation by affinity-based methods, such as immunomagnetic-based separation.

[0078] Electroporation can also be used.

[0079] In one embodiment of any one of compositions or methods provided herein, the nucleic acid sequence encoding tRNA is in a closed end form, for example, in the form of a plasmid, a nanoplasmid or a mini-ring. In one embodiment of any one of compositions or methods provided herein, the nucleic acid sequence encoding tRNA is in the form of a mini-ring or a microthread.

[0080] As used herein, the term "minicircle" refers to a small circular DNA fragment that is largely free of or completely free of non-essential prokaryotic elements. Minicircles include circular forms of DNA without prokaryotic elements and / or prokaryotic elements have been removed. Minicircles can be derived from parent plasmids from which bacterial DNA sequences have been excised. Minicircles can be in the form of any suitable recombinant plasmid containing heterologous nucleic acid sequences to be delivered to target cells in vitro or in vivo. The preparation of minicircles has been described in the art (e.g., in Nehlsen et al., Gene Ther. Mol. Biol. 10: 233-244, 2006; and Kay et al., Nature Biotechnology. 28: 1287-1289, 2010). The preparation can, for example, follow a two-step procedure: (i) producing a 'parent plasmid' (a bacterial plasmid with a eukaryotic insert); and (ii) inducing a site-specific recombinase at the end of the process. After these steps, the prokaryotic vector portion can be excised by the recombinase target sequence and recovered by capillary gel electrophoresis.

[0081] As a non-limiting example, minicircles can be produced as follows. An expression cassette containing a polynucleotide coding sequence and regulatory elements for its expression is flanked by a connection site for a recombinase. The sequence encoding the recombinase is located outside the expression cassette and contains elements for inducible expression (e.g., such as an inducible promoter). After inducing the expression of the recombinase, the vector DNA is recombined to produce two unique circular DNA molecules. One circular DNA molecule is relatively small and forms a minicircle containing a polynucleotide expression cassette; the minicircle DNA vector does not contain any bacterial DNA sequence. The second circular DNA sequence contains the remaining vector sequences, including bacterial sequences and sequences encoding recombinases. The minicircle DNA containing the polynucleotide sequence can then be separated and purified separately. In some embodiments, the minicircle DNA vector can be produced using plasmids similar to pBAD.φ.C31.hFIX and pBAD.φ.C31.RHB. See, for example, Chen et al. (2003) Mol.Ther.8:495-500, or as provided in other aspects herein.

[0082] Some examples of recombinases that can be used to generate minicircles include, but are not limited to, Streptomyces phage φ31 integrase, Cre recombinase, and λ integrase / DNA topoisomerase IV complex. Each of these recombinases catalyzes recombination between unique sites. For example, φ31 integrase catalyzes recombination between corresponding attP and attB sites, Cre recombinase catalyzes recombination between loxP sites, and λ integrase / DNA topoisomerase IV complex catalyzes recombination between phage λ attP and attB sites.

[0083] Published U.S. Application 20170342424 also describes a system utilizing a parent plasmid that is exposed to an enzyme that causes recombination at a recombination site, thereby forming (i) a minicircle comprising a polynucleotide sequence and (ii) a miniplasmid comprising the remainder of the parent plasmid. One recombination site is modified at the 5' end so that it reacts less efficiently with the enzyme than the wild-type site, and another recombination site is modified at the 3' end so that it reacts less efficiently with the enzyme than the wild-type site, and another recombination site is modified at the 3' end so that it reacts less efficiently with the enzyme than the wild-type site, and both modified sites are located in the minicircle after recombination.

[0084] Using the smallest possible excision site, removal of prokaryotic sequences should ideally be efficient while generating supercoiled DNA minicircles consisting only of gene expression elements, preferably mammalian control regions, where appropriate. Some techniques for minicircle generation use bacterial phage lambda (λ) integrase-mediated recombination to generate minicircle DNA. See, e.g., Darquet, et al. 1997 Gene Ther 4(12):1341-9; Darquet et al. 1999 Gene Ther 6(2):209-18; and Kreiss, et al. 1998 Appl Micbiol Biotechnol 49(5):560-7).

[0085] Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). For example, the MC-EasyTM (Cat#MN920A-1, SBI System Biosciences) minicircle DNA production kit can be used to obtain high-quality minicircle DNA. Information about minicircle DNA is provided in: Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 21 8, 1526-1535; and Hou et al., Molecular Therapy—Methods & Clinical Development, Article Number: 14062 (2015) doi: 10.1038 / mtm.2014.62. More information about minicircles is provided in Chen ZY, He CY, Ehrhardt A, Kay M A. Mol Ther. 2003 Sep;8(3):495-500, and minicircle DNA vectors achieve sustained expression as reflected by active chromatin and transcription levels. Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay M A. Mol Ther. 2013 Jan;21(1):131.

[0086] In some embodiments, the closed end form is a supercoiled spiral. DNA supercoil refers to the amount of distortion in a specific DNA chain. Supercoiled DNA can be positive supercoiled DNA or negative supercoiled DNA. "Supercoiled DNA" as used herein refers to such a DNA molecule or a fragment of a DNA molecule, wherein one or two DNA chains contain an increased distortion compared to the distortion amount of the DNA in a reference state called "relaxed B-form". In the "relaxed" double helical segment of DNA, two chains twist around the helical axis once in a sequence of every 10.4 to 10.5 base pairs. A given DNA chain can be "positive supercoiled" or "negative supercoiled" (i.e., more or less tightly wound). Supercoiled DNA produces torsional strain in a DNA chain. The amount of supercoiled chain affects many biological processes, such as compacting DNA and regulating access to the genetic code (this strongly affects DNA metabolism and possible gene expression). Certain enzymes (e.g., topoisomerases) can increase or decrease the amount of distortion (e.g., supercoiling) in a DNA chain to facilitate functions such as DNA replication and transcription. If a DNA segment under torsional strain is closed into a ring by connecting its two ends and then allowed to move freely, a supercoiled structure can be formed. Some examples of supercoiled structures of circular DNA molecules include, but are not limited to, figure-of-eight structures, structures of twisted wires, or ring structures.

[0087] In one embodiment of any one of the compositions or methods provided herein, the oligonucleotides described herein also include a promoter. Such oligonucleotides may be included in a vector. The promoter characteristically has a specific nucleotide sequence required for initiating transcription. In some embodiments, the oligonucleotides provided herein include a nucleic acid sequence that can instruct a specific nucleotide sequence to be expressed in a suitable cell, which may include a promoter operably connected to the target nucleotide sequence, and the nucleotide sequence may also be operably connected to a termination signal (or terminator). The oligonucleotides provided herein may be a recombinant form that can be used for heterologous expression. In certain embodiments, the promoter is an adjustable promoter. In certain embodiments, the promoter is a constitutive promoter. The promoter driving the expression of the sequence encoding the tRNA to be delivered may be any desired promoter selected according to known considerations (e.g., the expression level of the nucleic acid connected to the promoter function and the cell type of the vector to be used). The promoter may be an exogenous or endogenous promoter.

[0088] In one embodiment of any one of the compositions or methods provided herein, the size of the promoter can be (35 to 105 bp). The promoter can be any known promoter, including a natural tRNA leader sequence, the size of which can be about 50 to 60 bp. In one embodiment of any one of the compositions or methods provided herein, the promoter can be a shortened sequence (reduced-sequence) or a reconfigured promoter. Exemplary promoter sequences that can be included in any one of the oligonucleotides or vectors provided herein include, but are not limited to, any one of the sequences provided herein or otherwise known in the art.

[0089] Optionally, the oligonucleotides or vectors provided herein may also contain additional sequences (e.g., 3' tRNA tail or trailer sequence). Such sequences may be 2 to 20 bp in length. They may include native sequences or engineered variants having 3 to 10 consecutive "T" residues.

[0090] The present disclosure also provides such cells, which contain tRNA, oligonucleotides, vectors or other compositions of the present invention described herein. The cells can be mammalian, such as human. According to one aspect, a cell expression system is provided. The expression system comprises cells and oligonucleotides or groups thereof, or vectors, as provided herein. Expression cassettes include, but are not limited to, plasmids, viral vectors and other carriers for delivering heterologous genetic material to cells. The cell expression system can be formed in vivo.

[0091] In some embodiments of any of the tRNAs or other compositions of the invention provided herein, the tRNA or composition is a tRNA or composition that does not occur in nature.

[0092] The present invention is further illustrated by the following examples, which should in no way be construed as further limiting. The entire contents of all references cited throughout this application (including literature references, granted patents, published patent applications, and co-pending patent applications) are expressly incorporated by reference herein.

[0093] Example

[0094] Example 1. Design and generation of universal inhibitors

[0095] Anticodon-edited (ACE) tRNA inhibitor sequences were designed by replacing the anticodons of exemplary natural human tRNA sequences with engineered anticodons that match the TGA, TAG, and TAA stop codons. DNA sequences of these ACE tRNAs were synthesized and incorporated into plasmid constructs such as Figures 3 to 6 Described in . Figure 3Provides the design of plasmids carrying single tRNA sequences. Figure 4 For plasmids carrying two tRNA sequences, Figure 5 For plasmids carrying three tRNA sequences, and Figure 6 For plasmids carrying three tRNA sequences and also transfection reporter sequences. All plasmids are based on the pUC57 backbone, utilize the same DNA polymerase III promoter, have the same tRNA tailing / termination sequence, and contain the same DTS sequence. In any given design, the plasmid size varies slightly depending on the length of the encoded tRNA sequence. Table 1 provides a list of ACE tRNAs uniquely encoded by synthetic plasmids.

[0096] Table 1. Synthetic universal inhibitor plasmids and related control plasmids

[0097]

[0098] *Synthesized as a natural non-ACE to be used as a control sequence for plasmids carrying ACE-tRNA.

[0099] **No tRNA control plasmid hCR000.p014 is a pUC backbone plasmid in which the pol III promoter, tRNA, tRNA tail and DTS are absent and replaced by random non-coding sequences.

[0100] ^A tRNA composition classified as "universal" means that the composition comprises a mixture of suppressor tRNAs that can target all three stop codons TAG, TGA and TAA.

[0101] ^^A tRNA composition classified as "multiple" means that the composition comprises a mixture of suppressor tRNAs that can donate at least two different amino acids at different stop codons.

[0102] Table 2. tRNA sequences corresponding to Table 1

[0103]

[0104]

[0105] *The sequences in Table 2 are DNA sequences encoding the indicated tRNAs. The actual tRNAs have U where T is present in the coding sequences provided in Table 2.

[0106] Example 2. Rescue of TGA premature stop codon using universal inhibitors in HEK293T PTC reporter cell line

[0107] HEK293T cells were transfected with piggyBac plasmid and antibiotic selection to generate a stable cell line with a nanoluciferase (NLuc) gene with a TGA stop codon at N160 promoted by integrated CMV. The cells were trypsinized and counted using a DeNovix CellDrop instrument for cell concentration. Cells were seeded into 96-well plates at 10,000 cells per well in 100 μL of culture medium without penstrep. Cells were transfected with Lipofectamine 3000 according to the manufacturer's instructions at the concentrations listed in Table 3. Lipofectamine reagent was prepared and incubated for 15 minutes and then added to the cells. 10 μL of master mix was added to each well.

[0108] Table 3. HEK293T PTC NLuc cell treatment

[0109]

[0110] For the nanoluciferase assay, equilibrate the nanoluciferase reagent and substrate to room temperature. Incubate the cell plate at room temperature. Add 100 μL of reagent + substrate to each test well in the 96-well plate. Shake the plate for 10 minutes before reading the signal on the plate reader.

[0111] like Figure 7 As shown in, the universal inhibitor plasmid design provides a significant and dose-dependent nanoluciferase positive signal, indicating the restorative read-through of the TGA PTC. Strong signals were observed for the universal inhibitor plasmids based on serine (hCR004.p001 and hCR004.p002) and the multi-tRNA universal inhibitor design (hCR301.p001 and hCR301.p002) of the ACE arginine tRNA inhibitor comprising TGA. No significant signal was observed for the control plasmid design lacking the TGA inhibitor element (hCR201.p003, hCR201.p005 and hCR000.p014) or for untreated cells.

[0112] Example 3. Restoration of p53 PTC in HT1299 cancer cell line using universal inhibitory plasmid

[0113] H1299 cells were transfected with pCDNA-3.1 and puromycin antibiotic selection to generate a stable cell line with an integrated CMV-promoted p53 gene with a TGA stop codon at R213.

[0114] Transfect cells using Lipofectamine 3000 following the manufacturer's instructions at the concentrations listed in Table 4. Prepare Lipofectamine reagent and incubate for 15 minutes before adding to cells. Add 200 μL of the master mix to each well.

[0115] Table 4: H1299 p53 PTC design (6 wells)

[0116]

[0117] After transfection, the culture medium was removed relative to the cells. The wells were washed 3 times with PBS. 100 μL of RIPA buffer and protease / phosphatase inhibitors were added to each well. The cells were scraped and collected in 1.7 mL Eppendorf tubes. The cells were lysed on ice for 1 hour and clarified at 15,000 rpm for 20 minutes. Protein concentration was standardized by BCA. Western blotting was performed using approximately 15 to 30 μg of protein in a 10-well gel at 100 V for 60 minutes. The gel was transferred to a nitrocellulose membrane and blocked by iBlot2.0. The membrane was incubated overnight with 1 ° antibody and incubated with 2 ° antibody for 1 hour the next day. After incubation, the membrane was washed 3 times in TBST. Licor was used to image the membrane.

[0118] Western blot data ( Figure 8 ) showed that serine-based universal inhibitors (hCR004.p001 and hCR004.p002) and multi-tRNA universal inhibitors containing TGA arginine tRNA inhibitor of ACE (hCR301.p001 and hCR301.p002) essentially restored full-length p53 protein and reduced the presence of PTC truncated p53. The multi-ACE tRNA plasmid lacking the TGA inhibitor sequence (hCR201.p005) was used as a negative control and, like untreated cells, no significant full-length p53 protein appeared.

[0119] Based on the Western blot data, the p53 recovery rate relative to that of untreated cells and the retention rate of truncated p53 relative to that of untreated cells were calculated. Fig.9A and 9B The results shown in demonstrate that there is substantial expression of full-length p53 in cells treated with the universal inhibitor construct but not in the negative control.

[0120] Equivalent solution

[0121] While the foregoing description and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the appended claims.

[0122] All publications, patents and patent applications are incorporated herein by reference. Although the present invention has been described in conjunction with certain embodiments of the present invention in the foregoing description, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is susceptible to other embodiments and that considerable changes may be made to certain details described herein without departing from the basic principles of the present invention.

[0123] Unless otherwise indicated herein or obviously contradictory with the context, the undefined number of terms used in the context of describing the present invention and similar references will be interpreted as covering both the singular and the plural. Unless otherwise noted, the terms "comprising", "having", "including" and "containing" will be interpreted as open terms (that is, meaning "including but not limited to"). Unless otherwise indicated herein, the recording of the value range herein is only intended to be used as a shorthand method for referring to each individual value falling within the range, and each individual value is incorporated into the specification as it is recorded separately in this article. Unless otherwise indicated herein or obviously contradictory with the context in other cases, all methods described herein can be carried out in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary languages ​​(such as "for example") provided herein is only intended to better illustrate the present invention, and is not intended to limit the scope of the present invention. The language in the specification should not be interpreted as representing any unclaimed element necessary for the practice of the present invention.

[0124] Some embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, the changes of those embodiments may become obvious to those of ordinary skill in the art. The inventor expects that the technician will adopt such changes when appropriate, and the inventor hopes to practice the present invention in a manner other than that specifically described herein. Therefore, as allowed by applicable law, the present invention includes all modifications and equivalents of the subject matter recorded in the appended claims. In addition, unless otherwise indicated herein or otherwise clearly contradicted with the context, the present invention covers any combination of the above-mentioned elements in all possible changes thereof.

Claims

1. A composition comprising more than one tRNA, each of the tRNAs having an anticodon specific for a stop codon, wherein the composition can target at least two stop codons, and wherein each of the tRNAs can provide an amino acid for replacing its specific stop codon.

2. The composition of claim 1, wherein the composition comprises more than two tRNAs, each of which has an anticodon specific for a stop codon, and wherein the composition can target all three stop codons.

3. The composition of claim 1 or 2, wherein each of the tRNAs can provide the same amino acid for replacement of its specific stop codon.

4. The composition of any one of claims 1 to 3, wherein the amino acid used to replace the stop codon is serine.

5. The composition of any one of claims 1 to 3, wherein the amino acid used to replace the stop codon is arginine.

6. The composition of any one of claims 1 to 3, wherein the amino acid used to replace the stop codon is glutamine.

7. The composition of any one of claims 1 to 3, wherein the amino acid used to replace the stop codon is glutamic acid.

8. The composition of claim 1 or claim 2, wherein the tRNA can provide two different amino acids for replacing the at least two specific stop codons thereof.

9. The composition of claim 8, wherein the two amino acids used to replace the stop codon are selected from arginine, glutamine and glutamic acid.

10. The composition of claim 8, wherein the two amino acids used to replace the stop codon are arginine and glutamine.

11. The composition of claim 2, wherein the tRNA can provide three different amino acids for replacing the three specific stop codons thereof.

12. The composition of claim 8, wherein the amino acid used to replace the stop codon is arginine, glutamine and glutamic acid.

13. The composition of any one of claims 1 to 12, wherein the stop codon is selected from the group consisting of TGA, TAA and TAG.

14. The composition of any one of claims 1 to 13, wherein the composition further comprises a pharmaceutically acceptable carrier.

15. An oligonucleotide encoding the tRNA according to any one of claims 1 to 14. The oligonucleotide of claim 15 , comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 14.

17. A set of oligonucleotides that collectively encode the tRNA of any one of claims 1 to 14.

18. The set of oligonucleotides of claim 17, each of which comprises the nucleotide sequence of any one of SEQ ID NOs: 1 to 14.

19. The oligonucleotide or set of oligonucleotides of claim 15, 16 or 17, wherein the oligonucleotide is DNA.

20. The oligonucleotide or set of oligonucleotides of claim 15, 16 or 17, wherein the oligonucleotide is RNA.

21. An expression cassette comprising a promoter and a nucleic acid encoding the tRNA of any one of claims 1 to 14 or the oligonucleotide of any one of claims 15, 16, 19 and 20 or a group of oligonucleotides of any one of claims 18 to 20.

22. A set of expression cassettes, each of which comprises a promoter and a nucleic acid encoding at least one tRNA, wherein the set of expression cassettes together encodes all of the tRNAs of any one of claims 1 to 14 or a set of oligonucleotides of any one of claims 18 to 20.

23. The expression cassette of claim 21 or 22, further comprising a terminator.

24. A vector comprising the oligonucleotide of any one of claims 15 to 20 or the expression cassette of any one of claims 21 to 23.

25. The vector of claim 24, wherein the vector is a viral vector or a plasmid vector.

26. A composition comprising the tRNA of any one of claims 1 to 14, the oligonucleotide of any one of claims 15 to 20, the expression cassette of any one of claims 21 to 23, or the vector of claim 24 or 25, and a pharmaceutically acceptable carrier.

27. The composition of claim 26, wherein the composition is contained in particles, such as nanoparticles.

28. The composition of claim 27, wherein the particle is a liposome or a lipid nanoparticle.

29. The composition of claim 26, wherein the composition is contained in an exosome.

30. A cell comprising the tRNA of any one of claims 1 to 14, the oligonucleotide of any one of claims 15 to 20, the expression cassette of any one of claims 21 to 23, the vector of claim 24 or 25, or the composition of any one of claims 26 to 29.

31. A method of reading through one or more stop codons, comprising delivering to a cell the tRNA of any one of claims 1 to 14, the oligonucleotide of any one of claims 15 to 20, the expression cassette of any one of claims 21 to 23, the vector of claim 24 or 25, or the composition of any one of claims 26 to 29 in an amount effective to read through the one or more stop codons.

32. A method for restoring at least partial protein function, comprising delivering to a cell the tRNA of any one of claims 1 to 14, the oligonucleotide of any one of claims 15 to 20, the expression cassette of any one of claims 21 to 23, the vector of claim 24 or 25, or the composition of any one of claims 26 to 29 in an amount effective to restore at least partial protein function.

33. A method of reducing or inhibiting cell survival or proliferation, comprising contacting a cell with the tRNA of any one of claims 1 to 14, the oligonucleotide of any one of claims 15 to 20, the expression cassette of any one of claims 21 to 23, the vector of claim 24 or 25, or the composition of any one of claims 26 to 29 in an amount effective to reduce or inhibit cell survival or proliferation.

34. The method of any one of claims 31 to 33, wherein the cell is in vitro.

35. The method of any one of claims 31 to 33, wherein the cell is in vivo.

36. A method of treating a subject in need thereof, comprising administering to a cell the tRNA of any one of claims 1 to 14, the oligonucleotide of any one of claims 15 to 20, the expression cassette of any one of claims 21 to 23, the vector of claim 24 or 25, or the composition of any one of claims 26 to 29 in an amount effective to treat the subject.

37. The method of claim 36, wherein the subject has a disease caused by or attributed to one or more nonsense mutations or one or more premature stop codons.

38. The method of claim 36 or 37, wherein the subject suffers from a hyperproliferative disease or disorder.

39. The method of claim 38, wherein the hyperproliferative disease or disorder is cancer.

40. The method of any one of claims 36 to 39, wherein the administration is systemic administration or local administration.

41. The method of any one of claims 36 to 40, wherein the subject is a human.

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