Universal suppressor tRNAs and their uses

Abstract

The present invention pertains, at least in part, to tRNA therapeutic compositions that are specific for more than one stop codon.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 302,885, filed January 25, 2022, and entitled "UNIVERSAL SUPPRESSOR tRNAS," the entire contents of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (H101570007WO00-SEQ-ZJG.xml, size: 19,216 bytes, created on January 24, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background DNA molecules carry genetic information in the form of a sequence of nucleotide bases that make up the DNA polymer. In DNA, only four nucleotide bases are utilized: adenine, guanine, cytosine, and thymine. This information, in the form of a codon of three consecutive bases, is transcribed into messenger RNA (mRNA) and then translated by transfer RNA (tRNA) and ribosomes to form proteins. In RNA, four nucleotide bases are utilized: adenine, guanine, cytosine, and uracil. The genetic code is the relationship of triplet codons to specific amino acids. 64 possible codon triplets form the genetic code, in which three stop (also called "termination" or "nonsense") codons provide a signal to the translation machinery (the cellular ribosomes) to stop protein production at a particular codon. The other 61 codon triplets (also called "sense codons") correspond to one of the 20 standard amino acids. Summary of the Invention

[0004] SUMMARY OF THE PRESENT APPLICATION Premature termination codons (PTCs) caused by mutations such as nonsense mutations can cause premature termination of protein translation and thus can cause protein dysfunction and / or disease. Provided herein is an approach to provide tRNAs that can at least partially restore protein function. Such tRNAs can ameliorate or treat diseases or disorders caused by or associated with one or more PTCs.

[0005] The present invention relates, at least in part, to tRNA-mediated therapeutics that target nonsense mutations, where the substituted amino acid may or may not be the native amino acid (e.g., the amino acid of the wild-type protein) that would have been coded or coded in the absence of the nonsense mutation. That is, the tRNA-mediated therapeutics may provide a "read-through" of PTC and / or at least partial, if not complete, functional restoration of the gene product. Thus, the resulting gene product may or may not have the same amino acid sequence as the original wild-type or non-disease-associated gene product. It is proposed that tRNA compositions that target more than one possible premature stop codon and replace it with an amino acid such as serine are preferred aspects of the present invention. In one embodiment of any of the compositions or methods provided herein, the tRNA composition targets all three possible stop codons and replaces them with an amino acid such as serine.

[0006] While there are only three stop codons, there are 23 different nucleotide substitutions that lead to stop codons. For example, the UGA stop codon is one nucleotide away from the codons for arginine (CGA, AGA), glycine (GGA), leucine (UUA), serine (UCA), cysteine ​​(UGU, UGC), and tryptophan (UGG). The most frequent nonsense mutations are CGA (arginine) -> UGA and CAG (glutamine) -> UGA. Disease can result from protein truncation induced by premature stop codons. A disease-free or disease-reduced state can be induced by reading through the PTC with a suppressor tRNA that inserts an amino acid at the position miscoded by the PTC.

[0007] On the other hand, there are other disease states, including cancer, where the specific PTC or set of PTCs may be unknown, while certain rare genetic diseases caused by PTCs may have known specific nonsense mutations and therefore specific repair-treatment suppressor tRNAs. In these instances, treatment of the PTC(s) with suppressor tRNAs that provide read-through or functionally similar amino acid codes may be substantially therapeutic. It is noted that serine, as a hydrophilic amino acid of moderate size, is likely to provide similar structural value in many cases as, for example, arginine, leucine, glutamine, cysteine, or glycine. It is further noted that some aminoacyl-tRNA synthetases do not substantially bind to the anticodon region of tRNA, while others do. Alanine, serine, and leucine tRNAs are recognized by synthetases primarily through the non-anticodon elements of tRNA.

[0008] Thus, the use of tRNA compositions that target at least two, if not all three, stop codons to replace an amino acid, such as serine, can be used to treat disease without having to know the specific PTC(s) that cause or contribute to the disease.

[0009] Some aspects of the disclosure provide compositions comprising more than one tRNA, each with an anticodon specific for a stop codon, where the composition can target at least two stop codons, and where the tRNAs can each provide an amino acid to substitute in place of the stop codon for which it is specific.

[0010] In some embodiments, the composition comprises more than two tRNAs, each with an anticodon specific to a stop codon, and wherein the composition can target all three stop codons. In some embodiments, the tRNAs can each provide the same amino acid to substitute for the stop codon for which it is specific. In some embodiments, the amino acid to substitute for the stop codon is serine. In some embodiments, the amino acid to substitute for the stop codon is arginine. In some embodiments, the amino acid to substitute for the stop codon is glutamine. In some embodiments, the amino acid to substitute for the stop codon is glutamic acid.

[0011] In some embodiments, tRNA can provide two different amino acids to replace at least two stop codons that they are specific for.In some embodiments, the two amino acids to replace the stop codon are selected from arginine, glutamine and glutamic acid.In some embodiments, the two amino acids to replace the stop codon are arginine and glutamine.

[0012] In some embodiments, the tRNAs can provide three different amino acids to substitute for the three stop codons for which they are specific. In some embodiments, the amino acids to substitute for the stop codons are arginine, glutamine, and glutamic acid. In some embodiments, the stop codon is selected from TGA, TAA, and TAG. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier.

[0013] Further provided herein are oligonucleotides encoding the tRNAs described herein. In some embodiments, the oligonucleotides comprise the nucleotide sequence of any one of SEQ ID NOs: 1-14. Also provided are sets of oligonucleotides that together encode the tRNAs described herein. In some embodiments, each oligonucleotide in the set comprises the nucleotide sequence of any one of SEQ ID NOs: 1-14. In some embodiments, the oligonucleotide(s) is DNA. In some embodiments, the oligonucleotide(s) is RNA.

[0014] Further provided herein is an expression cassette comprising a promoter and a nucleic acid encoding a tRNA, an oligonucleotide, or a set of oligonucleotides.In some embodiments, the promoter is operably linked to such a nucleic acid, oligonucleotide, or set of oligonucleotides.In some embodiments, the set of expression cassettes encodes all the tRNAs described herein together.In some embodiments, one or more expression cassettes further comprises a terminator.In some embodiments, each expression cassette further comprises a terminator.

[0015] In some embodiments, a vector is provided that comprises the oligonucleotide(s) or expression cassette(s) described herein. In some embodiments, the vector is a viral vector or a plasmid vector.

[0016] Further provided herein is a composition comprising the tRNA, oligonucleotide(s), expression cassette(s), or vector described herein and a pharma- ceutically acceptable carrier.In some embodiments, the composition is comprised in a particle, such as a nanoparticle.In some embodiments, the particle is a liposome or lipid nanoparticle.In some embodiments, the composition is comprised in an exosome. Further provided herein is a cell comprising the tRNA, oligonucleotide(s), expression cassette(s), vector, or composition described herein.

[0017] Another aspect of the present disclosure provides a method of readthrough of one or more stop codons comprising delivering to a cell a tRNA, oligonucleotide(s), expression cassette(s), vector, or composition described herein in an amount effective to readthrough the one or more stop codons.

[0018] Another aspect of the present disclosure provides a method of restoring at least partial protein function comprising delivering to a cell a tRNA, oligonucleotide(s), expression cassette(s), vector, or composition described herein in an amount effective to restore at least partial protein function.

[0019] Another aspect of the disclosure provides a method of reducing or inhibiting cell viability or proliferation, comprising delivering to a cell a tRNA, oligonucleotide(s), expression cassette(s), vector, or composition described herein in an amount effective to reduce or inhibit cell viability or proliferation. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.

[0020] Another aspect of the present disclosure provides a method of treating a subject, comprising delivering to a cell a tRNA, oligonucleotide(s), expression cassette(s), vector, or composition described herein in an amount effective to treat the subject. In some embodiments, the subject has a disease caused or resulting from one or more nonsense mutations or one or more premature termination codons. In some embodiments, the subject has a genetic disease or genetic disorder. In some embodiments, the subject has a hyperproliferative disease or disorder. In some embodiments, the hyperproliferative disease or disorder is cancer. In some embodiments, the administration is systemic or local. In some embodiments, the subject is a human.

[0021] Each of the limitations of the invention may cover various aspects of the invention. Thus, it is anticipated that each of the limitations of the invention that encompass any one element or combination of elements may be encompassed in each aspect of the invention. The invention is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention can be practiced or carried out in other embodiments and in various manners. Additionally, the phraseology and terminology used in this disclosure are for purposes of explanation and should not be considered limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof in this disclosure are meant to encompass the items listed thereafter and equivalents thereof, as well as additional items. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0022] In any one of the embodiments 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 require the specified integer(s) or step(s), as well as those that do not substantially affect the characteristics or function of the claimed invention. As used herein, the term "consisting" is used to indicate that the recited integer (e.g., a feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., a feature, element, characteristic, property, method / process step, or limitation) is present alone. [Brief description of the drawings]

[0023] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1-2] Figure 1 provides a table of the genetic code. Figure 2 shows the general four-arm structure of a tRNA, including a T arm, a D arm, an anticodon arm, and an acceptor stem (or arm) (SEQ ID NO: 15). These regions are sometimes referred to throughout as "loops." [Diagram 3] FIG. 3 provides a map of a plasmid encoding one single tRNA. [Figure 4] FIG. 4 provides a map of the plasmid encoding the two tRNAs. [Diagram 5] FIG. 5 provides maps of the plasmids encoding the three tRNAs.

[0024] [Figure 6] FIG. 6 provides a map of the plasmid encoding three tRNAs (anticodon editing (ACE) or native) and in addition encoding a transfection reporter (superfolder GFP). [Figure 7]FIG. 7 provides a graph showing that a plasmid encoding a universal suppressor tRNA transfected into HEK293T PTC NLuc cells induced expression of nanoluciferase, while a control plasmid (hCR000.p014) lacking the coding sequence for the suppressor tRNA did not. [Figure 8] 8 provides a Western blot gel image showing increased expression of full-length p53 in HT1299 cancer cells containing an integrated CMV-promoted p53 gene with a premature TGA stop codon leading to a truncated p53 protein lacking the universal suppressor tRNA. A control plasmid (hCR000.p014) lacking the suppressor tRNA coding sequence did not induce increased expression of full-length p53.

[0025] [Figure 9A-9B] Figures 9A and 9B provide graphs showing the ratio of full-length p53 expression levels (Figure 9A) or truncated p53 expression levels (Figure 9B) 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, compared to the respective expression levels in untreated cells after normalization to total protein. Cells treated with a plasmid encoding a universal suppressor tRNA showed enhanced expression of full-length p53 and reduced expression of truncated p53, while cells treated with a control plasmid (hCR000.p014) lacking the suppressor tRNA coding sequence showed levels of full-length p53 and truncated p53 comparable to untreated cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description Provided herein are therapeutic agents for treatment, such as for the treatment of cancer, where the therapeutic agent provides one or more nonsense suppressor tRNAs specific for one or more, 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 the PTC(s). Nonsense suppressor tRNAs may not provide full and / or exact restoration of native or wild-type protein, but partially restored or even similar function can have therapeutic effects. In some embodiments, the tRNA can target the PTC(s) and replace with serine, which may be an effective replacement in many cases due to the nature of the amino acid and the recognition of the synthetic enzyme. In some embodiments, the tRNA can target the PTC(s) and replace with arginine. In some embodiments, the tRNA can target the PTC(s) and replace with glutamine. In some embodiments, the tRNA can target the PTC(s) and substitute with glutamic acid. In some embodiments, the tRNA can target the PTC(s) and substitute with more than one (e.g., two, three, or more) amino acid. In some embodiments, the more than one (e.g., two, three, or more) amino acid is selected from serine, arginine, glutamine, and glutamic acid.

[0027] Transfer RNA (tRNA) is the decoder of DNA and RNA "blueprints." DNA transcription results in messenger RNA (mRNA), which upon interaction with ribosomes and tRNA, codes for the major amino acid structures that can be post-transcriptionally modified and end up in folded or unfolded proteins. When the mRNA binds to the ribosome, the tRNA delivers the amino acids to the ribosome and forms a chain of amino acids based on the code in the mRNA (Figure 1).

[0028] tRNA has a general four-arm structure, including a T arm, a D arm, an anticodon arm, and an acceptor stem or arm (FIG. 2). The T arm is composed of a "T stem" and a "TΨC loop". Any one of the tRNAs provided herein can include this four-arm structure. The tRNA is, in some embodiments, about 100 nucleotides in length and can be easily introduced into cells.

[0029] RNA molecules can be modified to allow for the systematic "recoding" of the genetic code. It has been found that through molecular editing of the anticodon sequence within the tRNA, the tRNA can be altered. This approach allows unwanted stop codons to be reprogrammed and replaced with amino acids.

[0030] Provided herein are compositions and methods related to techniques for the introduction of one or more suppressor tRNAs that can read through one or more PTCs into diseased cells or tissues. Specifically, one or more suppressor tRNAs can act broadly at a variety of premature termination codons (PTCs). The tRNA compositions provided herein can be modified to target any one or more unwanted stop codons, such as any one or more of the stop codons provided herein. In some embodiments, the one or more amino acids that can be substituted are any amino acids suitable for the present disclosure. Without wishing to be bound by any theory, serine, as a hydrophilic amino acid of moderate size, is expected to provide similar structural value to other amino acids such as arginine, leucine, glutamine, cysteine, or glycine. As a result, serine is expected to be an effective amino acid substitute in many of the common PTCs. In some embodiments, one or more tRNAs can be used to suppress misencoded PTC(s) during the production of a polypeptide or protein and replace them with an amino acid (e.g., serine, arginine, glutamine, or glutamic acid). In some embodiments, one or more tRNAs can be used to suppress more than one type of misencoded PTC(s) during the production of a polypeptide or protein and replace them all with an amino acid (e.g., serine, arginine, glutamine, or glutamic acid) or with a different amino acid (e.g., an amino acid selected from serine, arginine, glutamine, or glutamic acid, or a combination thereof).

[0031] As used herein, "premature stop codon" or PTC refers to a single nucleotide mutation that converts a standard triplet nucleotide codon into one of three stop codons (e.g., TAG, TGA, or TAA). Currently, there are three stop codons known in the art, with nucleotide substitutions leading to 23 different stop codons. For example, the UGA stop codon can be derived from the codons for arginine (CGA, AGA), glycine (GGA), leucine (UUA), serine (UCA), cysteine ​​(UGU, UGC), and tryptophan (UGG). The presence of a stop or termination codon signals a stop in protein synthesis (i.e., the end of the translation process of the current protein). PTCs can cause protein truncation, resulting in abnormalities and disease.

[0032] In some embodiments, the present disclosure provides a composition comprising one or more tRNAs as described herein.In some embodiments, the composition described herein comprises more than one tRNA, each with an anticodon specific to a stop codon, wherein the composition can target at least two stop codons (e.g., two or three stop codons selected from TAA, TGA and TAG), and wherein each tRNA can provide an amino acid (e.g., the same amino acid or multiple different amino acids) to substitute for the stop codon for which it is specific.

[0033] In some embodiments, the composition comprises more than two tRNAs, each with an anticodon specific for a stop codon, and wherein the composition is capable of targeting all three stop codons (i.e., all of TAA, TGA, and TAG).

[0034] In some embodiments, the compositions described herein comprise more than one tRNA, where each tRNA can provide the same amino acid to substitute for the stop codon for which it is specific.In some embodiments, the amino acid to substitute for the stop codon is serine.In some embodiments, the amino acid to substitute for the stop codon is arginine.In some embodiments, the amino acid to substitute for the stop codon is glutamine.In some embodiments, the amino acid to substitute for the stop codon is glutamic acid.

[0035] In some embodiments, the compositions described herein comprise more than one tRNA, where the tRNA can provide two different amino acids to replace at least two stop codons that they are specific for.In some embodiments, the two amino acids to replace the stop codon are selected from arginine, glutamine, and glutamic acid.In some embodiments, the two amino acids to replace the stop codon are arginine and glutamine.

[0036] In some embodiments, the compositions described herein include more than one tRNA, where the tRNA can provide three different amino acids to substitute for the three stop codons for which they are specific. In some embodiments, the amino acids to substitute for the stop codons are arginine, glutamine, and glutamic acid.

[0037] Further provided herein is an oligonucleotide, or set of oligonucleotides, encoding any one or more of the tRNAs as provided herein. In some embodiments, the oligonucleotide comprises a nucleotide sequence represented by any one of SEQ ID NOs: 1-14. In some embodiments, the compositions described herein comprise an oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 1-14, or a tRNA encoded by the oligonucleotide.

[0038] In some embodiments, the present disclosure provides a composition comprising one or more nucleic acids or expression constructs encoding one or more tRNAs as described herein. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier. In some embodiments, the pharma- ceutically acceptable carrier can be any carrier known in the art and suitable for the present disclosure. In some embodiments, the present disclosure provides a cell comprising one or more tRNAs or one or more nucleic acids that they encode as described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0039] The present disclosure provides methods of treating or achieving read-through of one or more PTCs by using one or more suppressor tRNAs that can provide a functionally similar amino acid code (e.g., serine, arginine, glutamine, or glutamic acid). Insertion of a functionally similar amino acid into an amino acid codon erroneously coded by a PTC(s) can restore partial, if not complete, function of the gene product. In some embodiments, the restored gene product(s) may or may not be identical to the wild type or to the gene product(s) without the PTC(s).

[0040] The present disclosure provides a method for treating a subject with a disease or disorder. In some embodiments, the method comprises administering one or more tRNAs or compositions as disclosed herein to the subject. In some embodiments, the disease or disorder may include a genetic disease or disorder, or a hyperproliferative disease or disorder. In some embodiments, the disease or disorder may be cancer. In some embodiments, the disease or disorder may be any disease or disorder that can benefit from tRNA-based treatment as provided herein.

[0041] In some embodiments, the expression or function of the protein is at least partially restored compared to the control. As used herein, an example of a "control" can be a tRNA that is not a suppressor tRNA as provided herein, or a tRNA that is not expected to read through a PTC. As another example, the control can be the same tRNA, but unable to substitute a desired amino acid, such as serine, into the position erroneously encoded by the PTC(s). Comparison of the improvement, amelioration, or other characteristics of a disease or disorder can also be evaluated in vitro or in vivo using a test subject, as known to those skilled in the art.

[0042] In some embodiments, in a subject administered a tRNA(s) or composition provided herein, the disease or disorder is reduced or eliminated 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%, 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%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 109%, , 68%, 68%, 68%, 68%, 68%, 68%, 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 ​​in between).

[0043] As used herein, a "genetic disease or disorder" is a disease or disorder that is genetically inherited or caused by a mutation that occurs in the genetic code of a subject or that predisposes the subject to the disease or disorder.

[0044] Any one of the compositions or methods provided herein can be for treating or preventing genetic diseases or disorders. As used herein, "hyperproliferative disease or disorder" refers to any disease or disorder in which the proliferation rate of cells is abnormally high, due to rapid division, substantial hyperproliferation, and the like. Certain embodiments of the present disclosure provide a method for treating a disease or disorder, such as a hyperproliferative disease or disorder, in a subject, such as a mammal. In certain embodiments, the mammal is a human. Certain embodiments of the present disclosure provide the use of one or more tRNA(s) or compositions as described herein for preparing a medicament useful for treating a disease or disorder, such as a hyperproliferative disease or disorder, in a subject, such as a mammal, such as a human. In certain embodiments, the treatment has potential use for the treatment / management of a disease or disorder, such as a hyperproliferative disease or disorder, including tumors, cancers, and neoplastic tissues, as well as non-neoplastic or non-malignant hyperproliferative disorders. In certain embodiments, the hyperproliferative disease or disorder is cancer.

[0045] Nucleotide sequences encoding tRNAs and RNAs can be synthetically produced. Nucleotide sequences encoding hundreds of human tRNAs are also known and generally available to those skilled in the art through sources such as GenBank. In one embodiment of any of the compositions or methods provided herein, the tRNA(s) are selected from those provided in PCT / US2018 / 059065, the disclosure of which tRNAs and sequences is incorporated herein by reference in its entirety. In one embodiment of any of the compositions or methods provided herein, the tRNA(s) are selected from those provided in PCT / US2018 / 059065, which can be substituted with serine. The structure of tRNAs is generally highly conserved, and tRNAs can function across species. Thus, bacterial or other eukaryotic tRNA sequences are also potential sources of tRNAs provided herein. The determination of whether a particular tRNA or combination of tRNAs is functional as desired, such as in a desired mammalian cell, can be confirmed as described herein or through other experimentation apparent to those skilled in the art with the benefit of the teachings provided herein.

[0046] The tRNA(s) may be in any suitable form, such as any suitable recombinant plasmid containing a heterologous nucleic acid sequence for delivery to a target cell or subject, either in vitro or in vivo. The heterologous nucleic acid sequence encodes a gene product of interest (e.g., a tRNA) for any one of the uses provided herein, including, for example, disease treatment, and may optionally be in the form of an expression cassette. The term "recombinant" refers to a polynucleotide that is either not naturally occurring or is linked to another polynucleotide in an arrangement not found in nature. The term "heterologous" as used herein refers to a nucleic acid sequence obtained or derived from an entity that is genetically distinct from the rest of the entity to which it is compared.

[0047] The present disclosure provides a method for delivering one or more tRNA(s) or compositions provided herein to a subject or to a cell of a subject. The tRNA(s) or compositions as described herein can be delivered to a cell in vivo or in vitro. Administration to a cell can be accomplished by any means, including simply contacting the cell. Contacting the cell can be for any desired length of time. The cell can include any desired cell in humans and other large (non-rodent) mammals, such as primates, horses, sheep, goats, pigs, and dogs. Any one of the subjects provided herein can be a human or other mammal. The term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep.

[0048] In some embodiments, administration can be systemic. In some embodiments, administration can be local. In some embodiments, administration can be direct delivery to selected organs, oral, inhalation, intraocular, intravenous including facial vein injection and retro-orbital injection, intracerebroventricular (ICV), intracisternal (ICM) injection, intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. If desired, routes of administration can be combined. In some embodiments, tRNA as disclosed herein can be administered via any route suitable for this disclosure.

[0049] Suitable methods for delivery and introduction into a subject are also provided or otherwise understood in the art. In one embodiment, the pharmaceutical composition will contain sufficient genetic material to produce a therapeutically effective amount of the tRNA(s) of interest. The tRNA or genetic material encoding the tRNA(s) can be delivered in an effective amount and into a cell, such as with endogenous tRNA synthetase. A tRNA synthetase is considered to be "endogenous" to a cell when it is present in a cell into which a tRNA has been introduced according to the present invention. As will be apparent to those skilled in the art, a tRNA synthetase can be considered to be endogenous for these purposes, regardless of whether it is naturally found in the relevant type of cell, or whether the specific cell in question has been engineered or otherwise manipulated to contain or express it.

[0050] In some embodiments, the tRNA can be formulated in a pharmaceutical composition. The pharmaceutical composition may also contain a pharmaceutically acceptable excipient. Such excipients may include any pharmaceutical agent that can be administered without undue 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 can be included therein, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0051] As will be clear to those skilled in the art in view of the teachings herein, the effective amount of tRNA provided can be empirically determined. Administration can be done in a single dose, continuously or intermittently throughout the course of treatment. The method of determining the most effective administration means and dosage can vary depending on the composition of the treatment, the target cells, and the subject being treated, etc. Single and multiple administrations can be performed, and the level and pattern of administration are selected by the treating physician.

[0052] Vehicles including water, saline, artificial CSF, or other known substances can be employed with the present invention. To prepare the formulation, the purified composition can be isolated. The composition can then be adjusted to the appropriate concentration and packaged for use.

[0053] As used herein, the terms "treat" and "treatment" refer to both therapeutic treatments and measures that can alleviate symptoms or provide some benefit to a subject, where the objective is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as the growth, development or spread of cancer. For purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, alleviating symptoms, reducing the extent of disease, a stabilized (i.e., not worsening) disease state, a delay or slowdown in disease progression, alleviation or relief of disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean extending survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition, disease, or disorder.

[0054] The phrase "therapeutically effective amount" refers to an amount of one or more compounds or compositions of the present invention that (i) treats a specific disease, condition, or disorder, (ii) attenuates, alleviates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder, as described herein. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down to some extent, and preferably stop) the invasion of cancer cells into peripheral organs; inhibit (i.e., slow down to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or ameliorate to some extent one or more symptoms associated with cancer. To the extent that a drug may prevent proliferation and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0055] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancer cells. Examples of cancer include, but are not limited to, carcinomas, malignant tumors, and the like.

[0056] The tRNA(s) or compositions described herein may be administered to result in a reduction of at least one symptom associated with a disease or disorder, such as a genetic disease or disorder, or a hyperproliferative disease or disorder (e.g., cancer). The amount administered will vary depending on a variety of factors, including, but not limited to, the composition selected, the specific disease, the mammal's weight, physical condition, and age. Such factors can be readily determined by the clinician employing animal models or other test systems known in the art.

[0057] Administration of the tRNA(s) or compositions according to the invention may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic, and other factors known to the skilled physician. Administration may be essentially continuous over a preselected period of time, or may be in a series of spaced doses.

[0058] One or more suitable unit dosage forms having one or more tRNA(s) or compositions of the present invention may be formulated and administered by various routes. When the agents of the present invention are prepared for administration, they may be combined with a pharma- ceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredient in such a formulation comprises 0.1-99.9% by weight of the formulation. "Pharmaceutically acceptable" refers to a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutical formulations can be prepared by procedures known in the art using well-known and readily available ingredients. The agents of the present invention can also be formulated as a solution suitable for administration. Pharmaceutical formulations of the agents of the present invention can take the form of an aqueous or anhydrous solution or dispersion, or the form of an emulsion or suspension.

[0059] Thus, the drug(s) may be formulated for administration and presented in unit dosage form in ampoules, prefilled syringes, small volume infusion containers, or in multi-dose containers with added preservatives.The active ingredient may take such forms as suspension, solution, or emulsion in oily or aqueous vehicles, and may contain compounding agents such as suspending agents, stabilizing agents, and / or dispersing agents.Alternatively, the active ingredient may be in a suitable vehicle, for example, in sterile, pyrogen-free water, before use.It will be fully understood that the unit content of the active ingredient(s) contained in each dose of each dosage form does not necessarily constitute an effective amount for treating a specific indication or disease, since the required effective amount can be achieved by administering multiple dosage units.Moreover, the effective amount may be achieved either individually or in a series of administrations, using a dose smaller than the dose in the dosage form.

[0060] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharma- ceutically acceptable carriers, diluents, solubilizers or emulsifiers, and salts of the type well known in the art. Specific non-limiting examples of carriers and / or diluents useful in the pharmaceutical formulations of the present invention include water, and physiologically acceptable buffered saline, such as phosphate buffered saline pH 7.0-8.0, and water.

[0061] Any of the compositions provided herein can be contacted, administered, or introduced into cells using gene transfer methods such as transfection.Thus, any of the compositions provided herein can be included with or in gene delivery vehicles.The gene delivery vehicle can be any delivery vehicle known in the art, and can include naked nucleic acid that is facilitated by receptor and / or lipid-mediated transfection.

[0062] The compositions provided herein can be contacted with cells or delivered or administered to a subject in particles such as nanoparticles.Particles such as nanoparticles can be, but are not limited to, lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles whose structure is made up of mostly lipid materials), and / or particles using a combination of nanomaterials.Particles can be in a variety of different shapes, including, but are not limited to, spherical, rectangular, pyramidal, oblong, cylindrical, toroidal, and the like.

[0063] In some embodiments, the particles, such as nanoparticles, may comprise one or more lipids. In some embodiments, the particles, such as nanoparticles, may comprise liposomes. In some embodiments, the particles, such as nanoparticles, may comprise lipid bilayers. In some embodiments, the particles, such as nanoparticles, may comprise lipid monolayers. In some embodiments, the particles, such as nanoparticles, may comprise micelles.

[0064] The tRNA or compositions provided herein can be contacted with cells or delivered or administered to a subject using extracellular vesicles or exosomes. In general, exosomes are nano-sized extracellular vesicles (EVs) (diameter 30-150 nm) that can be formed and released by many mammalian cells. The EVs or exosomes can be loaded with a drug(s) of interest, for example, by pretreatment of cells with the drug and subsequent isolation of the loaded EVs or exosomes.

[0065] EVs or exosomes can be derived from human fetal kidney cells, bone marrow stem cells, immature dendritic cells, red blood cells, and milk. EVs or exosomes can be isolated and purified by a number of different techniques. EVs or exosomes can be isolated and purified by a number of different techniques. Electroporation may also be used.

[0066] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encoding the tRNA is in a closed-end form, such as in a plasmid, nanoplasmid or minicircle.In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encoding the tRNA is in the form of a minicircle or microthread.

[0067] The term "minicircle" as used herein refers to a small circular DNA fragment that is largely or completely free of non-essential prokaryotic elements. Minicircles encompass circular forms of DNA that are free of and / or have been removed from prokaryotic elements. Minicircles can be from a parental plasmid from which bacterial DNA sequences have been excised. Minicircles can be in the form of any suitable recombinant plasmid that contains a heterologous nucleic acid sequence that is delivered to a target cell either in vitro or in vivo. 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). Preparation can, for example, follow a two-step procedure: (i) production of a "parental plasmid" (bacterial plasmid with eukaryotic insert); and (ii) induction of a site-specific recombinase at the end of this process. These steps can be followed by excision of the prokaryotic vector portion via the recombinase target sequence and recovery by capillary gel electrophoresis.

[0068] As a non-limiting example, minicircles may be produced as follows: An expression cassette contains the polynucleotide coding sequence together with regulatory elements for its expression, flanked by attachment sites for the recombinase. The sequence encoding the recombinase is located outside the expression cassette and includes elements for inducible expression, such as an inducible promoter. When recombinase expression is induced, the vector DNA recombines, resulting in two separate circular DNA molecules. One of the circular DNA molecules is relatively small, forming a minicircle containing the expression cassette for the polynucleotide, and this minicircle DNA vector is free of bacterial DNA sequences. The second circular DNA sequence contains the remaining vector sequences, including bacterial sequences and sequences encoding the recombinase. The minicircle DNA containing the polynucleotide sequences can then be isolated and purified separately. In some embodiments, minicircle DNA vectors may be produced using plasmids similar to pBAD.φ.C31.hFIX and pBAD.φ.C31.RHB. See, e.g., Chen et al. (2003) Mol. Ther. 8:495-500, or as otherwise provided herein.

[0069] Examples of recombinases that may be used to create minicircles include, but are not limited to, Streptomyces bacteriophage φ31 integrase, Cre recombinase, and λ integrase / DNA topoisomerase IV complex. Each of these recombinases catalyzes recombination between distinct 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 bacteriophage λ attP and attB sites.

[0070] Published U.S. Application 20170342424 also describes a system that utilizes parental plasmids that are exposed to enzymes that cause recombination at recombination sites, thereby forming (i) a minicircle that encompasses a polynucleotide sequence, and (ii) a miniplasmid that includes the remainder of the parental plasmid, where one recombination site is modified at the 5' end such that it reacts with the enzyme less efficiently than the wild-type site, and the other recombination site is modified at the 3' end such that it reacts with the enzyme less efficiently than the wild-type site, and the other recombination site is modified at the 3' end such that it reacts with the enzyme less efficiently than the wild-type site, and both modified sites are located within the minicircle after recombination.

[0071] Removal of prokaryotic sequences should ideally be efficient, using the smallest possible excision sites while creating supercoiled DNA minicircles consisting only of gene expression elements under appropriate (preferably mammalian) regulatory regions. Some techniques for minicircle production use bacterial phage lambda (λ) integrase-mediated recombination to produce minicircle DNA. See, for example, 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.

[0072] Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). For example, high quality minicircle DNA can be obtained by using MC-EasyTM (Cat. No. MN920A-1, SBI System Biosciences) minicircle DNA production kit. Information on 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 No.:14062 (2015) doi:10.1038 / mtm.2014.62. Further information on 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 reflected by active chromatin and transcriptional level", Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay M A. Mol Ther. 2013 Jan;21(1):131.

[0073] In some embodiments, the closed-end form is a supercoiled helix. DNA supercoiling refers to the amount of twist in a particular DNA strand. Supercoiled DNA can be positively supercoiled or negatively supercoiled. As used herein, "supercoiled DNA" refers to a DNA molecule, or a fragment of a DNA molecule, in which one or both DNA strands contain an increased amount of twist compared to the amount of twist in a reference state known as "relaxed B-form" DNA. In a "relaxed" double helix segment of DNA, the two strands twist around the helical axis once every 10.4 to 10.5 base pairs of sequence. A given DNA strand may be "positively supercoiled" or "negatively supercoiled" (i.e., more or less tightly wound). Supercoiling creates torsional strain in the DNA strand. The amount of supercoiling of a strand affects numerous biological processes, such as compacting DNA and regulating access to the genetic code, which strongly influences DNA metabolism and possibly gene expression. Certain enzymes (e.g., topoisomerases) can increase or decrease the amount of twisting (e.g., supercoiling) in DNA strands to facilitate functions such as DNA replication and transcription. When a DNA segment under torsional strain is closed into a circle by connecting its two ends together and then allowed to move freely, it can form a supercoiled structure. Examples of supercoiled structures of circular DNA molecules include, but are not limited to, figure-of-eight structures, plectonemic structures, or toroidal structures.

[0074] In one embodiment of any one of the compositions or methods provided herein, the oligonucleotides described herein further comprise a promoter. Such oligonucleotides may be included in a vector. A promoter characteristically has a specific nucleotide sequence required to initiate transcription. In some embodiments, the oligonucleotides provided herein comprise a nucleic acid sequence capable of directing the expression of a certain nucleotide sequence in an appropriate cell, which may include a promoter operably linked to a nucleotide sequence of interest, which may also be operably linked to a termination signal (or terminator). The oligonucleotides as provided herein may be in a recombinant form useful for heterologous expression. In some embodiments, the promoter is a regulatable promoter. In some embodiments, the promoter is a constitutive promoter. The promoter driving the expression of the sequence encoding the tRNA(s) to be delivered can be any desired promoter, selected by known considerations, such as the expression level of the nucleic acid operably linked to the promoter and the cell type in which the vector is used. The promoter can be an exogenous or endogenous promoter.

[0075] In one embodiment of any one of the compositions or methods provided herein, the promoter may be between (35-105 bp in size). The promoter may be any known promoter, including native tRNA leader sequences, which may be 50-60 bp in size. In one embodiment of any one of the compositions or methods provided herein, the promoter may be a reduced-sequence or re-configured promoter. Examples of promoter sequences that may 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.

[0076] Optionally, the oligonucleotides or vectors provided herein may further include additional sequences, such as, for example, a 3' tRNA tail or trailer sequence. Such sequences may be between 2 and 20 bp in length. They may include naturally occurring sequences or engineered variants having 3 to 10 consecutive "T" residues.

[0077] The present disclosure also provides a cell containing the tRNA(s), oligonucleotide(s), vector, or other compositions of the present invention described herein. The cell may be mammalian, such as human. According to one aspect, a cell expression system is provided. The expression system includes a cell and an oligonucleotide, or a set thereof, or a vector, as provided herein. Expression cassettes include, but are not limited to, plasmids, viral vectors, and other vehicles for delivering heterologous genetic material to cells. The cell expression system can be formed in vivo. In some embodiments of any one of the tRNA or other compositions of the invention provided herein, the tRNA(s) or composition(s) is not found in nature.

[0078] The present invention is further illustrated by the following examples, which should not be construed as further limiting the present invention. The entire contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference.

[0079] example Example 1. Design and generation of a universal suppressor Anticodon editing (ACE) tRNA suppressor sequences were designed by replacing the anticodons of exemplary native human tRNA sequences with anticodons modified to match the TGA, TAG, and TAA stop codons. The DNA sequences of these ACE tRNAs were synthesized and incorporated into plasmid constructs as described in Figures 3-6. Figure 3 provides designs for a plasmid with a single tRNA sequence, Figure 4 for a plasmid with two tRNA sequences, Figure 5 for a plasmid carrying three tRNA sequences, and Figure 6 for a plasmid with three tRNA sequences and also a transfection reporter sequence. All plasmids are based on the pUC57 backbone, utilize the same DNA polymerase III promoter, have the same tRNA trailer / terminator sequences, and contain the same DTS sequences. The size of the plasmids varies slightly within a given design depending on the length of the encoded tRNA sequence. Table 1 lists the ACE tRNAs that are uniquely encoded by the synthesized plasmids. [Table 1] [Table 2]

[0080] Example 2. Rescue of a TGA premature stop codon using a universal suppressor in a HEK293T PTC reporter cell line HEK293T cells were transfected with piggyBac plasmid and antibiotic selection to generate a stable cell line with an integrated CMV-promoted nanoluciferase (NLuc) gene with a TGA stop codon at N160. Cells were trypsinized and cell concentration was measured using a DeNovix Cell Drop instrument. Cells were seeded into 96-well plates at 10,000 cells / well in 100 μL of medium without pen-strep. Lipofectamine 3000 was used according to the manufacturer's instructions at the concentrations listed in Table 3 to transfect cells. Lipofectamine reagent was made up and incubated for 15 minutes prior to adding to cells. 10 μL of master mix was added to each well. [Table 3]

[0081] For the nanoluciferase assay, the nanoluciferase reagent and substrate were equilibrated to room temperature. The cell plate was incubated at room temperature. 100 μL of the reagent + substrate was added to each test well of the 96-well plate. The plate was agitated for 10 minutes prior to reading the signal in the plate reader.

[0082] As shown in Figure 7, the universal suppressor plasmid designs provided significant and dose-dependent positive nanoluciferase signals, indicating restorative read-through of the TGA PTC. Strong signals were observed for the serine-based universal suppressor plasmids (hCR004.p001 and hCR004.p002) as well as for the multi-tRNA universal suppressor designs (hCR301.p001 and hCR301.p002) that contained the ACE arginine tRNA suppressor of TGA. No significant signals were observed for the control plasmid designs (hCR201.p003, hCR201.p005, and hCR000.p014) that lacked the TGA suppressor element or for untreated cells.

[0083] Example 3. Restoration of p53 PTC in HT1299 cancer cell lines using a universal suppressor plasmid H1299 cells were transfected using pCDNA-3.1 with puromycin antibiotic selection to generate stable cell lines with an integrated CMV-promoted p53 gene with a TGA stop codon at R213.

[0084] Lipofectamine 3000 was used to transfect the cells according to the manufacturer's instructions at the concentrations listed in Table 4. Lipofectamine reagent was made up and incubated for 15 minutes prior to adding to the cells. 200 μL of master mix was added to each well. [Table 4]

[0085] After transfection, media was removed from cells. Wells were washed 3X with PBS. 100 μL of RIPA buffer and protease / phosphatase inhibitors were added to each well. Cells were scraped and collected in 1.7 mL Eppendorf tubes. Cells were lysed on ice for 1 hour and clarified at 15,000 rpm for 20 minutes. Protein concentration was normalized via BCA. Western blots were run at 100V for 60 minutes using approximately 15-30 μg of protein in 10-well gels. Gels were transferred to nitrocellulose membranes via iBlot 2.0 and blocked. Membranes were incubated with 1° antibody overnight and 2° antibody for 1 hour the following day. Following incubation, membranes were washed 3 times in TBST. Membranes were imaged using Licor.

[0086] Western blot data (Figure 8) show that the serine-based universal suppressor (hCR004.p001 and hCR004.p002) and the multi-tRNA universal suppressor (hCR301.p001 and hCR301.p002) containing the ACE arginine tRNA suppressor of TGA substantially restored full-length p53 protein and reduced the presence of PTC-truncated p53. The multi-ACE tRNA plasmid lacking the TGA suppressor sequence (hCR201.p005) served as a negative control and, like untreated cells, did not evidence significant full-length p53 protein.

[0087] Based on the Western blot data, the percentage of p53 restoration relative to untreated cells and the percentage of truncated p53 retention relative to untreated cells were calculated. The results are shown in Figures 9A and 9B, respectively, and demonstrate substantial full-length p53 expression in cells treated with the universal suppressor construct and not in the negative control.

[0088] Equivalent The above specification and examples fully disclose and enable the invention, but are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0089] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with reference to specific embodiments, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and that certain details described herein may be varied considerably without departing from the underlying principles of the invention.

[0090] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the present invention shall be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprise", "have", "include", and "contain" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated herein. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is intended merely to serve as a shorthand method of individually referring to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. Unless otherwise indicated herein or otherwise clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (such as, for example, "etc.") provided herein is intended merely to better illuminate the invention and does not impose limitations on the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0091] Aspects of the present invention, including the best mode known to the inventors for carrying out the invention, are described herein. Variations of those aspects may become apparent to those skilled in the art upon reading the above description. The inventors anticipate that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is covered by the present invention, unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A composition comprising more than one tRNA, each having an anticodon specific for a stop codon, wherein the composition is capable of targeting at least two stop codons, and wherein each tRNA is capable of providing an amino acid to substitute in place of the stop codon for which it is specific.

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

3. 3. The composition of claim 1 or 2, wherein each tRNA is capable of providing the same amino acid to substitute in place of the stop codon for which it is specific.

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

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

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

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

8. 3. The composition of claim 1 or 2, wherein the tRNAs are capable of providing two different amino acids to substitute in place of at least two stop codons for which they are specific.

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

10. 9. The composition of claim 8, wherein the two amino acids substituted for the stop codon are arginine and glutamine.

11. 3. The composition of claim 2, wherein the tRNAs are capable of providing three different amino acids to substitute in place of the three stop codons for which they are specific.

12. 9. The composition of claim 8, wherein the amino acids substituted for the stop codon are arginine, glutamine, and glutamic acid.

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

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

15. An oligonucleotide encoding a tRNA according to any one of claims 1 to 14.

16. The oligonucleotide of claim 15, comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 14.

17. A set of oligonucleotides, which together encode a tRNA according to any one of claims 1 to 14.

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

19. 18. The oligonucleotide or set of oligonucleotides according to claim 15, 16 or 17, wherein the oligonucleotide or oligonucleotides are DNA.

20. 18. The oligonucleotide or set of oligonucleotides according to claim 15, 16 or 17, wherein the oligonucleotide(s) is RNA.

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

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

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

24. A vector comprising an oligonucleotide(s) according to any one of claims 15 to 20, or an expression cassette(s) according to 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 a tRNA according to any one of claims 1 to 14, an oligonucleotide(s) according to any one of claims 15 to 20, an expression cassette(s) according to any one of claims 21 to 23, or a vector according to claim 24 or 25, and a pharma- ceutically acceptable carrier.

27. 27. The composition of claim 26, wherein the composition is contained in a particle, such as a nanoparticle.

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

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

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

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

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

33. 30. A method for reducing or inhibiting cell survival or proliferation, comprising contacting a cell with a tRNA according to any one of claims 1 to 14, an oligonucleotide(s) according to any one of claims 15 to 20, an expression cassette(s) according to any one of claims 21 to 23, a vector according to claim 24 or 25, or a composition according to 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. 30. A method of treatment in a subject in need thereof, comprising administering to a cell a tRNA according to any one of claims 1 to 14, an oligonucleotide(s) according to any one of claims 15 to 20, an expression cassette(s) according to any one of claims 21 to 23, a vector according to claim 24 or 25, or a composition according to any one of claims 26 to 29 in an amount effective to treat the subject.

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

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

39. 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 or local.

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

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