Modified transfer RNA with improved activity

Abstract

The present invention relates, at least in part, to modified transfer RNAs (tRNAs) and related methods and compositions.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 301,930, filed January 21, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 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 between triplet codons and 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

[0003] SUMMARY OF THE PRESENT APPLICATION When tRNA is delivered to the body in an unmodified form, it may be more susceptible to degradation in vivo and therefore have reduced efficiency and / or function for effective therapeutic benefit. Provided herein are approaches to provide modified tRNAs that may have enhanced activity (e.g., efficacy).

[0004] In one aspect, compositions comprising tRNA with such modifications are provided herein, as well as related compositions and methods of their use.In some embodiments of any one of the compositions or methods provided herein, tRNA is modified at least one of any one of the specific positions provided herein and / or as provided in any one of the examples provided herein.Such tRNA can have an extended circulating half-life, and / or different affinity for other molecules, and / or increased presence or accumulation in one or more target tissues or cell types, and / or increased uptake in one or more target tissues or cell types, and / or increased functional readout in one or more target tissues or cell types.

[0005] In some embodiments of any one of the compositions or methods provided herein, the tRNA is modified such that at least one of any one of the specific positions provided herein can have or has a chemical modification. In some embodiments of any one of the compositions or methods provided herein, the modified tRNA has a 2' sugar modification (e.g., a 2'-O-methyl (2'-OMe) modification). In some embodiments of any one of the compositions or methods provided herein, the tRNA is modified such that at least one of any one of the specific positions provided herein can have or has a backbone modification. In some embodiments of any one of the compositions or methods provided herein, the modified tRNA has a phosphorothioate modification. In some embodiments of any one of the compositions or methods provided herein, the modified tRNA has a 2' sugar modification (e.g., a 2'-O-methyl (2'-OMe) modification) and a phosphorothioate modification.

[0006] In some embodiments of any one of the compositions or methods provided herein, the tRNA is any one of the modified tRNAs provided herein, in labeled or unlabeled form.In some embodiments of any one of the compositions or methods provided herein, the tRNA is any one of the modified tRNAs provided herein, with or with a label and / or quencher.

[0007] In some embodiments, compositions are provided that include modified tRNA(s). In some embodiments, oligonucleotides are provided that code for modified tRNA(s). In some embodiments, compositions are provided that include oligonucleotide(s) provided herein. The oligonucleotide may be an expression cassette or any vector from which modified tRNA(s) can be produced.

[0008] In some embodiments, the modified tRNA or composition provided herein can be used for administration to a subject, such as for treating a subject. In some embodiments, the modified tRNA or composition provided herein can be used to restore protein translation in a subject, modify the resulting protein, and so on. The modified tRNA or composition provided herein can be used to correct nonsense or missense mutations. The modified tRNA or composition provided herein can be used to replace stop codons or other codons in a protein or part thereof with alternative amino acids.

[0009] Methods of producing the modifications are also provided herein. Such methods can include any one or more of the specific steps provided herein. An active tRNA molecule can have conserved secondary and tertiary structures that are components of its activity, including, for example, specific recognition by aminoacyl-tRNA synthetases, specific recognition by elongation factors, compatibility with the a, p, and e sites of the ribosome (aminoacyl, peptidyl, and exit sites), and / or anticodon interaction with an mRNA codon, etc.

[0010] In some embodiments, the modification is at one or more positions within the tRNA sequence, hi some embodiments, the one or more positions are within the acceptor stem of the tRNA. Aspects of the present disclosure pertain to compositions comprising any one of the tRNA(s) described herein. In some embodiments, any one of the improved effects of the tRNA, such as targeting of the tRNA, is improved as compared to a control tRNA without the modification(s).

[0011] Aspects of the present disclosure relate to methods comprising administering to a subject in need thereof any one of the tRNAs described herein or any one of the compositions described herein, and optionally in an amount effective to achieve any one or more of the improved effects, such as in the subject, provided herein. In some embodiments, the administration is systemic or local administration. In some embodiments, the subject is a human.

[0012] Each of the limitations of the invention may encompass various aspects of the invention. Thus, it is anticipated that each of the limitations of the invention involving any one element or combination of elements may be included in each aspect of the invention. The invention is not limited in its application to the details of the configuration 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.

[0013] In any 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 steps, 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.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure, and may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this disclosure. The accompanying drawings are not intended to be drawn to scale. The drawings are merely illustrative and are not required to enable the present disclosure. For clarity, not every component is labeled in every drawing. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 provides a table of the genetic code. [Diagram 2] 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). These regions are also sometimes referred to throughout as "loops." [Diagram 3] FIG. 3 shows the activity of exemplary modified tRNAs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description Transfer RNA (tRNA) is the decoder of the DNA and RNA "blueprints." DNA transcription results in messenger RNA (mRNA), which may be post-transcriptionally modified and which codes for the primary amino acid structure that ultimately becomes a folded or unfolded protein upon interaction with the ribosome and tRNA. Once the mRNA engages the ribosome, the tRNA delivers the amino acid to the ribosome and forms a chain of amino acids based on the code in the mRNA (Figure 1).

[0017] 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. In some embodiments, the tRNA is approximately 100 nucleotides in length and can be easily introduced into cells. Engineered tRNAs have the potential to correct defects in damaged or mutated mRNA molecules by delivering the correct amino acid where the wrong amino acid would normally be delivered. Engineered tRNAs can also have the potential to reduce the amount of protein in a cell by delivering the wrong amino acid where the right amino acid would normally be delivered, making the resulting amino acid defective and unfoldable and / or susceptible to proteolysis.

[0018] The engineered or therapeutic tRNA delivered to the body in unmodified form without the benefit of delivery agents, vectors, electromechanical facilitation, penetration enhancers or the like is expected to be rapidly degraded to a large extent and lack significant capacity for cellular uptake, thereby providing very limited potential therapeutic benefits.Immune responses directed against tRNA may also occur.Therefore, modified engineered tRNA has the potential to be effectively delivered to cells, which may increase therapeutic efficacy.

[0019] Provided herein are compositions and methods related to techniques based on the modification of tRNA. In some embodiments, tRNA can be engineered to include one or more backbone modifications. In some embodiments, one or more modified nucleotides are located at one or more positions of tRNA. In some embodiments, tRNA can be engineered to include one or more nucleoside modifications. In some embodiments, tRNA can be engineered to include one or more nucleoside modifications and one or more backbone modifications. Such tRNAs (such as those in the examples and in Figure 3) have been found to have increased activity compared to unmodified tRNAs.

[0020] In some embodiments, the backbone modification includes, but is not limited to, phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl intersugar linkage, or short chain heteroatom and heterocyclic intersugar linkage. In some embodiments, the modified tRNA can include a phosphorothioate backbone; a heteroatom backbone such as a methylene (methylimino) or MMI backbone; an amide backbone; a morpholino backbone; or a peptide nucleic acid (PNA) backbone. In some embodiments, the modified backbone can include any modification(s) suitable for the present disclosure.

[0021] In some embodiments, modified nucleosides can include 2'O-methyl, 2'-fluoro, 2'-amino and / or 2'-hydroxyl. In some embodiments, modified nucleosides can include any one or more of 2'O-methyl, 2'-fluoro, 2'-amino and 2'-hydroxyl. For example, tRNA can be modified to include 2'O-methyl and / or 2'-fluoro modifications. In some embodiments, 2'O-methyl, 2'-fluoro and / or 2'-amino can be substituted at the 2'-hydroxyl position of the ribose sugar. In some embodiments, modified nucleosides can include any modification(s) suitable for the present disclosure.

[0022] As used herein, "phosphorothioate substitution" refers to a chemical modification of the tRNA backbone in which the single non-bridging oxygen of the phosphate is replaced with a sulfur atom. Phosphorothioate substitutions can stabilize the tRNA molecule by protecting it from hydrolysis.

[0023] As used herein, "2'-fluoro or 2'-O-methyl substitution at the 2'-hydroxyl position of the ribose sugar" refers to a 2'-sugar modification, such as a 2'-fluoro or 2'-O-methyl substitution at the 2'-hydroxyl position of the ribose sugar in RNA, which in some embodiments can increase the stability of the RNA in solution. Without wishing to be bound by any theory, a 2'-fluoro substitution at the 2'-hydroxyl position of the ribose sugar in an RNA backbone can be generated when the 2'-hydroxyl position (2'-OH) of the ribose sugar is replaced by a fluorine atom. A 2'-O-methyl substitution at the 2'-hydroxyl position of the ribose sugar in an RNA backbone can be generated when the 2'-hydroxyl position (2'-OH) of the ribose sugar is replaced by an O-methyl (O-Me, or O-CH3) group.

[0024] In some embodiments, the tRNAs described herein may further comprise one or more covalent conjugations, and the one or more modifications can include those made covalently. In some embodiments, the disclosure provides compositions comprising one or more tRNAs as described herein.

[0025] The present disclosure provides a method for increasing the half-life of tRNA, such as in vivo. The present disclosure provides a method for increasing the stability of tRNA, such as in vivo. In some embodiments, the methods described herein extend the circulation of tRNA, such as in vivo, by reducing susceptibility to nucleases. In some embodiments, the circulation of extended tRNA can affect the association affinity of tRNA with other molecules, resulting in improved availability of tRNA.

[0026] In some embodiments, increasing the half-life or stability of tRNA in vivo comprises modifying the nucleoside and / or backbone of tRNA. In some embodiments, the half-life or stability of tRNA is increased compared to a control. As used herein, "control" refers to a tRNA that is not modified in this way. A control may be the same tRNA as provided herein, but without modification. The comparison of half-life, stability, or any other characteristic can be assessed in vitro as known to those skilled in the art, or in vivo using a test subject.

[0027] In some embodiments, the half-life or stability of the modified tRNA is 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%, 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%, 108%, 109%, 109%, 100%. , 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% (inclusive of all values ​​therebetween).

[0028] In some embodiments, compositions are provided that include modified tRNA(s). In some embodiments, oligonucleotides are provided that encode modified tRNA(s). In some embodiments, compositions are provided that include oligonucleotide(s) provided herein. The oligonucleotide may be an expression cassette or any vector from which tRNA can be produced.

[0029] The present disclosure provides a method of treating a subject in need thereof, such as one with a disease or disorder. In some embodiments, the method comprises administering a tRNA or composition 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. tRNA can also be used to disrupt protein expression and / or function, preferably of a protein encoded by an oncogene or tumor suppressor gene. tRNA can also be used where there is a benefit to cell survival or mobility. tRNA can also be used to restore protein expression and / or function. In some embodiments, the disease or disorder may be any disease or disorder that can benefit from tRNA treatment.

[0030] In some embodiments, in subjects administered the tRNA, the disease or disorder is reduced 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%, 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%, 108%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 109%, 109%, 109%, 109%, 109%, 109%, 1 improved (e.g., less severe) by 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).

[0031] As used herein, a "genetic disease or disorder" is a disease or disorder that is inherited genetically or is caused by a mutation that occurs in the genetic code of a subject, or that predisposes the subject to the disease or disorder. Any one of the compositions or methods provided herein can be for treating or preventing a genetic disease or disorder.

[0032] As used herein, "hyperproliferative disease or disorder" refers to any disease or disorder in which cells have an abnormally high rate of proliferation, such as by 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 a use of a tRNA or composition 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.

[0033] The present disclosure provides a method for improving cellular uptake of a tRNA by administering a tRNA to a subject. In some embodiments, the cellular uptake is improved 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%, 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%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... improve by 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% (inclusive of all values ​​in between).

[0034] The present disclosure provides a method of increasing on-target activity by administering a tRNA to a subject. In some embodiments, the on-target activity is 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%, 67%, 68%, 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%, 108%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 109%, 109%, 109%, 109 %, 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% (inclusive of all values ​​therebetween).

[0035] The present disclosure provides a method of reducing immunogenicity caused by a tRNA by administering the tRNA to a subject. In some embodiments, the immunogenicity is reduced 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%, 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%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... 8%, 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% (inclusive of all values ​​therebetween).

[0036] The tRNA and the nucleotide sequences encoding the tRNA can be produced synthetically. Any one of the tRNAs provided herein can have the sequence of any tRNA, such as those provided herein or otherwise known in the art, but with modifications as provided herein. Nucleotide sequences encoding human tRNAs are known and generally available to those skilled in the art through sources such as GenBank. The structure of tRNAs is highly conserved, and tRNAs can function across species. Thus, bacterial or other eukaryotic tRNA sequences are also potential sources of tRNAs of the present invention, but with anticodons as provided herein. Determination of whether a particular tRNA is functional as desired, such as in a desired mammalian cell, can be confirmed as described herein or through other experiments that will be apparent to those skilled in the art with the benefit of the teachings provided herein. In one embodiment of any of the compositions or methods provided herein, the tRNA sequence can be, for example, any of the sequences provided in PCT / US2018 / 059065, WO2019 / 090154, WO2019 / 090169, and Lueck et al., Nature Communications 10, 822, 2019, which sequences are incorporated herein by reference, with the anticodons as provided herein.

[0037] The tRNA(s) may be in any form suitable for delivery to a target cell, either in vitro or in vivo (e.g., any suitable recombinant plasmid containing a heterologous nucleic acid sequence). 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.

[0038] The tRNA(s) or compositions as described herein can be delivered to cells in vivo or in vitro. Administration to cells can be accomplished by any means, including simply contacting the cells. Contacting the cells can be for any desired length of time. The cells can include any desired cells 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.

[0039] Methods for determining the amount of any of the modified tRNA molecules described herein effectively delivered to cells in vitro compared to the amount of control tRNA molecules delivered to cells in vitro are also provided or otherwise understood in the art.In some embodiments, the amount of modified and control tRNA molecules effectively delivered to cells in vitro can be determined by sequencing, measuring protein production rate, protein analysis, or any other technique otherwise understood in the art.In some embodiments, the verification of the increased level of modified tRNA molecules compared to control tRNA molecules represents such activity in vivo.

[0040] 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.

[0041] 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 of interest.The tRNA or composition 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 if it is present in the cell into which the tRNA is introduced.As will be clear 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.

[0042] In some embodiments, the tRNA or composition 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).

[0043] As will be clear to those skilled in the art in view of the teachings herein, the effective amount of the provided tRNA or composition 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 done, and the level and pattern of administration are selected by the treating physician. Vehicles including water, saline, artificial CSF, or other known substances can be employed. To prepare the formulation, the purified composition can be isolated. The composition can then be adjusted to the appropriate concentration and packaged for use.

[0044] 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 purpose is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as the growth, development or spread of cancer. Beneficial or desired 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 of 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.

[0045] The phrase "therapeutically effective amount" refers to an amount of a compound 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).

[0046] 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.

[0047] The tRNA or composition can 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 using animal models or other test systems known in the art.

[0048] Administration of the tRNA or composition 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 of the tRNA or composition may be essentially continuous over a preselected period of time, or may be in a series of spaced doses.

[0049] One or more suitable unit dosage forms having the tRNA or composition 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 containing the tRNA or composition can be prepared by procedures known in the art using well-known and readily available ingredients. The tRNA or composition can also be formulated as a solution suitable for administration. The pharmaceutical formulations of the tRNA or composition can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0050] Thus, the tRNA or composition may be formulated for administration and presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers, or in multi-dose containers with added preservatives. The active ingredient may take such forms as suspensions, solutions, or emulsions 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.

[0051] 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.

[0052] 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.

[0053] The tRNA or composition provided herein can be contacted with cells or delivered or administered to a subject in a particle such as a nanoparticle. The particle such as a nanoparticle can be, but is not limited to, a lipid-based nanoparticle (also referred to herein as lipid nanoparticle, i.e., a nanoparticle whose structure is made up of a majority of lipids), and / or a particle using a combination of nanomaterials. The particle can be a variety of different shapes, including, but not limited to, a sphere, a rectangular prism, a pyramid, an oblong, a cylindrical, a toroidal, and the like. In some embodiments, the particle such as a nanoparticle can include one or more lipids. In some embodiments, the particle such as a nanoparticle can include a liposome. In some embodiments, the particle such as a nanoparticle can include a lipid bilayer. In some embodiments, the particle such as a nanoparticle can include a lipid monolayer. In some embodiments, the particle such as a nanoparticle can include a micelle.

[0054] 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) (30-150 nm in diameter) that can be formed and released by many mammalian cells. The EVs or exosomes can be loaded with an agent of interest, for example, by pretreatment of the cells with the agent and subsequent isolation of the loaded EVs or exosomes. The EVs or exosomes can be derived from human fetal kidney cells, bone marrow stem cells, immature dendritic cells, red blood cells, and milk. The EVs or exosomes can be isolated and purified by a number of different techniques. Such methods include, but are not limited to, ultracentrifugation, ultrafiltration, size exclusion chromatography (SEC), precipitation with polymers, and isolation by affinity-based methods such as immunomagnetic-based isolation.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Electroporation may also be used. In one embodiment of any one of the compositions or methods provided herein, an oligonucleotide is provided that encodes any one of the modified tRNAs.In one embodiment of any one of the compositions or methods provided herein, the oligonucleotide described herein further comprises a promoter.Such an oligonucleotide may be contained in a vector.

[0064] 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 a suitable cell, which may comprise 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 delivered tRNA 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.

[0065] 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.

[0066] 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.

[0067] The present disclosure also provides a cell containing the tRNA, oligonucleotide, or vector 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 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.

[0068] In some embodiments of any one of the tRNAs provided herein, the tRNA is not found in nature. In some of the above, such tRNA is engineered or modified from that found in nature. In some of the above, such tRNA is a recombinant tRNA. In other embodiments of any one of the tRNAs provided herein, the tRNA is selected for improved activity as provided herein and can be used in any one of the methods provided herein.

[0069] The present invention is further illustrated by the following examples, which should not be construed as further limiting in any way. 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.

[0070] example Example 1. Evaluation of modified tRNA activity material and method Design and generation of modified ACE-tRNA The human ACE-tRNA sequence was used as the backbone for the generation of modified tRNAs tRNAArg(CCT)-2, tRNAArg(CCT)-3, tRNAArg(CCT)-4, and tRNAArg(TCT)-1. Specifically, the anticodon sequence of each of the tRNAs was altered to be complementary to the UGA stop codon. The ribose of the nucleotide at the beginning of the acceptor stem (N1, N2, N3, and / or N4) of the sequence was modified with a 2'-O-methyl group. The phosphate backbone between such nucleotides was changed to a phosphorothioate backbone. Some sequences contained both 2'-O-ME modifications and phosphorothioates. All sequences were synthesized by Trilink BioTechnologies and delivered as lyophilized powders. The tRNAs were resuspended in water to a concentration of 10 mg / mL.

[0071] cell culture HEK293T cells (from Dr. Chris Ahern, University of Iowa) stably expressing pNluc-UGA (nanoluciferase with UGA stop codon at position 160) were cultured in Dulbecco's Modified Eagle Medium (Gibco Catalog No. 10566-016) containing Glutamax and 4.5g / L glucose, and supplemented with 10% FBS and 1x penicillin / streptomycin. Cells were cultured in T75 tissue culture treated flasks in a 5% CO2 incubator at 37°C. For cell passaging, cells were first washed with PBS, then 3ml of 0.25% trypsin-EDTA was added to the flask, which was then incubated at 37°C for approximately 5 minutes. The flask was then returned from the incubator and 7ml of DMEM medium was added. Cells were counted and divided for different purposes.

[0072] Transient tRNA transfection 24 hours prior to transfection, HEK293T cells were seeded at 10,000 cells / well in 100 μl / well of DMEM medium containing Glutamax and 4.5 g / L glucose supplemented with 10% FBS without antibiotics in clear-bottom tissue culture treated 96-well plates. Cells were transfected with tRNA mutants in triplicate at 300 ng / ml, 600 ng / ml, 1,000 ng / ml, 3,000 ng / ml, and 10,000 ng / ml using Lipofectamine 3000 (Invitrogen Cat. No. L3000001), avoiding the wells at the perimeter of the plate. 24 hours after transfection, the medium was changed to DMEM medium containing Glutamax and 4.5 g / L glucose, and supplemented with 10% FBS and 1x penicillin / streptomycin.

[0073] Nanoluciferase detection assay 48 hours after transfection, the 96-well plates were removed from the incubator and tRNA activity was measured as follows: 100 μl of reconstituted nano-glo Dual Luciferase Reporter Assay System (Promega Cat# N1130) (200 μl of substrate in 10 mL of buffer) was added to each well and the plate was left on the bench for 10 minutes to allow complete dissolution. Luminescence was then measured using a Varioskan LUX plate reader (Thermo Fisher Cat# VL0000D0). The fold change in luminescence relative to the untreated signal was obtained by averaging the values ​​from each triplicate treatment and dividing the values ​​by the average background measurement from untreated wells.

[0074] result Surprisingly, it is found that the addition of 2'-O-methyl group to the first nucleobase of ACE-tRNA significantly improves its activity.Furthermore, it is found that the incorporation of phosphorothioate backbone into the chain of such nucleobase also improves its activity.Finally, the combination of both the incorporation of 2'-O-methyl group and phosphorothioate can improve the activity even further.

[0075] Equivalent While the foregoing specification and examples fully disclose and enable the invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto. All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with respect to specific embodiments thereof, 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 adaptable to additional embodiments, and that certain of the details set forth herein may be varied considerably without departing from the underlying principles of the invention.

[0076] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the present invention should be construed to cover both the singular and the plural, unless otherwise indicated herein or otherwise clearly contradicted by context. The terms "comprising", "having", "including" and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein is merely intended 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 set forth 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 (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation 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.

[0077] 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 foregoing description. The inventors expect that those skilled in the art will adopt such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, all combinations of the above-described elements in all possible variations thereof are covered by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A transfer RNA (tRNA), wherein the tRNA comprises one or more nucleoside modifications and / or one or more backbone modifications.

2. The tRNA of claim 1, wherein the one or more nucleoside modifications include a 2' sugar modification (e.g., 2'O-methyl).

3. 3. The tRNA of claim 1 or 2, wherein the one or more backbone modifications comprise a phosphorothioate internucleotide substitution.

4. The tRNA of any one of claims 1 to 3, wherein the modification is in the acceptor stem of the tRNA, optionally in one or more (e.g., all four) of the first four nucleotides / nucleobases.

5. 10. An oligonucleotide comprising a sequence encoding a modified tRNA according to any one of the preceding claims.

6. The oligonucleotide of claim 5, wherein the oligonucleotide is DNA or RNA.

7. The oligonucleotide of claim 5 or 6, further comprising a promoter and / or a terminator.

8. The oligonucleotide according to any one of claims 5 to 7, wherein the oligonucleotide is in plasmid or closed-ended form.

9. A vector, such as a viral vector, comprising the oligonucleotide according to any one of claims 5 to 8.

10. A composition comprising a tRNA according to any one of claims 1 to 4, an oligonucleotide according to any one of claims 5 to 8, or a vector according to claim 9, optionally further comprising a pharma- ceutically acceptable carrier.

11. A particle, such as a nanoparticle, comprising a tRNA according to any one of claims 1 to 4, an oligonucleotide according to any one of claims 5 to 8, or a vector according to claim 9.

12. The particle of claim 11 , wherein the particle is a liposome or a lipid nanoparticle.

13. 13. A composition comprising a particle according to claim 11 or 12, or a population of such particles, optionally further comprising a pharma- ceutically acceptable carrier.

14. A cell comprising a tRNA according to any one of claims 1 to 4, an oligonucleotide according to any one of claims 5 to 8, a vector according to claim 9, a particle according to claim 11 or 12, or a composition according to claim 10 or 13.

15. A method for increasing the activity of a tRNA (in vitro or in vivo), comprising modifying the tRNA to contain one or more nucleoside modifications and / or one or more backbone modifications.

16. A method for increasing the half-life or stability of a tRNA (in vitro or in vivo), comprising modifying the tRNA to contain one or more nucleoside modifications and / or one or more backbone modifications.

17. 17. The method of claim 15 or 16, wherein the one or more nucleoside modifications comprise 2'O-methyl.

18. 18. The method of any one of claims 15 to 17, wherein the one or more backbone modifications comprise a phosphorothioate internucleotide substitution.

19. The method of any one of claims 15 to 18, wherein the modification is in the acceptor stem of the tRNA.

20. The method of any one of claims 15 to 19, wherein the half-life or stability of the tRNA is increased compared to a control tRNA without the modification(s).

21. 21. The method of claim 20, wherein the half-life or stability is measured in vitro.

22. 21. The method of claim 20, wherein the half-life or stability is measured in a test subject, in a sample therefrom, or the like.

23. A method comprising administering to a subject in need thereof a tRNA according to any one of claims 1 to 4, an oligonucleotide according to any one of claims 5 to 8, a vector according to claim 9, a particle according to claim 11 or 12, or a composition according to claim 10 or 13.

24. 24. The method of claim 23, wherein the administration is systemic or local.

25. 25. The method of claim 23 or 24, wherein the subject is a human.

Images