Modified transport RNA with improved activity

Abstract

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

Description

[0001] Related Applications

[0002] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Application Serial No. 63 / 301,930, filed on January 21, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] DNA molecules carry genetic information in the form of nucleotide base sequences that constitute DNA polymers. Only four nucleotide bases are used in DNA: adenine, guanine, cytosine and thymine. This information in the form of codons of three consecutive bases is transcribed into messenger RNA (messenger RNA, mRNA), and then translated by transfer RNA (transfer RNA, tRNA) and ribosomes to form proteins. Four nucleotide bases are used in RNA: adenine, guanine, cytosine and uracil. The genetic code is the relationship between triplet codons and specific amino acids. Sixty-four possible codon triplets form the genetic code, of which three stop (also known as "terminating" or "nonsense") codons provide signals for the translation machinery (cellular ribosomes) to stop protein production at specific codons. Another sixty-one codon triplets (also known as "sense codons") correspond to one of the 20 standard amino acids. Summary of the invention

[0004] tRNA delivered to the body in an unmodified form may be more susceptible to degradation in vivo and thus may be less efficient and / or functional for effective therapeutic benefit. Provided herein are methods for providing modified tRNAs that may have enhanced activity (eg, potency).

[0005] In one aspect, compositions and related compositions and methods of use thereof comprising tRNAs with such modifications are provided herein. In some embodiments of any of the compositions or methods provided herein, at least one of any of the specific positions provided herein and / or as provided in any of the examples provided herein is modified to tRNA. Such tRNAs may have an extended circulation half-life and / or have different affinities for other molecules and / or have an increased presence or accumulation in one or more target tissues or cell types and / or have an increased uptake in one or more target tissues or cell types and / or have an increased functional readout in one or more target tissues or cell types.

[0006] In some embodiments of any of the compositions or methods provided herein, the tRNA has been modified so that at least one of any of the specific positions provided herein may have a chemical modification. In some embodiments of any of the compositions or methods provided herein, the modified tRNA has a 2' sugar modification (e.g., 2'-O-methyl (2'-O-methyl, 2'-OMe) modification). In some embodiments of any of the compositions or methods provided herein, the tRNA has been modified so that at least one of any of the specific positions provided herein may have a backbone modification. In some embodiments of any of the compositions or methods provided herein, the modified tRNA has a thiophosphate modification. In some embodiments of any of the compositions or methods provided herein, the modified tRNA has a 2' sugar modification (e.g., 2'-O-methyl (2'-OMe) modification) and a thiophosphate modification.

[0007] In some embodiments of any of the compositions or methods provided herein, the tRNA is any of the modified tRNAs provided herein in labeled or unlabeled form. In some embodiments of any of the compositions or methods provided herein, the tRNA is any of the modified tRNAs provided herein with or without a label and / or quencher.

[0008] In some embodiments, compositions comprising modified tRNA are provided. In some embodiments, oligonucleotides encoding modified tRNA are provided. In some embodiments, compositions comprising oligonucleotides provided herein are provided. The oligonucleotides can be expression cassettes or any vector from which modified tRNA can be produced.

[0009] In some embodiments, the modified tRNA or composition provided herein can be used for administration to a subject, for example, for treating a subject. In some embodiments, the modified tRNA or composition provided herein can be used to restore protein translation in a subject, for changing the resulting protein, etc. 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 a stop codon or other codon with an alternative amino acid in a protein or portion thereof.

[0010] Also provided herein are methods of producing modifications. Such methods may include any one or more of the specific steps provided herein.

[0011] Active tRNA molecules can have conserved secondary and tertiary structures as components of their activities, including, for example, specific recognition by aminoacyl-tRNA synthetases, specific recognition by elongation factors, coordination with ribosomal a, p, and e sites (aminoacyl, peptidyl, and exit sites), and / or interaction with the anticodon of an mRNA codon, etc.

[0012] In some embodiments, the modification is at one or more positions in the tRNA sequence. In some embodiments, the one or more positions are in the acceptor stem of the tRNA.

[0013] Some aspects of the disclosure relate to compositions comprising any of the tRNAs described herein.

[0014] In some embodiments, any of the improved effects of the tRNA (e.g., targeting of the tRNA) is improved compared to a control tRNA without the modification.

[0015] Some aspects of the disclosure relate to methods comprising administering to a subject in need thereof any of the tRNAs described herein or any of the compositions described herein, and optionally, in an effective amount to achieve any one or more of the improved effects provided herein, e.g., in the subject. In some embodiments, the administration is systemic or local administration. In some embodiments, the subject is a human.

[0016] Each limitation of the present invention can cover multiple embodiments of the present invention. Therefore, it is expected that each limitation of the present invention involving any element or combination of elements can be included in each aspect of the present invention. The present invention is not limited to the details of the arrangement and construction of the components set forth in the following description or illustrated in the accompanying drawings in terms of its application. The present invention can have other embodiments and can be practiced or implemented in a variety of ways. In addition, the wording and terminology used in this disclosure are for descriptive purposes and should not be considered as restrictive. "Including", "comprising", or "having", "containing", "involving" and its variants used in this disclosure are meant to cover the items listed thereafter and their equivalents and additional items. Unless otherwise expressly provided, nouns used in this specification and the appended claims that are not modified by quantifiers include plural indicators.

[0017] In some embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" is used herein to claim the specified integers or steps and those that do not materially affect the characteristics or functions of the claimed invention. As used herein, the term "consisting of" is used to indicate that only the enumerated integers (e.g., features, elements, properties, attributes, methods / processing steps, or limitations) or groups of integers (e.g., features, elements, properties, attributes, methods / processing steps, or limitations) are present. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present disclosure, which can be better understood by referring to one or more of these drawings in combination with the detailed description of some specific embodiments given in the present disclosure. The drawings are not intended to be drawn to scale. The drawings are merely illustrative and are not necessary to implement the present disclosure. For the purpose of clarity, not every component may be labeled in every drawing. In the drawings:

[0019] Figure 1 A table of the genetic code is provided.

[0020] Figure 2 The general four-arm structure of tRNA is shown, comprising a T arm, a D arm, an anticodon arm, and an acceptor stem (or arm). These regions as a whole may also be referred to as a "loop".

[0021] Figure 3 The activities of exemplary modified tRNAs are shown. DETAILED DESCRIPTION

[0022] Transfer RNA (tRNA) is the decoder of the DNA and RNA "blueprint". DNA transcription produces messenger RNA (mRNA) that encodes the primary amino acid structure, which can be post-transcriptionally modified and ultimately become a folded or unfolded protein after interacting with the ribosome and tRNA. Once the mRNA is attached to the ribosome, the tRNA delivers the amino acid to the ribosome and forms an amino acid chain based on the mRNA code ( Figure 1 ).

[0023] tRNA has a general four-arm structure, which includes a T arm, a D arm, an anticodon arm, and an acceptor stem or arm ( Figure 2). The T arm is composed of a "T stem" and a "TΨC loop". Any of the tRNAs provided herein may comprise this four-arm structure. In some embodiments, the tRNA is about 100 nucleotides in length and can be easily introduced into cells. The modified tRNA has the potential to correct defects in damaged or mutant mRNA molecules by delivering the correct amino acid where the incorrect amino acid would normally be delivered. The modified tRNA may also have the potential to reduce the amount of protein in the cell by delivering the incorrect amino acid where the correct amino acid would normally be delivered, thereby making the subsequent amino acid defective and unable to fold and / or susceptible to protein degradation.

[0024] It is expected that without the benefit of delivery agents, carriers, electromechanical facilitation, penetration enhancers, etc., engineered or therapeutic tRNAs delivered to the body in an unmodified form would be rapidly and extensively degraded and lack significant cellular uptake capacity, thereby presenting very limited potential for therapeutic benefit. Immune responses to tRNAs may also occur. Therefore, modified, engineered tRNAs have the potential to be effectively delivered to cells, which can improve therapeutic efficacy.

[0025] Compositions and methods related to tRNA-modified technologies are provided herein. In some embodiments, tRNAs can be modified to include one or more backbone modifications. In some embodiments, one or more modified nucleotides are located at one or more positions of the tRNA. In some embodiments, tRNAs can be modified to include one or more nucleoside modifications. In some embodiments, tRNAs can be modified to include one or more nucleoside modifications and one or more backbone modifications. Such tRNAs (e.g., Examples and Figure 3 Those with modified tRNAs have increased activity compared to unmodified tRNAs.

[0026] In some embodiments, the backbone modifications include, but are not limited to: phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl sugar inter-linkages or short chain heteroatom and heterocyclic sugar inter-linkages. In some embodiments, the modified tRNA may 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 may include any modification suitable for the present disclosure.

[0027] In some embodiments, modified nucleosides may include 2'O-methyl, 2'-fluoro, 2'-amino and / or 2'-hydroxyl. In some embodiments, modified nucleosides may include any one or more of 2'O-methyl, 2'-fluoro, 2'-amino and 2'-hydroxyl. For example, tRNA may be modified to include 2'O-methyl and / or 2'-fluoro modifications. In some embodiments, 2'O-methyl, 2'-fluoro and / or 2'-amino may be replaced in the 2'-hydroxyl position of ribose sugar. In some embodiments, modified nucleosides may include any modification suitable for the present disclosure.

[0028] As used herein, "phosphorothioate substitution" refers to a chemical modification of the tRNA backbone in which a single non-bridging oxygen of a phosphate is replaced by a sulfur atom. Phosphorothioate substitutions can stabilize tRNA molecules by preventing hydrolysis.

[0029] As used herein, "2'-fluoro or 2'-O-methyl replacement at the 2'-hydroxyl position of ribose sugar" refers to a 2'-sugar modification (e.g., 2'-fluoro or 2'-O-methyl replacement) at the 2'-hydroxyl position of ribose sugar in RNA, which in some embodiments can improve the stability of RNA in solution. Without wishing to be bound by any theory, a 2'-fluoro replacement at the 2'-hydroxyl position of ribose sugar in the RNA backbone can occur when the 2'-hydroxyl position (2'-OH) of the ribose sugar is replaced by a fluorine atom. A 2'-O-methyl replacement at the 2'-hydroxyl position of ribose sugar in the RNA backbone can occur when the 2'-hydroxyl position (2'-OH) of the ribose sugar is replaced by an O-methyl (O-Me or O-CH3) group.

[0030] In some embodiments, the tRNA described herein may further comprise one or more covalent conjugates and one or more modifications may comprise those that are covalently constituted.

[0031] In some embodiments, the disclosure provides compositions comprising one or more tRNAs as described herein.

[0032] The present disclosure provides methods for increasing the half-life of tRNA (e.g., in vivo). The present disclosure provides methods for increasing the stability of tRNA (e.g., in vivo). In some embodiments, the methods described herein extend the circulation of tRNA (e.g., in vivo) by reducing susceptibility to nucleases. In some embodiments, the extended circulation of tRNA can affect the binding affinity of tRNA to other molecules, so that the tRNA produces improved efficacy.

[0033] In some embodiments, improving the half-life or stability of tRNA in vivo includes modifying the nucleosides and / or the backbone of the tRNA. In some embodiments, the half-life or stability of the tRNA is improved compared to the control. "Control" as used herein refers to a tRNA that has not been modified in this way. The control can be the same tRNA but without the modification provided herein. The comparison of half-life, stability or any other characteristics can be evaluated in vitro as known to those of ordinary skill or evaluated in vivo using a test subject.

[0034] In some embodiments, the half-life or stability of the modified tRNA is increased by at least

[0035] 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 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.

[0036] In some embodiments, compositions comprising modified tRNA are provided. In some embodiments, oligonucleotides encoding modified tRNA are provided. In some embodiments, compositions comprising oligonucleotides provided herein are provided. The oligonucleotides can be expression cassettes or any vector from which tRNA can be produced.

[0037] The present disclosure provides methods for treating a subject in need thereof (e.g., a subject suffering from a disease or disorder). In some embodiments, the method comprises administering to the subject a tRNA or composition as disclosed herein. In some embodiments, the disease or disorder may comprise a genetic disease or disorder or a hyperproliferative disease or disorder. In some embodiments, the disease or disorder may be cancer. tRNA may also be used to disrupt protein expression and / or function, preferably protein expression and / or function of proteins encoded by oncogenes or tumor suppressor genes. tRNA may also be used where cell survival or movement is beneficial. tRNA may also be used to restore protein expression and / or function. In some embodiments, the disease or disorder may be any disease or disorder that may benefit from tRNA treatment.

[0038] In some embodiments, the disease or disorder in a subject administered the tRNA is improved (e.g., less severe) by at least

[0039] 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%.

[0040] 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,

[0041] Includes all values ​​in between.

[0042] As used herein, a "genetic disease or disorder" is a disease or disorder caused by an inherited mutation or mutations in the genetic code of a subject or mutations that predispose a subject to the disease or disorder. Any of the compositions or methods provided herein can be used to treat or prevent a genetic disease or disorder.

[0043] As used herein, "hyperproliferative disease or disorder" refers to any disease or disorder in which there is an abnormally high rate of cell proliferation caused by rapid division, excessive proliferation, etc. Certain embodiments of the present disclosure provide methods for treating a disease or disorder (e.g., a hyperproliferative disease or disorder) in a subject (e.g., a mammal). In certain embodiments, the mammal is a human. Certain embodiments of the present disclosure provide the use of tRNA or compositions as described herein for preparing a drug that can be used to treat a disease or disorder (e.g., a hyperproliferative disease or disorder) in a subject (e.g., a mammal (e.g., a human)). In certain embodiments, treatment has potential uses for treating / managing a disease or disorder, such as a hyperproliferative disease or disorder, including tumors, cancers, and neoplastic tissues, as well as non-neoplastic tissues or non-malignant hyperproliferative disorders. In certain embodiments, a hyperproliferative disease or disorder is cancer.

[0044] The present disclosure provides methods for improving cellular uptake of tRNA by administering tRNA to a subject. In some embodiments, the cellular uptake is improved by at least

[0045] 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 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.

[0046] The present disclosure provides methods for increasing on-target activity by administering tRNA to a subject. In some embodiments, the on-target activity is increased by at least 1:1 when compared to a control tRNA.

[0047] 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 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.

[0048] The present disclosure provides methods for reducing the immunogenicity caused by a tRNA by administering the tRNA to a subject. In some embodiments, the immunogenicity is reduced by at least

[0049] 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 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.

[0050] tRNA and nucleotide sequences encoding tRNA can be produced synthetically.

[0051] Any of the tRNA provided herein can have the sequence of any tRNA (e.g., those provided herein or otherwise known in the art), but with modifications as provided herein. The nucleotide sequence encoding human tRNA is known and can be obtained by a person skilled in the art through sources such as GenBank. The structure of tRNA is highly conservative, and tRNA can be functional between species. Therefore, bacteria or other eukaryotic tRNA sequences are also potential sources for the tRNA of the present invention, but with anticodons as provided herein. Whether a specific tRNA has the functionality as desired (e.g., in a desired mammalian cell) can be determined as described herein or by other experiments that are obvious to those of ordinary skill in the art who benefit from the teachings provided herein. In one embodiment of any of the compositions or methods provided herein, the tRNA sequence can be any of the sequences provided in PCT / US2018 / 059065, WO2019 / 090154, WO2019 / 090169, and Lueck et al., Nature Communications 10, 822, 2019 (the sequences of which are incorporated herein by reference), for example, but with an anticodon as provided herein.

[0052] The tRNA can be in any form suitable for delivery to a target cell in vitro or in vivo (e.g., any suitable recombinant plasmid comprising a heterologous nucleic acid sequence). For purposes such as any of the uses provided herein (including disease treatment), the heterologous nucleic acid sequence encodes a target gene product (e.g., tRNA) and is optionally in the form of an expression cassette. The term "recombinant" refers to a polynucleotide that does not exist in nature or a polynucleotide that is connected to another polynucleotide in an arrangement that does not exist in nature. The term "heterologous" as used herein refers to a nucleic acid sequence obtained or derived from an entity that is genetically different from the rest of the entity being compared.

[0053] As described herein, tRNA or compositions can be delivered to cells in vivo or in vitro. Administration to cells can be accomplished by any means, including simple contact with the cells. Contact with cells can last for any desired length of time. Cells can include any desired cells in humans and other large (non-rodent) mammals (e.g., primates, horses, sheep, goats, pigs, and dogs). Any of the objects provided herein can be humans or other mammals. The term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cattle, pigs, and sheep.

[0054] Also provided is a method for determining the amount of any of the modified tRNA molecules described herein that have been effectively delivered to cells in vitro compared to the amount of control tRNA molecules delivered to cells in vitro, or as otherwise understood in the art. In some embodiments, the amount of modified tRNA molecules and control tRNA molecules that have been effectively delivered to cells in vitro can be determined by sequencing, protein production rate measurement, protein analysis, or any other technique otherwise understood in the art. In some embodiments, verification of increased levels of modified tRNA molecules compared to control tRNA molecules indicates such activity in vivo.

[0055] In some embodiments, administration can be performed systemically. In some embodiments, administration can be performed locally. In some embodiments, administration can be delivered directly to selected organs by oral, inhalation, intraocular, intravenous, including facial vein injection and retro-orbital injection, intraventricular (intracerebroventricular, ICV), intracerebellomedullary cisterna (intracisterna magna, ICM) injection, intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral and other parenteral administration routes. If desired, the administration routes can be combined. In some embodiments, tRNA as disclosed herein can be administered by any route suitable for the present disclosure.

[0056] Also provided is a suitable method for delivery and introduction into a subject, or otherwise understood in the art. In one embodiment, the pharmaceutical composition will include enough 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 enter into a cell, such as together with an endogenous tRNA synthetase. If a tRNA synthetase is present in a cell into which the tRNA has been introduced, it is considered that the tRNA synthetase is "endogenous" to the cell. It is obvious to one of ordinary skill in the art that, for these purposes, a tRNA synthetase can be considered to be endogenous, whether it is naturally present in a cell of the relevant type, or the specific cell in question has been transformed or otherwise artificially manipulated to contain or express it.

[0057] In some embodiments, tRNA or composition can be formulated in a pharmaceutical composition. The pharmaceutical composition may also include a pharmaceutically acceptable excipient. Such an excipient includes any medicament that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids (e.g., water, saline, glycerol, and ethanol). Pharmaceutically acceptable salts may be included, for example: inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., and organic acid salts such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may be present in such carriers. A comprehensive discussion of pharmaceutically acceptable excipients can be obtained in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0058] In view of the teachings of this specification, it is obvious to those skilled in the art that the effective amount of the tRNA or composition provided can be determined empirically. It can be administered continuously or intermittently in one dose throughout the treatment process. The method of determining the most effective means and dosage of administration can vary according to the therapeutic composition, target cell and treatment object, etc. Single and multiple administrations can be performed according to the mode and dosage level selected by the treating physician.

[0059] Carriers include water, aqueous saline, artificial CSF or other known substances that can be used. In order to prepare the preparation, the purified composition can be isolated. The composition can then be adjusted to an appropriate concentration and packaged for use.

[0060] The term "treatment" and its variations used herein refer to both therapeutic treatment and measures that can alleviate symptoms or provide some benefit to the subject, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as the growth, progression or spread of cancer. Beneficial or desired clinical results include, but are not limited to: symptom relief, weakened disease extent, stable disease state (i.e., no worsening), delayed or slowed disease progression, improved or alleviated disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" also means prolonged survival compared to expected survival without treatment. Those with treatment needs include those who already suffer from illness, disease or disorder.

[0061] The phrase "therapeutically effective amount" means an amount of a compound of the invention that (i) treats a specific disease, condition, or disorder described herein, (ii) attenuates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder described herein, or (iii) prevents one or more symptoms of a specific disease, condition, or disorder described herein or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow down and preferably stop to some extent) cancer cell infiltration into peripheral organs, inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that a drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer treatment, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the responder rate (RR).

[0062] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. A "tumor" contains one or more cancerous cells. Some examples of cancer include, but are not limited to, carcinomas, malignancies, etc.

[0063] The tRNA or composition can be administered so as to result in the alleviation 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 selected composition, the specific disease, the weight, physical condition, and age of the mammal. Such factors can be readily determined by a clinician using animal models or other test systems known in the art.

[0064] Administration of the tRNA or composition can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic, and other factors known to skilled practitioners. Administration of the tRNA or composition can be substantially continuous over a preselected period of time, or can be a series of spaced doses.

[0065] One or more suitable unit dosage forms with tRNA or compositions of the present invention can be prepared and can be administered by a variety of routes. When preparing the medicaments of the present invention for administration, they can be combined with pharmaceutically acceptable carriers, diluents or excipients to form pharmaceutical preparations or unit dosage forms. The total active ingredients in such preparations contain 0.1% to 99.9% by weight of the preparation. "Pharmaceutically acceptable" is a carrier, diluent, excipient and / or salt that is compatible with the other ingredients of the preparation and harmless to its recipient. Pharmaceutical preparations containing tRNA or compositions can be prepared using well-known and readily available ingredients by procedures known in the art. tRNA or compositions can also be formulated into solutions suitable for administration. Pharmaceutical preparations of tRNA or compositions can also take the form of aqueous or anhydrous solutions or dispersions, or alternatively in the form of emulsions or suspensions.

[0066] Therefore, tRNA or composition can be formulated for administration, and can be present in an ampoule with an added preservative, a prefilled syringe, a small volume infusion container, or a multi-dose container in a unit dosage form. The active ingredient can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain a formulation (formulatoryagent) such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the active ingredient can be placed in a suitable carrier (e.g., sterile, pyrogen-free water) before use. It should be understood that the unit content of one or more active ingredients contained in a single dose of each dosage form does not itself need to constitute an effective amount for treating a specific indication or disease, because the required effective amount can be achieved by administering multiple dosage units. In addition, the effective amount can be achieved using less than the dosage in the dosage form alone or in a series of administrations.

[0067] The pharmaceutical preparation of the present invention may include, for example, optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers and salts of types well known in the art. Some specific non-limiting examples of carriers and / or diluents that can be used in the pharmaceutical preparation of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions and water at pH 7.0 to 8.0.

[0068] Any of the compositions provided herein can be contacted with cells, applied to cells or introduced into cells using a genetic transfer method (e.g., transfection). Therefore, any of the compositions provided herein can be included with a gene delivery vehicle or in a gene delivery vehicle. The gene delivery vehicle can be any delivery vehicle known in the art, and can include a naked nucleic acid promoted by a receptor and / or lipid-mediated transfection.

[0069] The tRNA or compositions provided herein can be contacted with a cell, or delivered to or applied to a subject in a particle (e.g., nanoparticle). The particle (e.g., nanoparticle) can be, but is not limited to, a lipid-based nanoparticle (also referred to herein as a lipid nanoparticle, i.e., a nanoparticle whose majority of the material constituting its structure is a lipid) and / or a particle having a combination of nanomaterials. The particle can be a variety of different shapes, including but not limited to spherical, cubic, pyramidal, rectangular, cylindrical, annular, etc. In some embodiments, the particle (e.g., nanoparticle) may comprise one or more lipids. In some embodiments, the particle (e.g., nanoparticle) may comprise a liposome. In some embodiments, the particle (e.g., nanoparticle) may comprise a lipid bilayer. In some embodiments, the particle (e.g., nanoparticle) may comprise a lipid monolayer. In some embodiments, the particle (e.g., nanoparticle) may comprise a micelle.

[0070] The tRNA or composition provided herein can be contacted with a cell or delivered to or applied to a subject together with an extracellular vesicle or an exosome. In general, an exosome is an extracellular vesicle (EV) of nanometer size (diameter is 30 to 150nm), which can be formed and released by many mammalian cells. EV or exosomes can be loaded with a target agent, for example, by pretreating cells with an agent, and then separating the loaded EV or exosomes. EV or exosomes can be derived from human embryonic kidney cells, bone marrow stem cells, immature dendritic cells, erythrocytes, and from milk. EV or exosomes can be separated and purified with many different techniques. Such methods include, but are not limited to, ultracentrifugation, ultrafiltration, size exclusion chromatography (SEC), polymer precipitation, and separation by affinity-based methods (e.g., separation based on immunomagnetism).

[0071] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encoding tRNA is a closed end form, such as a plasmid, a nanoplasmid or a mini-ring. In one embodiment of any one of the compositions or methods provided herein, the nucleic acid sequence encoding tRNA is a mini-ring or micro-thread (microthread) form.

[0072] The term "minicircle" as used herein refers to a small circular DNA fragment that is mostly or completely free of non-essential prokaryotic elements. Minicircles include DNA in a circular form without prokaryotic elements and / or in which prokaryotic elements have been removed. Minicircles can be from parent plasmids, in which bacterial DNA sequences have been excised. Minicircles can be in the form of any suitable recombinant plasmid, which contains heterologous nucleic acid sequences to be delivered to target cells in vitro or in vivo. The preparation of minicircles has been described in the art (e.g., in Nehlsen et al., Gene Ther. Mol. Biol. 10: 233-244, 2006; and Kay et al., Nature Biotechnology. 28: 1287-1289, 2010). The preparation can, for example, follow the following two-step procedure: (i) produce a "parent plasmid" (a bacterial plasmid with a eukaryotic insert); and (ii) induce a site-specific recombinase at the end of the process. These steps can be followed by excision of the prokaryotic vector portion by targeting the sequence of the recombinase and recovery by capillary gel electrophoresis.

[0073] As a non-limiting example, minicircles can be produced as follows. An expression cassette comprising a polynucleotide coding sequence and regulatory elements for its expression is flanked by attachment sites for a recombinase. The sequence encoding the recombinase is located outside the expression cassette and comprises elements for inducible expression (e.g., such as an inducible promoter). When the recombinase is induced to express, the vector DNA is recombined to produce two different circular DNA molecules. One of the circular DNA molecules is relatively small and forms a minicircle containing an expression cassette of a polynucleotide; the minicircle DNA vector lacks any bacterial DNA sequence. The second circular DNA sequence contains the remaining vector sequences, including bacterial sequences and sequences encoding a recombinase. The minicircle DNA containing the polynucleotide sequence can then be separated and purified separately. In some embodiments, plasmids similar to pBAD.φ.C31.hFIX and pBAD.φ.C31.RHB can be used to produce minicircle DNA vectors. See, for example, Chen et al. (2003) Mol.Ther.8:495-500, or as otherwise provided herein.

[0074] Some examples of recombinases that can be used to generate minicircles include, but are not limited to, Streptomyces phage φ31 integrase, Cre recombinase, and λ integrase / DNA topoisomerase IV complex. Each of these recombinases catalyzes recombination between different sites. For example, φ31 integrase catalyzes recombination between corresponding attP and attB sites, Cre recombinase catalyzes recombination between loxP sites, and λ integrase / DNA topoisomerase IV complex catalyzes recombination between phage λattP and attB sites.

[0075] Published U.S. Application 20170342424 also describes a system utilizing a parent plasmid that is exposed to an enzyme that causes recombination at a recombination site, thereby forming (i) a minicircle comprising a polynucleotide sequence and (ii) a miniplasmid comprising the remainder of the parent plasmid. One recombination site is modified at the 5' end so that it reacts less efficiently with the enzyme than the wild-type site, and another recombination site is modified at the 3' end so that it reacts less efficiently with the enzyme than the wild-type site, and another recombination site is modified at the 3' end so that it reacts less efficiently with the enzyme than the wild-type site, and both modified sites are located in the minicircle after recombination.

[0076] Using the smallest possible excision site, removal of prokaryotic sequences should ideally be efficient while generating a supercoiled DNA minicircle consisting only of gene expression elements under appropriate (preferably mammalian) control regions. Some techniques for minicircle generation use bacterial phage lambda (λ) integrase-mediated recombination to generate minicircle DNA. See, e.g., Darquet, et al. 1997 Gene Ther 4(12):1341-9; Darquet et al. 1999 Gene Ther 6(2):209-18; and Kreiss, et al. 1998 Appl Micbiol Biotechnol 49(5):560-7).

[0077] Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). For example, the MC-EasyTM (Catalog No. MN920A-1, SBI System Biosciences) minicircle DNA production kit can be used to obtain high-quality minicircle DNA. Information about minicircle DNA is provided in Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 21 8, 1526-1535 and Hou et al., Molecular Therapy—Methods & Clinical Development, Article number: 14062 (2015) doi: 10.1038 / mtm.2014.62. More information about minicircles is provided in Chen ZY, He CY, Ehrhardt A, Kay M A. Mol Ther. 2003 September; 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 January; 21(1): 131.

[0078] In some embodiments, the closed end form is a supercoiled coil. DNA supercoiled refers to the amount of distortion in a specific DNA chain. Supercoiled DNA can be positive supercoiled DNA or negative supercoiled DNA. "Supercoiled DNA" used herein refers to a DNA molecule or a fragment of a DNA molecule, wherein one or two DNA chains contain increased distortion compared to the amount of distortion in a reference state called "relaxed B-form" DNA. In the "relaxed" double helix segment of DNA, the two chains are twisted around the helical axis, once every 10.4 to 10.5 sequence base pairs. A given DNA chain can be a "positive supercoil" or a "negative supercoil" (i.e., more or less tightly wound (wound)). Supercoiled produces a twisted chain in a DNA chain. The amount of supercoiled chain affects many biological processes, such as compressing DNA and regulating access to genetic codes (which strongly affects DNA metabolism and possible gene expression). Certain enzymes (e.g., topoisomerases) can increase or decrease the amount of distortion (e.g., supercoiled) in a DNA chain to promote functions such as DNA replication and transcription. If a DNA segment under twisting strain is closed into a loop by ligating its two ends and then allowed to move freely, it can form a superhelical structure. Some examples of superhelical structures of circular DNA molecules include, but are not limited to, figure-of-eight structures, plectonemic structures, or toroidal structures.

[0079] Electroporation may also be used.

[0080] In one embodiment of any one of the compositions or methods provided herein, oligonucleotides encoding any modified tRNA are provided. In one embodiment of any one of the compositions or methods provided herein, the oligonucleotides described herein also include a promoter. Such oligonucleotides can be included in a vector.

[0081] Promoter characteristically has specific nucleotide sequence required for initiating transcription.In some embodiments, oligonucleotide as provided herein comprises a nucleotide sequence capable of instructing specific nucleotide sequence to be expressed in suitable cells, which may comprise a promoter operably connected to a target nucleotide sequence, which may also be operably connected to a termination signal (or terminator). Oligonucleotide as provided herein may be a recombinant form that can be used for heterologous expression.In certain embodiments, promoter is an adjustable promoter.In certain embodiments, promoter is a constitutive promoter. The promoter driving the expression of the sequence encoding the tRNA to be delivered may be any desired promoter, which is selected by known considerations, such as the expression level of the nucleic acid connected to the promoter function and the cell type of the carrier to be used. Promoter may be an exogenous or endogenous promoter.

[0082] In one embodiment of any one of the compositions or methods provided herein, the promoter can be (size is 35 to 105bp). The promoter can be any known promoter, which includes a natural tRNA leader sequence, the size of which can be about 50 to 60bp. In one embodiment of any one of the compositions or methods provided herein, the promoter can be a reduced sequence or a reconstructed promoter. Exemplary promoter sequences that can be included in any one of the oligonucleotides or vectors provided herein include, but are not limited to, any one of the sequences provided herein or otherwise known in the art.

[0083] Optionally, the oligonucleotides or vectors provided herein may also contain additional sequences (e.g., 3' tRNA tail or trailer sequence). Such sequences may be 2 to 20 bp in length. They may contain native sequences or engineered variants having 3 to 10 consecutive "T" residues.

[0084] The disclosure also provides cells comprising tRNA, oligonucleotides or vectors as described herein. The cells can be mammalian, such as human. According to one aspect, a cell expression system is provided. The expression system comprises cells and oligonucleotides or vectors as provided herein. Expression cassettes include, but are not limited to, plasmids, viral vectors and other carriers for delivering heterologous genetic material to cells. The cell expression system can be formed in vivo.

[0085] In some embodiments of any of the tRNAs provided herein, the tRNA is a tRNA that does not exist in nature. In some of the foregoing embodiments, such tRNA is transformed or modified from a tRNA that exists in nature. In some of the foregoing embodiments, such tRNA is a recombinant tRNA. In other embodiments of any of the tRNAs provided herein, the tRNA is selected for improved activity, as provided herein, and can be used in any of the methods provided herein.

[0086] The present invention is further illustrated by the following examples, which should in no way be construed as further limiting. The entire contents of all references cited throughout this application (including literature references, granted patents, published patent applications, and co-pending patent applications) are expressly incorporated by reference herein.

[0087] Example

[0088] Example 1. Evaluation of modified tRNA activity

[0089] Materials and methods

[0090] Design and generation of modified ACE-tRNA

[0091] The human ACE-tRNA sequence is used as a backbone for producing modified tRNA, tRNAArg (CCT) -2, tRNAArg (CCT) -3, tRNAArg (CCT) -4, tRNAArg (TCT) -1. In particular, the anticodon sequence of each tRNA is changed to be complementary to the UGA stop codon. The ribose of the nucleotides (N1, N2, N3 and / or N4) at the beginning of the acceptor stem of the sequence is modified with 2'-O-methyl. The phosphate backbone between such nucleotides is changed to a thiophosphate backbone. Some sequences include both 2'-O-ME modifications and thiophosphates. All sequences are synthesized by Trilink Bio Technologies and delivered as lyophilized powders. The tRNA is resuspended in water to a concentration of 10 mg / ml.

[0092] Cell culture

[0093] 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's medium (Gibco catalog number 10566-016) containing Glutamax and 4.5g / L glucose and supplemented with 10% FBS and 1× penicillin / streptomycin. The cells were cultured in a T75 tissue culture treated bottle at 37°C in a 5% CO2 incubator. For cell passage, the cells were first washed with PBS, and then 3ml of 0.25% trypsin-EDTA was added to the bottle, which was then incubated at 37°C for about 5 minutes. The bottle was then removed from the incubator and 7ml of DMEM medium was added. Cells were counted and split for different purposes.

[0094] Transient tRNA transfection

[0095] 24 hours before transfection, HEK293T cells were seeded in 100 μl / well DMEM culture medium with 10,000 cells / well in a treated white transparent bottom tissue culture 96-well plate, the culture medium containing Glutamax and 4.5g / L glucose, supplemented with 10% FBS and without antibiotics. Using Lipofectamine 3000 (Invitrogen catalog number L3000001), cells were transfected in triplicate with 300ng / ml, 600ng / ml, 1,000ng / ml, 3,000ng / ml and 10,000ng / ml tRNA variants, but avoiding the holes on the periphery of the plate. 24 hours after transfection, the culture medium was replaced with a DMEM culture medium containing Glutamax and 4.5g / L glucose and supplemented with 10% FBS and 1× penicillin / streptomycin.

[0096] Nano-luciferase detection assay

[0097] 48 hours after transfection, 96 orifice plates were taken out from incubator, and the activity of tRNA was measured in the following manner. The nano-glo dual luciferase reporter assay system (Promega catalog number N1130) (200 μl substrates were added to 10 ml buffer) reconstructed by 100 μl was added to each well, and the plate was placed on the bench for 10 minutes to allow complete lysis. Then Varioskan LUX plate reader (Thermo Fisher catalog number VL0000D0) was used to measure luminescence. By averaging the value from each triplicate process and dividing the value by the average background measurement value from untreated holes, the luminescence multiple of untreated signal was obtained.

[0098] result

[0099] It was unexpectedly found that adding a 2'-O-methyl to the first nucleobase of ACE-tRNA significantly improved its activity. In addition, it was found that incorporating a phosphorothioate backbone into a chain of such nucleobases also improved its activity. Finally, combining the 2'-O-methyl and incorporating phosphorothioates can even further improve activity.

[0100] Equivalent solution

[0101] While the foregoing description and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the appended claims.

[0102] All publications, patents and patent applications are incorporated herein by reference. Although the present invention has been described in conjunction with certain embodiments of the present invention in the foregoing description, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is susceptible to other embodiments and that considerable changes may be made to certain details described herein without departing from the basic principles of the present invention.

[0103] Unless otherwise indicated herein or obviously contradictory with context, otherwise the nouns modified by the use of quantifiers in the context of describing the present invention will be interpreted as one / kind or more / kind. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" will be interpreted as open terms (that is, meaning "including but not limited to"). Unless otherwise indicated herein, the recording of the value range herein is only intended to be used as a shorthand method for referring to each individual value falling within the range respectively, and each individual value is incorporated into the specification as it is recorded separately in this article. Unless otherwise indicated herein or obviously contradictory with context, all methods described herein can be carried out in any suitable order. Unless otherwise indicated, the use of any and all examples or exemplary languages ​​(such as "for example") provided herein is only intended to better illustrate the present invention, and is not intended to limit the scope of the present invention. The language in the specification should not be interpreted as representing any unclaimed element necessary for the practice of the present invention.

[0104] Some embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, the changes of those embodiments may become obvious to those of ordinary skill in the art. The inventor expects that the technician will adopt such changes when appropriate, and the inventor wishes to practice the present invention in a manner other than that specifically described herein. Therefore, as allowed by applicable law, the present invention includes all modifications and equivalents of the subject matter recorded in the appended claims. In addition, unless otherwise specified herein or otherwise clearly contradicted with the context, the present invention encompasses any combination of the above-mentioned elements in all possible changes thereof.

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 comprise a 2' sugar modification (eg, 2' O-methyl).

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

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 (eg, in all four) of the first four nucleotides / nucleobases.

5. An oligonucleotide comprising a sequence encoding the 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 according to claim 5 or 6, further comprising a promoter and / or a terminator.

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

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

10. A composition comprising the tRNA of any one of claims 1 to 4, the oligonucleotide of any one of claims 5 to 8, or the vector of claim 9, optionally further comprising a pharmaceutically acceptable carrier.

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

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

13. A composition comprising the particles of claim 11 or 12, or a population of such particles, optionally further comprising a pharmaceutically acceptable carrier.

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

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

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

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

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

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.

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

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

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

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

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

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