Novel use of aspirin compounds in increasing nucleic acid expression

Abstract

Provided is the use of an aspirin compound in promoting the delivery and / or expression of exogenous nucleic acids.

Description

Technical Field

[0001] The present disclosure relates to the use of aspirin compounds in promoting the delivery and / or expression of exogenous nucleic acids. Background Art

[0002] For many years, genetic disorders and gene-related diseases have been responsible for high mortality rates and reduced quality of life. Some congenital anomalies manifest very early, and these children have little hope of survival, which causes great distress to their families because treatment options are limited and there is little they can do to modify the disease. Gene therapy is a new form of molecular medicine that involves the transduction of fully functional foreign genes into an individual's cells or tissues to replace defective genes and modify genetic diseases. Gene therapy has the potential to correct genetic diseases such as hemophilia, familial hypercholesterolemia, Parkinson's disease, and Alzheimer's disease.

[0003] Although gene therapy has its advantages, it also has disadvantages such as low expression levels and short expression duration. Therefore, it is necessary to overcome the problems of existing exogenous gene transduction technology (especially viral transduction technology, more preferably AAV transduction technology) and provide a method for promoting exogenous gene expression. Summary of the Invention

[0004] In one aspect, the present disclosure provides a method of preparing a cell for delivery of an exogenous nucleic acid, the method comprising administering an aspirin compound to the cell prior to or concurrently with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivery.

[0005] In one aspect, the present disclosure provides a method for expressing an exogenous nucleic acid in a cell, the method comprising: delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein the cell has been or is being simultaneously administered with an aspirin compound, and wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted into double-stranded DNA in the cell after delivery.

[0006] In one aspect, the present disclosure provides a method for expressing an exogenous nucleic acid in a cell, the method comprising: a) administering an aspirin compound to the cell; and b) delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein step a) is performed before or simultaneously with step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted into double-stranded DNA in the cell after delivery.

[0007] In one aspect, the present disclosure provides a method for increasing the expression level of an exogenous nucleic acid in a cell, the method comprising administering an aspirin compound to the cell before or simultaneously with delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivery, and thereby increasing the expression level of the exogenous nucleic acid as compared to a control expression level obtained in a control cell not administered the aspirin compound.

[0008] In one aspect, the present disclosure provides a method for increasing the expression level of an exogenous nucleic acid in a cell, the method comprising: delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein the cell has been or is being concurrently administered with an aspirin compound, wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the cell after delivery, and thereby increasing the expression level of the exogenous nucleic acid as compared to a control expression level obtained in a control cell not administered with the aspirin compound.

[0009] In one aspect, the present disclosure provides a method for increasing the expression level of an exogenous nucleic acid in a cell, the method comprising: a) administering an aspirin compound to the cell; and b) delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein step a) is performed before or simultaneously with step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the cell after delivery, and wherein the expression level of the exogenous nucleic acid is increased compared to a control expression level obtained in a control cell not subjected to step a).

[0010] In one aspect, the present disclosure provides a method for extending the duration of expression of an exogenous nucleic acid in a cell, the method comprising: administering an aspirin compound to the cell before or simultaneously with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the cell after delivery, and wherein the duration of expression of the exogenous nucleic acid in the cell is increased compared to a control duration of expression obtained in a control cell not administered the aspirin compound.

[0011] In one aspect, the present disclosure provides a method for extending the duration of expression of an exogenous nucleic acid in a cell, the method comprising: delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein the cell has been or is being simultaneously administered an aspirin compound, wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the cell after delivery, and wherein the duration of expression of the exogenous nucleic acid in the cell is increased compared to a control duration of expression obtained in a control cell not administered the aspirin compound.

[0012] In one aspect, the present disclosure provides a method for extending the duration of expression of an exogenous nucleic acid in a cell, the method comprising: a) administering an aspirin compound to the cell; and b) delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein step a) is performed before or simultaneously with step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the cell after delivery, and thereby extending the duration of expression of the exogenous nucleic acid compared to a control duration of expression obtained in a control cell not administered the aspirin compound.

[0013] In certain embodiments, the cell is in vitro, ex vivo or in vivo. In certain embodiments, the aspirin compound is administered to the cell at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days before the nucleic acid is delivered. In certain embodiments, the aspirin compound is administered once or repeatedly (for example, twice, three times, four times, etc.) to the cell before the nucleic acid is delivered. In certain embodiments, the aspirin compound is administered in an amount sufficient to increase the expression of the exogenous nucleic acid in the cell or the subject by at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more. In certain embodiments, the expression level is based on mRNA level or protein level. In some embodiments, the expression level is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 700%, 800%, or 900%. In some embodiments, the expression level is determined over the duration of expression of the exogenous nucleic acid. In some embodiments, the duration of expression is the time period during which the exogenous nucleic acid is expressed at a detectable level or at a physiologically effective level. In some embodiments, the duration of expression is extended by at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0014] In some embodiments, the exogenous nucleic acid comprises double-stranded DNA, and wherein the double-stranded DNA comprises a double-stranded DNA viral vector, a double-stranded plasmid or a double-stranded artificial chromosome. In some embodiments, the exogenous nucleic acid can be converted into double-stranded DNA after delivery to a cell or subject, and wherein the exogenous nucleic acid comprises single-stranded DNA, a retroviral vector or a lentiviral vector. In some embodiments, the exogenous nucleic acid comprises a viral vector (e.g., an adeno-associated virus (AAV) vector, a lentiviral vector, a retroviral vector or an adenoviral vector), a plasmid or an exosome or is included in the viral vector, plasmid or exosome. In some embodiments, the viral vector comprises an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector comprises an AAV viral particle. In some embodiments, the AAV vector comprises a cap gene encoding a capsid protein. In some embodiments, the AAV vector comprises an AAV viral particle, and the AAV viral particle comprises a natural or recombinant capsid protein. In some embodiments, the capsid protein can be modified, chimeric or synthetic. In some embodiments, the cap gene or the capsid protein is derived from two or more AAV serotypes. In some embodiments, the cap gene or the capsid protein can have specific tropism characteristics.

[0015] In certain embodiments, the exogenous nucleic acid comprises a coding sequence encoding a protein of interest or a portion thereof, or encoding a functional RNA or a portion thereof. In certain embodiments, the protein of interest comprises a therapeutic protein, an immunogenic protein, a reporter protein, a nuclease or a therapeutic target protein, and / or the functional RNA comprises an antisense oligonucleotide, a ribozyme, an RNA that affects spliceosome mediation / original splicing, an interfering RNA (RNAi) or other non-translated functional RNA, such as a guide RNA and a single guide RNA. In certain embodiments, the exogenous nucleic acid is delivered to a subject or cell under conditions suitable for expression. In certain embodiments, the coding sequence is operably connected to one or more regulatory sequences.

[0016] In another aspect, the present disclosure provides a method for preparing a subject for a condition treatable by an exogenous nucleic acid or its expression product, the method comprising: administering to the subject an effective amount of an aspirin compound before or simultaneously with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid of the present disclosure comprises double-stranded DNA, or can be converted into double-stranded DNA in the subject after delivery.

[0017] In another aspect, the present disclosure provides a method for treating or preventing a condition that can be treated or prevented by an exogenous nucleic acid or an expression product thereof, the method comprising: delivering a therapeutically effective amount of the exogenous nucleic acid to the subject, wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted into double-stranded DNA in the subject after delivery, and wherein the subject has been or is being concurrently administered an aspirin compound.

[0018] In another aspect, the present disclosure provides a method for treating or preventing a condition that can be treated or prevented by an exogenous nucleic acid or its expression product, the method comprising: a) administering an effective amount of an aspirin compound to the subject; and b) delivering a therapeutically effective amount of the exogenous nucleic acid to the subject, wherein step a) is performed before or simultaneously with step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted into double-stranded DNA in the cell after delivery.

[0019] In some embodiments, the condition is characterized by the absence of one or more functional genes or functional proteins. In some embodiments, the condition is a monogenic disorder. In some embodiments, the monogenic disorder is autosomal dominant, autosomal recessive, X-linked, Y-linked, or mitochondrial.

[0020] In certain embodiments, the treatable condition is a CNS disorder. In certain embodiments, the CNS disorder is selected from the group consisting of Parkinson's disease, Alzheimer's disease, mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, Batten disease, spinocerebellar ataxia, spinal muscular atrophy, Canavan disease, and Friedreich's ataxia.

[0021] In certain embodiments, the exogenous nucleic acid comprises a sequence encoding a protein of interest or a portion thereof, wherein the protein of interest is selected from the group consisting of: Tau, MeCP2, NGF, APOE, GDNF, SUMF, SGSH, AADC, CD, p53, ARSA arylsulfatase A, ABCD1, SMN1, NAGLU, SOD1, C9ORF72, TARDBP, FUS, HTT, LRRK2, PARIS, PARKIN, GAD, and α-synuclein. In some embodiments, the exogenous nucleic acid comprises an AAV vector, optionally comprising an AAV viral particle. In certain embodiments, the exogenous nucleic acid comprises an AAV vector of the AAV9 serotype (e.g., an AAV viral particle of the AAV9 serotype). In some embodiments, the therapeutically effective amount is 10 6 vg / kg to 10 14 In certain embodiments, the therapeutically effective amount is no more than 10 14 vg / kg (e.g. no more than 10 13 vg / kg, 10 12.5 vg / kg, 10 12 vg / kg, 10 11 vg / kg or even lower).

[0022] In certain embodiments, the aspirin compound and / or the exogenous nucleic acid are administered systemically (eg, intravenously, intramuscularly, subcutaneously), or by an intraparenchymal, intracerebroventricular, or intrathecal route.

[0023] In some embodiments, the therapeutically effective amount is a subtherapeutic amount.

[0024] In another aspect, the present disclosure provides a method for reducing side effects or improving tolerance to an exogenous nucleic acid in a subject, the method comprising: delivering to the subject a subtherapeutic amount of the exogenous nucleic acid for treating or preventing a condition, wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the subject after delivery, and wherein the subject has been or is being concurrently administered with an effective amount of an aspirin compound.

[0025] In another aspect, the present disclosure provides a method for reducing side effects or improving tolerance to exogenous nucleic acids in a subject, the method comprising: a) administering an effective amount of an aspirin compound to the subject; and b) delivering to the subject a subtherapeutic amount of the exogenous nucleic acid for treating or preventing a condition, wherein step a) is performed before or simultaneously with step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the cell after delivery.

[0026] In some embodiments, the side effect is dose-dependent on the exogenous nucleic acid delivered to the subject.

[0027] In some embodiments, the subtherapeutic amount is no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, no more than 2% or no more than 1% of the conventional amount of the same exogenous nucleic acid that would be required without administration of the aspirin compound.

[0028] In some embodiments, the exogenous nucleic acid comprises an AAV vector, optionally comprising an AAV viral particle. In some embodiments, the subtherapeutic amount of the AAV vector or the AAV viral particle is no more than 10 7 vg / kg (vector genome / kg), not more than 10 8 vg / kg, not more than 10 9 vg / kg, not more than 10 10 vg / kg, not more than 10 11 vg / kg, not more than 10 12 vg / kg, not more than 10 13 vg / kg or not more than 10 14 vg / kg.

[0029] In some embodiments, the aspirin compound is administered to the subject in an amount of no more than 30 mg / kg, no more than 50 mg / kg, no more than 100 mg / kg, no more than 110 mg / kg, no more than 120 mg / kg, no more than 120 mg / kg, no more than 130 mg / kg, no more than 140 mg / kg, no more than 150 mg / kg, no more than 160 mg / kg, no more than 170 mg / kg, no more than 180 mg / kg, no more than 190 mg / kg or no more than 200 mg / kg.

[0030] In some embodiments, the aspirin compound is administered to the subject at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to delivering the exogenous nucleic acid to the subject, and / or is administered once or repeatedly (e.g., twice, three times, four times, etc.) prior to delivering the nucleic acid. In some embodiments, the aspirin compound and / or the exogenous nucleic acid is administered to the subject by a parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, cardiac, subcutaneous, intraparenchymal, intracerebroventricular, or intrathecal route of administration.

[0031] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a subtherapeutic amount of an exogenous nucleic acid and a pharmaceutically acceptable carrier, optionally further comprising an aspirin compound, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted into double-stranded DNA in the cell after delivering the exogenous nucleic acid to the cell.

[0032] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an exogenous nucleic acid, an aspirin compound, and a pharmaceutically acceptable carrier, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted into double-stranded DNA in the cell after delivering the exogenous nucleic acid to the cell.

[0033] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise instructions for use indicating that the aspirin compound is to be administered prior to or concurrently with the administration of the pharmaceutical composition.

[0034] In some embodiments, the exogenous nucleic acid comprises an AAV vector, optionally comprising an AAV viral particle. In some embodiments, the pharmaceutical composition is in a unit dose and contains no more than 10 10 vg, 10 10.5 vg, 10 11 vg, 10 11.5 vg, 10 12 vg, 10 12.5 vg, 10 13 vg, 10 13.5 vg, 10 14 vg, 10 14.5 vg, 10 15 vg, 10 15.5 vg or 10 16 vg AAV viral particles.

[0035] In another aspect, the present disclosure provides a kit comprising: a) a first composition comprising an aspirin compound; and b) a second composition comprising an exogenous nucleic acid, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivering the exogenous nucleic acid to the cell.

[0036] In some embodiments, the kit further comprises instructions for use, the instructions for use indicating that the first composition is to be administered before or simultaneously with the second composition. In some embodiments, the first composition and the second composition can be easily mixed prior to use to provide a combined composition. In some embodiments, the second composition comprises a subtherapeutic amount of the exogenous nucleic acid.

[0037] In an additional aspect, the present disclosure provides a kit comprising a composition comprising an aspirin compound and an exogenous nucleic acid, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivering the exogenous nucleic acid to the cell.

[0038] In yet another aspect, the present disclosure provides a composition comprising: a combination of an aspirin compound and an exogenous nucleic acid, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivering the exogenous nucleic acid to the cell.

[0039] The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shown is AAV-mediated expression of the luciferase gene in mice treated with different concentrations of aspirin.

[0041] Figure 2 Shown are the IFN-α levels 3 days after AAV injection by treatment with different concentrations of aspirin.

[0042] Figures 3A-3C Shown are the effects of AAV8 injection in mice treated with aspirin and in the control group. Figure 3A )、AAV9( Figure 3B ) and AAV843( Figure 3C ) after luciferase expression.

[0043] Figures 4A-4B Figure 2 shows the expression of IFN-α ( Figure 4A ) and IFN-β( Figure 4B )level.

[0044] Figures 5A-5B Figure 2 shows the expression of IFN-α ( Figure 5A ) and IFN-β( Figure 5B )level.

[0045] Figures 6A-6B Figure 2 shows the expression of IFN-α ( Figure 6A ) and IFN-β( Figure 6B )level.

[0046] Figures 7A-7DThe mRNA levels of Gluc in the brain or liver tissues of mice that received AAV9-CB-Gluc and were pre-injected with aspirin, injected with aspirin at the same time, or not injected with aspirin are shown ( Figure 7A 、 7C ) and enzyme activity levels ( Figure 7B 、 7D ).

[0047] Figure 8 The results show that in MPSII mice, the 13 vg / kg or 1×10 14 vg / kg or 3×10 13 IDS enzyme activity in the brain after treatment with 50 mg / kg of AAV9-CB-IDS vector combined with 50 mg / kg of aspirin pretreatment.

[0048] Figure 9 All sequences disclosed in this disclosure are shown. DETAILED DESCRIPTION

[0049] In the following detailed description, reference is made to the accompanying drawings, which form a part of the detailed description. In the drawings, similar symbols generally identify similar components unless the context indicates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein.

[0050] It will be appreciated that certain features of the present disclosure that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described for brevity in the context of a single embodiment may also be provided individually or in any suitable subcombination. For illustration, if a specific embodiment disclosed herein comprises components A, B, and C, it will be appreciated that the present disclosure is also intended to encompass embodiments comprising A, B, or C alone, or any combination of A, B, or C.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0052] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0053] As used herein, "a / an" or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a plurality of cells.

[0054] As used herein, unless expressly stated otherwise, the word "or" is used in the inclusive sense of "and / or" rather than the exclusive sense of "either / or."

[0055] Unless expressly stated otherwise, numerical ranges described herein may include every number within the range and every subrange.

[0056] As used herein, the term "about" when referring to a measurable value such as dosage, time, temperature, activity or other biological activity, is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

[0057] The present invention is based, at least in part, on the discovery that administration of an aspirin compound to a cell or subject prior to or concurrently with delivery of an exogenous nucleic acid can significantly increase expression of the exogenous nucleic acid in the cell or in the subject.

[0058] In another aspect, the present disclosure provides a method for increasing the expression level or extending the duration of expression of an exogenous nucleic acid in a cell or in a subject, the method comprising: administering an aspirin compound to the cell or the subject before or simultaneously with delivering the exogenous nucleic acid to the cell or the subject, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivery, and thereby increasing or extending the expression level or the expression duration of the exogenous nucleic acid, respectively, as compared to a control expression level or control expression duration obtained without administration of the aspirin compound.

[0059] In another aspect, the present disclosure provides a method for expressing an exogenous nucleic acid in a cell or a subject or increasing the expression level of an exogenous nucleic acid or extending the duration of expression of an exogenous nucleic acid, the method comprising: delivering the exogenous nucleic acid to the cell or the subject under conditions suitable for expression, wherein the cell or the subject has been or is being simultaneously administered an aspirin compound; and wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted into double-stranded DNA in the cell or the subject after delivery.

[0060] In another aspect, the present disclosure provides a method for expressing an exogenous nucleic acid in a cell or a subject, or increasing the expression of an exogenous nucleic acid, or extending the duration of expression of an exogenous nucleic acid, the method comprising:

[0061] a) administering an aspirin compound to the cell or the subject; and

[0062] b) delivering the exogenous nucleic acid to the cell or the subject under conditions suitable for expression,

[0063] wherein said step a) is performed before or simultaneously with said step b), wherein said exogenous nucleic acid comprises double-stranded DNA, or can be converted into double-stranded DNA in said cell or said subject after delivery.

[0064] In one aspect, the present disclosure provides a method of preparing a cell for delivery of an exogenous nucleic acid, the method comprising administering an aspirin compound to the cell prior to or concurrently with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivery.

[0065] In another aspect, the present disclosure provides a method for preparing a subject for a condition treatable by an exogenous nucleic acid or its expression product, the method comprising administering an aspirin compound to the subject prior to or concurrently with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the subject after delivery.

[0066] In another aspect, the present disclosure provides a method for treating or preventing a condition that can be treated or prevented by an exogenous nucleic acid or its expression product, the method comprising: delivering the exogenous nucleic acid to the subject, wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the subject after delivery, and wherein the subject has been or is being concurrently administered an aspirin compound.

[0067] In another aspect, the present disclosure provides a method for treating or preventing a condition that can be treated or prevented by an exogenous nucleic acid or its expression product, the method comprising: a) administering an aspirin compound to the subject; and b) delivering the exogenous nucleic acid to the subject, wherein step a) is performed before or simultaneously with step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or can be converted to double-stranded DNA in the subject after delivery.

[0068] As used herein, the term "aspirin compound" includes aspirin, its analogs and derivatives. Aspirin is also known as acetylsalicylic acid, and its chemical structure is shown below:

[0069] Derivatives of aspirin include, but are not limited to, salts (e.g., pharmaceutically acceptable salts), esters, solvates, and prodrugs of aspirin, which can provide acetylsalicylic acid or any active form thereof, for example, after hydrolysis or metabolic processes in vivo. Exemplary salts of aspirin include, but are not limited to, aspirin-arginine (which is a double salt formed from L-arginine and acetylsalicylic acid, also known as arginine aspirin), lithium acetylsalicylate (e.g., ), sodium acetylsalicylate (e.g., ), calcium acetylsalicylate (e.g., ) and magnesium acetylsalicylate (e.g. ). Exemplary esters of aspirin include, but are not limited to, ethanolamides, glycolic acid esters, (acyloxy)methyl esters, alkyl esters, and aryl esters of acetylsalicylic acid.

[0070] The analogue of aspirin is a compound that is a functional equivalent of aspirin, but does not have the chemical structure of aspirin and is not a derivative of aspirin. Compared with aspirin, the analogue of aspirin can have a similar (although not identical) structure and also shares the same biological activity as aspirin as provided herein.

[0071] exogenous nucleic acids

[0072] As used herein, the term "exogenous nucleic acid" is a nucleic acid to be delivered to a cell or subject. The exogenous nucleic acid can be linear or circular and can be in the form of naked nucleic acid or can be in a packaged form, such as a viral particle. The exogenous nucleic acid can contain sequences that are not naturally present in the cell or subject. The exogenous nucleic acid comprises double-stranded DNA or can be converted into double-stranded DNA in the cell after the exogenous nucleic acid is delivered to the cell.

[0073] In certain embodiments, the exogenous nucleic acid comprises double-stranded deoxyribonucleic acid (DNA). The exogenous nucleic acid can be composed of double-stranded DNA over its entire length, for example, as a double-stranded DNA (dsDNA) viral vector, dsDNA viral particles, double-stranded plasmids, double-stranded artificial chromosomes or exosomes, or double-stranded naked DNA. Alternatively, the exogenous nucleic acid can comprise a portion of dsDNA, for example, the exogenous nucleic acid can be a single-stranded DNA having a secondary double-stranded structure over a portion of the sequence.

[0074] In certain embodiments, the exogenous nucleic acid comprises a nucleic acid that is not dsDNA but can be converted to dsDNA in the cell after delivery. Examples of such nucleic acids include, but are not limited to, single-stranded DNA (which can be converted to dsDNA by, for example, DNA polymerase) and RNA (which can be reverse transcribed to dsDNA by reverse transcriptase), such as retroviral vectors, retroviral particles, lentiviral vectors, and lentiviral particles. In certain embodiments, such nucleic acids can be converted to dsDNA in the cytoplasm of the cell.

[0075] In certain embodiments, the exogenous nucleic acid comprises a vector. The term "vector" means any nucleic acid molecule (whether naked or packaged) used to clone and / or transfer nucleic acids into cells. Vectors include both viral nucleic acid molecules and non-viral (e.g., plasmids, exosomes) nucleic acid molecules for introducing nucleic acids into cells in vitro, ex vivo, and / or in vivo. In some embodiments, a vector can be recombinant because it contains one or more heterologous nucleotide sequences, such as a transgene or a heterologous regulatory sequence.

[0076] In certain embodiments, the exogenous nucleic acid comprises a plasmid. As used herein, the term "plasmid" refers to a construct comprising extrachromosomal genetic material, typically a circular duplex of DNA that can replicate independently of chromosomal DNA. Plasmids are commonly used in gene transfer as a medium that can introduce DNA fragments into a host organism.

[0077] In certain embodiments, the exogenous nucleic acid comprises a viral vector or is included in a viral vector. As used herein, "viral vector" refers to a single-stranded or double-stranded nucleic acid vector having a 5' viral terminal repeat sequence and / or a 3' viral terminal repeat sequence at the 5' end and / or 3' end of a nucleic acid sequence of interest (e.g., an expression construct encoding a protein of interest). A viral vector may comprise a pair of TRs or a single TR. The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and mediates desired functions such as replication, viral packaging, integration, and / or provirus rescue. For example, a 5' viral terminal repeat sequence and a 3' viral terminal repeat sequence may contain a replication origin and allow DNA synthesis to initiate at one viral terminal repeat and continue to the other viral terminal repeat. Examples of viral terminal repeats include, but are not limited to, inverted terminal repeats (ITRs) (e.g., those contained in AAV), long terminal repeats (LTRs) (e.g., those contained in retroviruses), etc. A viral vector may comprise one or more sequences heterologous to the viral genome between the ITRs.

[0078] As used herein, viral vectors may also encompass viral particles produced by one or more vectors containing viral nucleic acid sequences. As used herein, "viral particle" means a viral genome packaged within a viral capsid. The viral genome in the viral particle may be a modified viral genome such that it may lack some native viral sequences and / or may contain some sequences that are heterologous to the native viral genome.

[0079] Various viral vectors are known in the art to be suitable for delivering nucleic acid to cells or to subjects such as people. The most commonly used viral vectors include those derived from adenovirus, adeno-associated virus (AAV) and retroviruses, including slow viruses, such as human immunodeficiency virus (HIV). Retroviral vectors, adenovirus and AAV provide an effective and useful method for effectively introducing and expressing foreign genes in mammalian cells. These vectors have a wide range of hosts and cell types, and stably and effectively express genes. The safety of these vectors is well understood in the art. Other viral vectors that can be used to transfer genes into subjects include herpes simplex virus papovavirus, such as JC, SV40, polyomavirus; Epstein-Barr virus (Epstein-Barr Virus, EBV); papillomavirus, such as bovine papillomavirus type I (BPV); poliovirus and other human and animal viruses.

[0080] In certain embodiments, the exogenous nucleic acid comprises an AAV vector. AAV is a single-stranded human DNA parvovirus with a genome size of about 4.7 kb. The AAV genome contains two main genes: the rep gene, which encodes rep proteins (Rep 76, Rep 68, Rep 52, and Rep 40); and the cap gene, which encodes AAV structural proteins (VP-1, VP-2, and VP-3), flanked by 5' inverted terminal repeats (ITRs) and 3' ITRs. As used herein, the term "AAV vector" encompasses any viral vector comprising one or more heterologous sequences flanking at least one or two AAV inverted terminal repeats. As is well understood in the art, the term "AAVITR" is a sequence of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of ITR can exist in either of two alternative orientations, thereby generating heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several short self-complementary regions, allowing intrastrand base pairing to occur within this portion of the ITR.

[0081] AAV ITRs can be derived from any AAV, including but not limited to AAV serotype 1 (AAV 1), AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, as well as any other AAV now known or later discovered. For more information, see, for example, BERNARD NF et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers), Gao et al., (2004) J. Virol. 78: 6381-6388. The nucleotide sequences of the AAV ITR regions are known. See, e.g., Kotin, RM (1994) Human Gene Therapy 5:793-801; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd ed. (BN Fields and DMKnipe, eds.). An early description of the terminal repeat sequences of AAV1, AAV2, and AAV3 is provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration", PhD dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporated herein in its entirety).

[0082] The AAV ITRs may be native AAV ITRs, or alternatively may be altered from native AAV ITRs, e.g., by mutation, deletion, or insertion, so long as the altered ITRs can still mediate the desired biological function, such as replication, viral packaging, integration, etc. The 5' and 3' ITRs flanking the selected nucleotide sequence in the AAV vector do not have to be identical or derived from the same AAV serotype, so long as they function as intended, e.g., allowing excision and rescue of the sequence of interest and integration into the recipient cell genome.

[0083] The genome sequence of AAV and the AAV rep gene and cap gene are known in the art and can be found in the literature and public databases such as the GenBank database. Table 1 below shows some exemplary sequences of AAV genomes or AAV capsid sequences, and more is reviewed in Bernard NF et al., Virology, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers); Gao et al., (2004) Journal of Virology 78: 6381-6388; Naso MF et al., BioDrugs. 2017; 31(4): 317-334.

[0084] Table 1.

[0085]

[0086] In some embodiments, the AAV vector can be recombinant. The recombinant AAV vector can comprise one or more heterologous sequences of different viral origin (e.g., from a non-AAV virus, or from an AAV of a different serotype, or from a partially or completely synthetic sequence). In certain embodiments, the heterologous sequence is flanked by at least one AAV ITR.

[0087] In certain embodiments, the AAV vectors provided herein have a size suitable for packaging into AAV virus particles. For example, the size of the AAV vector can reach the size limit of the genome size of the AAV to be used, for example, up to 5.2 kb. In certain embodiments, the size of the AAV vector is no more than 5.2 kilobases (kb), no more than about 5 kb, no more than about 4.5 kb, no more than about 4 kb, no more than about 3.5 kb, no more than about 3 kb, and a size of no more than about 2.5 kb, see, for example, Dong, JY et al. (November 10, 1996).

[0088] Due to the packaging size limitations of a single AAV, for heterologous sequences that exceed the packaging capacity of a single AAV vector, two or more AAV vectors can be constructed in a manner that allows reconstruction as a complete sequence or expression cassette in cells co-transfected with these AAV vectors. Methods for constructing such AAV vectors, such as overlapping binary vectors, trans-splicing vector pairs, and hybrid vector systems, are known in the art, and more detailed information can be found in Chamberlain K et al., Hum Gene Ther Methods. 2016 Feb 1; 27(1): 1–12, and U.S. Patent No. 6,596,535.

[0089] AAV vectors can be constructed using methods known in the art. The general principles of rAAV vector construction are known in the art. See, for example, Carter, 1992, Current Opinion in Biotechnology, 3:533-539; and Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:97-129. For example, heterologous sequences can be directly inserted between the ITRs of the AAV genome, wherein the Rep gene and / or the Cap gene are deleted. As long as enough ITR portions are retained to allow replication and packaging functions, other parts of the AAV genome may also be deleted. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press). Press); Carter, BJ (1992) Current Reviews in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiology and Immunology 158:97-129; Kotin, RM (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

[0090] Alternatively, the AAV ITRs can be excised from the viral genome or from an AAV vector containing the viral genome and fused 5' and 3' to the heterologous sequence using standard ligation techniques such as those described in Sambrook et al., supra. AAV vectors containing AAV ITRs are commercially available and have been described, for example, in U.S. Patent No. 5,139,941.

[0091] In certain embodiments, the AAV vector comprises recombinant AAV virus particles. AAV virus particles can be produced by AAV expression vectors. AAV particles can be produced by using known techniques, such as by transfection, introducing an AAV expression vector together with other necessary mechanisms such as plasmids encoding AAV cap / rep genes and auxiliary genes provided by adenovirus or herpes virus into suitable host cells (see, for example, MF Naso et al., Biopharmaceuticals, 31 (4): 317-334 (2017), the entire contents of which are incorporated herein). AAV expression vectors can be expressed in host cells and packaged into virus particles.

[0092] In some embodiments, the AAV vector further comprises a cap gene encoding a capsid protein. In some embodiments, the AAV vector comprises an AAV virus particle, and the AAV virus particle comprises a natural or recombinant capsid protein. In some embodiments, the capsid protein can be modified or chimeric or synthetic. The modified capsid can comprise modifications such as insertions, additions, deletions or mutations. For example, the modified capsid can be incorporated into a detection or purification tag. Chimeric capsids comprise parts of two or more capsid sequences. Synthetic capsids comprise synthetic or artificially designed sequences. The capsid structure of AAV is also known in the art and is described in more detail in the above-mentioned Bernard NF et al. In some embodiments, the cap gene or the capsid protein is derived from two or more AAV serotypes. As used herein, the term "serotype" with respect to AAV refers to the reactivity of the capsid protein with a defined antiserum. It is known in the art that the various AAV serotypes are functionally and structurally related, even at the genetic level (see, for example, Blacklow, JR Pattison, ed., "Parvoviruses and Human Disease," pp. 165-174 (1988); and Rose, Comprehensive Virology 3: 1, 1974). However, AAV virus particles of different serotypes can have different tissue tropisms (for details, see Nonnenmacher M et al., "Gene Therapy," June 2012; 19(6): 649–658), and can be appropriately selected for gene therapy of target tissues. In some embodiments, the cap gene or the capsid protein can have a specific tropism property. The term "tropism property" refers to the transduction pattern of one or more target cells, tissues, and / or organs. For example, the capsid protein can have specific tropism for the liver (e.g., hepatocytes), brain, eye, muscle, lung, kidney, intestine, pancreas, salivary gland, or synovium, or any other suitable cell, tissue, or organ.

[0093] In some embodiments, the cap gene or the capsid protein is derived from any suitable AAV capsid gene or protein, such as, but not limited to, AAV capsid genes or proteins derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcyS, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVhu43, AAVhu44, AAVhu46, AAVhu19, AAVhu20, AAVhu23, AAVhu22, AAVhu24, AAVhu21, AAVhu27, AAVhu28, AAVhu29, AA Vhu63, AAVhu64, AAVhu13, AAVhu56, AAVhu57, AAVhu49, AAVhu58, AAVhu34, AAVhu45, AAVhu47, AAVhu51, AAVhu52, AAVhu T41, AAVhu S17, AAVhu T88, AAVhu T71, AAVhu T70, AAVhu T40, AAVhuT32, AAVhu T17, AAVhu LG15, AAVhu9, AAVhu10, AAVhu11, AAVhu53, AAVhu55, AAVhu54, AAVhu7, AAVhu18, AAVhu15, AAVhu16, AAVhu25, AAVhu60, AAVch5, AAVhu3, AAVhu 1. AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAV rh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, AAVpi2, AAVpi3, AAVrh57, AAVrh50, AAVrh49, AAVhu39, AAVrh58, AAVrh61, AAVrh52, AAVrh53, AAVrh51, AAVhu14, AAVhu31, AAVhu32, AAVrh34, AAVrh33, AAVrh32, avian AAV ATCC VR-865, avian AAV strain DA-1, or bovine AAV.

[0094] The capsid of AAV843 is identical to the synthetic capsid AAVXL32 disclosed in WO 2019241324A1 (the entire contents of which are incorporated herein), and AAV843 is also disclosed in, for example, Xu J. et al., Int J Clin Exp Med, 2019; 12(8): 10253-10261. In certain embodiments, the capsid protein of AAV843 has the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the capsid gene encoding the capsid protein of AAV843 has a nucleic acid sequence that is at least 90%, 92%, 95%, 97% or 98% identical to SEQ ID NO: 6 or is a variant of SEQ ID NO: 6 with degenerate codon substitutions. Degenerate codon substitutions, also known as synonymous nucleotide substitutions, can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues.

[0095] In certain embodiments, the capsid protein of AAV8 has the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the capsid gene encoding the capsid protein of AAV8 has a nucleic acid sequence that is at least 90% or 95% identical to SEQ ID NO: 4, or is a variant of SEQ ID NO: 4 with degenerate codon substitutions.

[0096] In certain embodiments, the capsid protein of AAV9 has the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the capsid gene encoding the capsid protein of AAV9 has a nucleic acid sequence that is at least 90% or 95% identical to SEQ ID NO: 5, or is a variant of SEQ ID NO: 5 having degenerate codon substitutions.

[0097] More examples of AAV capsid gene sequences and protein sequences can be found in the GenBank database, see GenBank accession numbers: AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 001358, NC 001540、AF513851、AF513852、AY530579、AY631965、AY631966; AF063497、AF085716、AF513852、AY530579、AAS99264.1、AY243022、A Y243015, AY530560, AY530600, AY530611, AY530628, AY530553, AY530606, AY530583, AY530555, AY530607, AY530580, AY530569, NC 006263, NC 005889, NC 001862, AY530609, AY530581, AY530563, AY530591, AY530562, AY530584, AY530622, AY530601, AY530586, AY243021, AY530570, AY530589, AY5305 95. AY530572, AY530588, AY530575, AY530565, AY530590, AY530602, AY530566, AY530587, AY530585, AY530564, AY530592, AY530623, AY530574, AY 530593, AY530560, AY530594, AY530573, AF513852, AY530624, AY530561, AY242997, AY530625, AY530567, AY530556, AY530578, AY530568, AY5306 18. AY243020, AY530579, AY530619, AY530596, AY530612, AY243000, AY530597, AY530620, AY242998, AY530598, AY242999, AY530599, AY243016, NC 001729, NC 001401, AY243018, NC 001863, AY530608, AY243019, NC001829, AY530610, AY243017, AY243001, AY530613, AY243013, AY243002, AY530614, AY243003, AY695378, AY530558 , AY530626, AY695376, AY695375, AY530605, AY695374, AY530603, AY530627, AY695373, AY695372, AY530604, AY695 371, AY530600, AY695370, AY530559, AY695377, AY243007, AY243023, AY186198, AY629583, NC004828, AY530629, AY530576, AY243015, AY388617, AY530577, AY530582, AY530615, AY530621, AY530617, AY530557, AY530616, or AY530554.

[0098] In certain embodiments, the AAV vector comprises a cap gene from one AAV serotype and an AAV ITR from a second serotype. In certain embodiments, the AAV vector comprises an AAV viral particle comprising a pseudotyped AAV. "Pseudotyped" AAV refers to an AAV containing a capsid protein from one serotype and a viral genome comprising 5'-3' ITRs of a second serotype. Pseudotyped AAV is expected to have the cell surface binding properties of the serotype from which the capsid protein is derived, as well as genetic properties consistent with the serotype from which the ITRs are derived.

[0099] In certain embodiments, the exogenous nucleic acid comprises an adenovirus vector. Adenoviruses have a double-stranded linear DNA genome that cannot be integrated into the host genome. Illustrative examples of adenovirus vectors include, but are not limited to, first-generation adenovirus vectors (e.g., adenovirus vectors with a deletion of the E1a gene and the E1b gene, and adenovirus vectors with a deletion of the E1 gene and the E3 gene), second-generation adenovirus vectors (e.g., adenovirus vectors with a deletion of the E1 gene and the E2 gene, adenovirus vectors with a deletion of the E1 gene and the E4 gene), and gutless adenovirus vectors in which all viral coding sequences are deleted (also known as helper-dependent adenovirus vectors).

[0100] In certain embodiments, the exogenous nucleic acid comprises a retroviral vector. A retrovirus has an RNA genome and can be replicated in a host cell by a reverse transcriptase to produce DNA from the RNA genome. Illustrative examples of retroviral vectors include, but are not limited to, vectors derived from avian leukosis virus, mouse mammary tumor virus, murine leukemia virus, bovine leukemia virus, walleye dermal sarcoma virus, HIV-1 (human immunodeficiency virus), HIV-2, SIV (simian immunodeficiency virus), EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus), CAEV (caprine arthritis encephalitis virus), VMV (visna / maedi virus), human foamy virus, Moloney murine leukemia virus, Rous sarcoma virus, feline leukemia virus, human T-lymphotropic virus, and simian foamy virus.

[0101] In certain embodiments, the exogenous nucleic acid comprises a lentiviral vector. Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Illustrative examples of lentiviral vectors include, but are not limited to, vectors derived from HIV-1, SIV, FIV, CAEV, VMV, and EIAV.

[0102] In certain embodiments, the exogenous nucleic acid may comprise a coding sequence encoding a protein of interest or a portion thereof. In certain embodiments, the coding sequence of the protein of interest may be divided or split into two or more exogenous nucleic acid sequences (e.g., two or more plasmids, or two or more viral particles) in a manner that allows the divided coding sequences to be joined together after delivery to the cell (e.g., by homologous recombination or by some viral packaging processes). This will allow coding sequences whose length exceeds the delivery capacity of a vector (e.g., an AAV vector or plasmid) to be delivered and expressed.

[0103] The protein of interest can be any protein whose expression in a cell or in a subject is of interest. In certain embodiments, the protein of interest can be a therapeutic protein (e.g., for medical or veterinary use), an immunogenic protein (e.g., for a vaccine), a reporter protein, a nuclease, or a therapeutic target protein.

[0104] Therapeutic proteins can be expressed in vitro to provide a therapeutic composition for delivery to a subject in need, or can be expressed in vivo to provide a therapeutic benefit. Examples of such therapeutic proteins include, but are not limited to, antibodies (e.g., monoclonal or bispecific or multispecific), insulin, glucagon-like peptide-1, peptide hormones, growth factors, erythropoietin (EPO), cytokines, coagulation factors, antihemophilic factor, interferon, Fc fusion proteins (e.g., CTLA-4 Fc fusion protein, VEGFR Fc fusion protein), therapeutic enzymes (e.g., lysosomal hydrolases and sulfatases). Alternatively, the therapeutic protein can be expressed in vivo in a subject in need. Examples of such therapeutic proteins include, but are not limited to, survival motor neuron 1 (SMN1, gene ID: 6066), α-N-acetylglucosaminidase (NAGLU, gene ID: 4669), N-sulfoglucosamine sulfonylhydrolase (SGSH, gene ID: 6448), iduronate 2-sulfatase (IDS, gene ID: 3423), coagulation factor VIII (FVIII, gene ID: 2157), coagulation factor IX (FIX, gene ID: 2158), Bruton's tyrosine kinase (B ... kinase, BTK, gene ID: 695), ATP-binding cassette subfamily D member 1 (ABCD1, gene ID: 215), acyl-CoA dehydrogenase very long chain (ACADVL, gene ID: 37), androgen receptor repeat instability region (AR, gene ID: 109504725), hemoglobin subunit beta (HBB, gene ID: 3043), sodium voltage-gated channel alpha subunit 1 (SCN1A, gene ID: 6323), CF transmembrane conductance regulator (CFTR, gene ID: 1080), colony-stimulating factor 2 receptor subunit alpha (CSF2RA, gene ID: 1438), interleukin 2 receptor subunit alpha (IL2AG, gene ID: 3559), phenylalanine hydroxylase (PHA, gene ID: 5053), serine / threonine kinase 1 1 (STK11, Gene ID: 6794), phosphatidylinositol glycan anchor biosynthesis class A (PIGA, Gene ID: 5277), ornithine transcarbamylase (OTC, Gene ID: 5009), N-acetylglutamate synthetase deficiency (NAGS, Gene ID: 162417), DM1 protein kinase (DMPK, Gene ID: 1760), CCHC-type zinc finger nucleic acid binding protein (CNBP, Gene ID: 7555), acyl-CoA dehydrogenase medium chain (ACADM, Gene ID: 34), GNAS complex locus (GNAS, Gene ID: 2778), fibrillin 1 (FBN1, Gene ID: 2200), lipase A, lysosomal acid type (LIPA, Gene ID: 3988), solute carrier family 7 member 7 (SLC7A7,acyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA, gene ID: 3030), growth hormone receptor (GHR, gene ID: 2690), isovaleryl-CoA dehydrogenase (IDV, gene ID: 3712), alkaline phosphatase, biomineralization-related (ALPL, gene ID: 249), solute carrier family 25 member 15 (SLC25A15, gene ID: 10166), huntingtin (HTT, gene ID: 3064), carboxylase synthetase (HCS, gene ID: 3141), notch receptor 3 (NOTCH3, gene ID: 4854), aldolase, fructose bisphosphate B (ALDOB, gene ID: 229), ATPase copper transporter beta (ATP7B, gene ID: 540), glucosidase alpha, acid (GAA, gene ID: 2548), glutaryl-CoA dehydrogenase (GCDH, gene ID: 2639), solute carrier family 12 member 3 (SLC12A3, gene ID: 6559), glucosylceramidase beta (GBA, gene ID: 2629), familial Mediterranean fever ( Mediterranean Fever (MEFV, Gene ID: 4210), galactosidase alpha (GLA, Gene ID: 2717), chloride voltage-gated channel 1 (CLCN1, Gene ID: 1180), nuclear receptor subfamily 0 group B member 1 (NR0B1, Gene ID: 190), argininosuccinate synthetase 1 (ASS1, Gene ID: 445), solute carrier family 25 member 13 (SLC25A13, Gene ID: 10165), solute carrier family 22 member 5 (SLC22A5, Gene ID: 84), sodium voltage-gated channel alpha subunit 5 (SCN5A, Gene ID: 6331), biotinidase (BTD, Gene ID: 686), acetyl-CoA acetyltransferase 1 (ACAT1, Gene ID: 38), arginase (ARG1, Gene ID: 383), cytochrome P450 family 21 subfamily A member 2 (CYP21A2, Gene ID: 1589). In yet another embodiment, the therapeutic protein can be expressed ex vivo, for example, on T cells to be transplanted into a subject. Such therapeutic proteins include, for example, chimeric antigen receptors (CARs).

[0105] In certain embodiments, the protein of interest can be an immunogenic protein. The immunogenic protein or immunogen can be any polypeptide suitable for protecting a subject from a disease, including but not limited to infectious diseases such as microbial, bacterial, protozoan, parasitic, fungal and viral diseases, and cancer. For example, the immunogen can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or an influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as an HIV or SIV envelope GP160 protein, an HIV or SIV matrix / capsid protein, and HIV or SIV gag, pol and env gene products). The immunogen can also be an arenavirus immunogen (e.g., a Lassa fever virus immunogen, such as a Lassa fever virus nucleocapsid protein gene and a Lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia, such as a vaccinia L1 or L8 gene), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen or a Marburg virus immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a severe acute respiratory syndrome (SARS) immunogen, such as an S [S1 or S2], M, E, or N protein or an immunogenic fragment thereof, or a COVID-19 immunogen). The immunogen may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, or hepatitis C) immunogen, or any other vaccine immunogen known in the art. For another example, the immunogen may be a tumor or cancer antigen expressed on the surface of a tumor or cancer cell. Exemplary tumor or cancer antigens include, but are not limited to, b-catenin, BRCA1 gene products, BRCA2 gene products, EpCAM, EGFR, Her2, VEGFR, CD19, PSMA, and the like.

[0106] In certain embodiments, the protein of interest can be a reporter protein. Reporter protein can be expressed in cells to provide engineered cells for bioassays. Examples of reporter proteins include, but are not limited to, fluorescent proteins (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), enzymes that produce detectable products, such as luciferase (e.g., from Gaussia, Renilla, or Pho), β-galactosidase, β-glucuronidase, alkaline phosphatase, and chloramphenicol acetyltransferase genes or proteins that can be directly detected. Almost any protein can be directly detected by using, for example, specific antibodies for protein. Additional markers (and related antibiotics) suitable for positive or negative selection of eukaryotic cells are disclosed in Sambrook and Russell (2001), Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley & Sons, including regular updates.

[0107] In certain embodiments, the protein of interest can be a nuclease. As used herein, the term "nuclease" refers to an enzyme that can cut a phosphodiester bond within a polynucleotide chain. The nucleases provided herein can be naturally occurring or modified. Examples of nucleases useful in the present disclosure include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas family proteins (e.g., Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas 1, Csf2, Csf3, Csf4, Cpf1, homologs thereof, or modified versions thereof). Such nucleases may be useful, for example, in genetic engineering or gene editing in a host genome.

[0108] In certain embodiments, the protein of interest can be a therapeutic target protein. Examples of therapeutic target proteins include, but are not limited to, GPCR, CTLA-4, HER2, fibronectin-4, sclerostin, P-selectin, VEGF, RSV F, VEGFR2, CD79, IL23p19, vWF, IFN-γ, C5, PD-1, PD-L1, CGRP, CD3, CD11a, CD20, CD22, CD30, CD33, CD38, CD40, CD52, IgE, KLK, CCR4, FGF-23, IL-6R, IL-5, IL- 23p19, IL-2R, IL-17R, IL-17, CD4, FIX / FX, IL-12, IL-23, IL-1β, IL-5R, IL-6R, IL-4 / IL-13, PDGF-α, dabigatran, SLAMF7, EGFR, PCSK9, GD2, CD3, CD19, α4β7 integrin, α4β1 integrin, PA, BLyS, RANK, TNF-α, EpCAM, GGTA1, endostatin and angiostatin. Expression of therapeutic target proteins in recombinant cells can be useful, for example, in generating recombinant cell lines for bioassays, or for screening or identifying potential therapeutic agents that can target or interact with therapeutic target proteins.

[0109] Alternatively, in certain embodiments, the exogenous nucleic acid may comprise a coding sequence encoding a functional RNA. Functional RNA may be a non-translated RNA that acts on a biological target nucleic acid sequence and regulates the target, for example, inhibits or reduces the expression or activity of the target. For example, functional RNA may be an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Patent No. 5,877,022), an RNA that affects spliceosome-mediated / primary splicing (see, Puttaraju et al., (1999) Nature Biotechnology, Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No. 6,083,702), interfering RNA (RNAi) comprising small interfering RNA (siRNA) that mediates gene silencing (see, Sharp et al., (2000) Science 287:2431), microRNA or other non-translated functional RNA, such as guide RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No. 5,869,248 of Yuan et al.), single guide RNA for CRISPR technology (see, for example, US10,266,850, US8,697,359, US20160298134, Adli M et al., Nat Commun. 9, 1911 (2018)), etc. Potential biological targets of functional RNA may include, but are not limited to, multiple drug resistance (MDR) protein targets, tumor targets (e.g., VEGF, Her2, EGFR, PD-L1, etc.), pathogen targets such as viral surface antigens (e.g., hepatitis B surface antigen gene), defective gene products (mutated dystrophin), or therapeutic targets as disclosed herein (e.g., myostatin).

[0110] The coding sequence encoding a protein of interest or encoding a functional RNA can be operably linked to one or more regulatory sequences in an exogenous nucleic acid. As used herein, the term "operably linked" means that a coding sequence is directly or indirectly linked or associated with one or more regulatory sequences in an exogenous nucleic acid in a manner that allows expression of a protein of interest from a coding sequence in a cell. The coding sequence together with the regulatory sequence can be referred to as an expression cassette in this article. In certain embodiments, the exogenous nucleic acid can be in the form of an expression vector. Examples of transcriptional regulatory elements include one or more promoters and / or enhancers, and optionally polyadenylation sequences and / or one or more introns inserted between the exons of the protein coding sequence.

[0111] As used herein, the term "regulatory sequence" refers to any nucleotide sequence that is necessary or advantageous for the expression of a coding sequence. Regulatory sequences may include, but are not limited to, one or more promoters, enhancers, transcription terminators, polyadenylation sequences, internal ribosome entry sites, and / or one or more introns inserted between exons of a protein coding sequence.

[0112] As used herein, the term "promoter" refers to a polynucleotide sequence that can control the transcription of a coding sequence. The promoter sequence contains specific sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. In addition, the promoter sequence may contain sequences that regulate the recognition, binding, and transcription initiation activity of RNA polymerase. A promoter can affect the transcription of a gene located on a nucleic acid molecule identical to itself or a gene located on a nucleic acid molecule different from itself. Depending on the nature of the regulation, the function of the promoter sequence can be constitutive or induced by stimulation. A "constitutive" promoter refers to a promoter that is used to continuously activate gene expression in a host cell. An "inducible" promoter refers to a promoter that activates gene expression in a host cell in the presence of a certain stimulus or stimuli. In some embodiments, the promoter is a tissue-specific promoter or a cell-specific promoter. As used herein, the term "tissue-specific promoter" refers to a promoter that is used to preferentially or exclusively activate gene expression in a certain tissue and has no activity or reduced activity in other tissues. In one embodiment, the promoter is a CNS-specific promoter. Examples of CNS-specific promoters include promoters isolated from the genes for myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and synaptophysin (SYN). Liver-specific promoters include, but are not limited to, thyroxine-binding globulin (TBG), apolipoprotein E (APOE), albumin (ALB), and alpha-1 antitrypsin (hAAT). Muscle-specific promoters include, but are not limited to, Unc-45 myosin chaperone B (UNC45B) and RIEG / PITX homeobox 3 (PITX3).

[0113] Examples of suitable promoters include, but are not limited to, pol II promoters, such as CMV (e.g., CMV immediate early promoter (CMV promoter)), chicken β-actin promoter, pol III promoter, adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, Epstein-Barr virus (EBV) promoter, human immunodeficiency virus (HIV) promoter (e.g., HIV long terminal repeat (LTR) promoter), Moloney virus promoter, mouse mammary tumor virus (MMTV) promoter, mouse mammary tumor virus LTR promoter, Rous sarcoma virus (RSV) promoter, SV40 early promoter, promoters from human genes, such as human myosin promoter, human hemoglobin promoter, human synaptophysin promoter, human muscle creatine promoter, human metallothionein β-actin promoter, human ubiquitin C promoter (UBC), mouse phosphoglycerate kinase 1 promoter (PGK), human thymidine kinase promoter (TK) , human elongation factor 1 alpha promoter (EF1A), cauliflower mosaic virus (CaMV) 35S promoter, E2F-1 promoter (promoter of E2F1 transcription factor 1), alpha-fetoprotein promoter, cholecystokinin promoter, carcinoembryonic antigen promoter, C-erbB2 / neu oncogene promoter, cyclooxygenase promoter, CXC-chemokine receptor 4 (CXCR4) promoter, human epididymis protein 4 (HE4) promoter, hexokinase type II promoter, L-plastin promoter, mucin-like glycoprotein (MUC1) promoter, prostate-specific antigen (PSA) promoter, survivin promoter, tyrosinase-related protein (TRP1) promoter and tyrosinase promoter, synthetic promoters, hybrid promoters, etc. Such promoter sequences are commercially available, for example, from Stratagene (San Diego, CA).

[0114] As used herein, the term "enhancer" refers to a nucleotide sequence that increases the transcription and / or translation of a coding sequence. The enhancer can be operably linked to the 5' end or the 3' end of the coding sequence. Any enhancer that functions in eukaryotic cells can be used in the present disclosure. Illustrative examples of enhancers include, but are not limited to, the simian virus 40 (SV40) early gene enhancer, the enhancer derived from the long terminal repeat (LTR) of Rous sarcoma virus, and the enhancer derived from human cytomegalovirus (CMV).

[0115] As used herein, the term "transcription terminator" refers to a nucleotide sequence that is recognized by eukaryotic RNA polymerase to terminate transcription. The terminator sequence can be operably linked to the 3' end of the coding sequence. In certain embodiments, the terminator can include a signal for cleaving RNA so that a polyadenylation site on the RNA can be exposed. Any terminator sequence that works in eukaryotic cells can be used in the present disclosure. Illustrative examples of terminator sequences include, but are not limited to, terminator sequences derived from viruses, such as the SV40 terminator, and terminator sequences derived from known genes, such as the bovine growth hormone terminator sequence.

[0116] As used herein, the term "polyadenylation sequence" refers to a nucleotide sequence that, when transcribed, is recognized by a eukaryotic cell as a signal for adding polyadenosine residues to a transcribed mRNA. The polyadenylation sequence can be operably linked to the 3' end of the coding sequence. Any polyadenylation sequence that functions in eukaryotic cells can be used in the present disclosure. Illustrative examples of polyadenylation sequences include, but are not limited to, AAUAAA and SV40 polyadenylation signals.

[0117] In certain embodiments, the exogenous nucleic acid may comprise two sequences encoding two proteins of interest. In such embodiments, the two coding sequences may be separated by an internal ribosome entry site (IRES), which allows translation to begin in the middle of the mRNA sequence, and therefore separates the translation of two or more coded products. The IRES may be operably connected at a position after the 3' end of the first coding sequence and before the 5' end of the second coding sequence. Any IRES sequence that works in eukaryotic cells may be used in the present disclosure. Illustrative examples of IRES may include, but are not limited to, picornavirus IRES, pestivirus IRES, foot-and-mouth disease virus IRES, hepatitis A IRES, and hepatitis C IRES.

[0118] Delivery of exogenous nucleic acids

[0119] The cells to be delivered with the exogenous nucleic acid can be in vitro, ex vivo, or in vivo. In certain embodiments, the cells are in vitro, e.g., cells adapted or engineered to be suitable for in vitro culture, such as cells of an established cell line. In certain embodiments, the cells are ex vivo, e.g., primary cells isolated from a subject or derived from a subject, such as T cells. In certain embodiments, the cells are in vivo cells in a living subject.

[0120] The subject to be delivered with exogenous nucleic acid can be a non-human animal or a human. In some embodiments, the subject is a warm-blooded mammal, e.g., a primate, dog, cat, cow, horse, sheep, goat, rabbit, rat, and mouse. In some embodiments, the subject is a primate, e.g., a human.

[0121] Exogenous nucleic acids can be delivered to a cell or subject using a variety of techniques available for such delivery. For delivery to cells in vitro or ex vivo, exogenous nucleic acids can be transfected into cells by calcium chloride-, lithium chloride-, lithium acetate / polyethylene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated transfection (Graham et al. (1973) Virology. 52:456-467; Mannino et al. (1988) BioTechniques 6:682-690; Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751), direct microinjection (Capecchi, MR (1980) Cell 22:479-488), or by using high-speed tungsten microparticles (Klein et al. (1987) Nature 327:70-73). When the exogenous nucleic acid is in the form of a viral vector, it can be transfected into the cell by viral infection. Transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory, New York, Davis et al. (1986); Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981) Gene 13:197.

[0122] In order to deliver exogenous nucleic acid to cells in vivo or to experimenter, exogenous nucleic acid can be applied to experimenter whole body or in local treatment area.In certain embodiments, described exogenous nucleic acid is delivered to described experimenter by parenteral, oral, enteral, oral cavity, nasal cavity, local, rectal, vaginal, through mucosa, epidermal, transdermal, corium, eye, lung, heart, subcutaneous, intraparenchymal, intraventricular or intrathecal administration route.In certain embodiments, described exogenous nucleic acid is delivered to described experimenter orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intrathecal, intraventricular (ICV) or rectum.

[0123] Administration of aspirin compounds

[0124] An aspirin compound can be administered to a cell or a subject prior to or concurrently with delivery of the exogenous nucleic acid to the cell or the subject.

[0125] In certain embodiments, the exogenous nucleic acid is delivered to a cell or subject that has been or is being concurrently administered an aspirin compound.

[0126] In certain embodiments, the aspirin compound is administered to the cell or subject prior to delivery of the exogenous nucleic acid in a manner that prepares the cell or subject for such delivery. In certain embodiments, the aspirin compound is administered to the cell or subject at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to delivery of the exogenous nucleic acid. In certain embodiments, the aspirin compound is administered once or repeatedly (e.g., twice, three times, four times, etc.) to the cell or subject prior to the delivery of the nucleic acid.

[0127] In some embodiments, the aspirin compound is administered to the cell or the subject simultaneously with the exogenous nucleic acid. As used herein, the term "simultaneously" refers to an arrangement in which exogenous nucleic acid and aspirin compound are co-delivered to a cell or subject (in vitro, in vitro, or in vivo). Such co-delivery can be achieved by, for example, combining the exogenous nucleic acid and aspirin compound in a combined composition, or in a separate composition but delivered at substantially the same time by the same or different routes of administration, or in a separate composition at a controlled time such that the exogenous nucleic acid and aspirin compound are expected to act substantially simultaneously in a cell or subject.

[0128] In some embodiments, the aspirin compound is administered in an amount sufficient to effectively increase the expression of the exogenous nucleic acid in the cell or the subject. In certain embodiments, the aspirin compound is administered in an effective amount to increase the expression of the exogenous nucleic acid in the cell or the subject by at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more.

[0129] In certain embodiments, aspirin compounds can be added or introduced into the culture medium of cell in vitro or in vitro. In certain embodiments, aspirin compounds are delivered to the experimenter by parenteral, oral, enteral, oral cavity, nasal cavity, topical, rectal, vaginal, mucosal, epidermal, transdermal, dermal, eye, lung, heart, subcutaneous, intraparenchymal, intracerebroventricular or intrathecal administration route. In certain embodiments, aspirin compounds can be applied to the experimenter by any suitable approach, such as but not limited to oral, intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intracerebroventricular (ICV) or rectum. In certain embodiments, aspirin compounds are delivered to the experimenter by intravenous administration.

[0130] Preparation method

[0131] In one aspect, the present disclosure provides a method of preparing a cell or a subject for delivery of an exogenous nucleic acid.

[0132] As used herein, the term "priming" refers to any pretreatment of a cell or subject prior to delivery of an exogenous nucleic acid to prepare the cell or subject to be in a state that is receptive to and expresses the exogenous nucleic acid. In certain embodiments, the primed cell or subject can be in a desired state for receiving and expressing the exogenous nucleic acid, e.g., less immune responsiveness to the exogenous nucleic acid, compared to an unprimed cell or subject.

[0133] Method for expressing or increasing the expression level or extending the duration of expression

[0134] On the other hand, the present disclosure provides a method for expressing exogenous nucleic acids in cells. On the other hand, the present disclosure also provides a method for increasing the expression level of exogenous nucleic acids in cells or in subjects. In yet another aspect, the present disclosure provides a method for extending the expression duration of exogenous nucleic acids in cells or in subjects.

[0135] As used herein, the term "expression" ("expression" or "expressing") refers to the transcription of a coding DNA sequence into mRNA and / or the translation of a coding DNA sequence into a peptide or protein. As used herein, the phrase "expression of exogenous nucleic acid" refers to the expression of a coding sequence (e.g., a sequence encoding a protein of interest) included in an exogenous nucleic acid. In certain embodiments, the expression level of an exogenous nucleic acid is determined based on mRNA levels or protein levels. In certain embodiments, the expression level is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 700%, 800% or 900% relative to a control expression level. The control expression level can be an expression level determined under comparable conditions in the absence of treatment with an aspirin compound.

[0136] In certain embodiments, the expression level of the exogenous nucleic acid is determined on day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, or day 15 after delivery of the exogenous nucleic acid to the cell or subject.

[0137] In certain embodiments, the expression level of the exogenous nucleic acid is determined collectively over the duration of expression.

[0138] As used herein, "duration of expression" is the period of time during which an exogenous nucleic acid is expressed in a cell or subject at a detectable level or at a physiologically or therapeutically effective level. In certain embodiments, the duration of expression is the period of time during which a protein of interest expressed from an exogenous nucleic acid is at or above a detectable level or at a physiologically or therapeutically effective level in a cell or subject.

[0139] In some embodiments of the methods provided herein, the duration of expression is extended by at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days relative to a control duration. A control duration can be a duration of expression determined under comparable conditions in the absence of treatment with an aspirin compound.

[0140] Treatment or prevention methods

[0141] In another aspect, the present disclosure provides a method of treating a condition treatable by an exogenous nucleic acid or its expression product.

[0142] In some embodiments, the subject is suffering from a condition that can be treated by an exogenous nucleic acid or its expression product. In some embodiments, the subject is in need of treatment (e.g., the subject will benefit biologically or medically from treatment).

[0143] As used herein, "a condition treatable by an exogenous nucleic acid or its expression product" refers to any disease or condition susceptible to treatment with an exogenous nucleic acid or its expression product.

[0144] In some embodiments, such conditions are characterized by lacking one or more functional genes or functional proteins. In some embodiments, such conditions are suitable for gene therapy. The exogenous nucleic acid delivered to the subject can be useful for replacing or repairing the missing or dysfunctional molecular elements (e.g., genes) in the DNA of the subject's living cells, or alternatively, the function of the missing or dysfunctional gene in the cell can be provided or enhanced by introducing and expressing a functional gene in the cell.

[0145] In some embodiments, the condition is a monogenic disorder. A monogenic disorder is a condition caused by one or more abnormalities in the genome that affect one or both copies of a single gene. The genomic abnormality can disrupt a gene and result in a loss or underactivity of the endogenous protein encoded by the disrupted gene. The symptoms of a monogenic disorder are caused by a loss or underactivity of the endogenous protein.

[0146] In some embodiments, the monogenic disorder is autosomal dominant, autosomal recessive, X-linked, Y-linked, or mitochondrial.

[0147] Examples of autosomal dominant monogenic disorders include, but are not limited to, Brugada syndrome, myotonic dystrophy type 1, myotonic dystrophy type 2, hereditary multi-infarct dementia, Huntington's disease, neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome, familial hypercholesterolemia (FH), polycystic kidney disease, hereditary spherocytosis, hereditary nonpolyposis colorectal cancer, hereditary multiple exostoses, tuberous sclerosis, von Willebrand disease and acute intermittent porphyria, Dravet syndrome, Peutz-Jeghers syndrome, achondroplasia, primary combined immunodeficiency, familial adenomatous polyposis (FAP), spinocerebellar ataxia, multiple endocrine neoplasia.

[0148] Examples of autosomal recessive single gene disorders include, but are not limited to, beta-ketothiolase deficiency, biotinidase deficiency, hepatolenticular degeneration, spinal muscular atrophy, N-acetylglutamate synthetase deficiency, lysosomal acid lipase deficiency, lysinuric protein intolerance, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, Laron syndrome, isovaleric acidemia, hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, carboxylase synthetase deficiency, hereditary fructose intolerance, glycogen storage disease type II, glutaric acidemia type I, Gitelman syndrome, Gaucher disease, and glutaric acidemia type II. Disease), familial Mediterranean fever, myotonia congenita, citrullinemia I, citrullinemia II, primary carnitine deficiency, arginase deficiency, medium-chain acyl-CoA dehydrogenase deficiency, sickle cell anemia, cystic fibrosis, Tay–Sachs disease, phenylketonuria, lysosomal acid lipase deficiency, glycogen storage diseases, galactosemia, Niemann-Pick disease, spinal muscular atrophy (SMA), Roberts syndrome, very long-chain acyl-CoA dehydrogenase deficiency, cystic fibrosis, Gaucher disease, Werner syndrome, Fanconi anemia, and mucopolysaccharidoses (I, IIIA, IIIB, IVA, IVB, VI, VII, IX).

[0149] Examples of X-linked monogenic disorders include, but are not limited to, fragile X syndrome, adrenal hypoplasia congenita, Duchenne muscular dystrophy and hemophilia A, hemophilia B, Fabry disease, X-linked agammaglobulinemia, X-linked adrenoleukodystrophy, spinal and bulbar muscular atrophy, ornithine transcarbamylase deficiency and mucopolysaccharidosis II, adrenoleukodystrophy (ALD), chronic granulomatous disease.

[0150] Other examples of monogenic disorders include McCune-Albright syndrome, paroxysmal nocturnal hemoglobinuria, ADA immunodeficiency, amyotrophic lateral sclerosis (ALS), glucose-galactose, muscular dystrophy, azoospermia, Ehlers-Danlos Syndrome, retinitis pigmentosa, hemochromatosis, melanoma, retinoblastoma, Alzheimer's disease, amyloidosis, myotonic dystrophy, giant axonal neuropathy, alpha-1 antitrypsin, Parkinson's disease, severe combined immunodeficiency (ADA-SCID / X-SCID).

[0151] Examples of polygenic disorders include, for example, heart disease, cancer (eg, leukemia, particularly acute lymphoblastic leukemia), diabetes, schizophrenia, and Alzheimer's disease.

[0152] As used herein, the terms "treating" or "treatment" of a condition, as used herein, include alleviating the condition, slowing the rate of onset or development of the condition, relieving, alleviating or ending the symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0153] In another aspect, the present disclosure provides a method for preventing a condition that is preventable by an exogenous nucleic acid or its expression product.

[0154] As used herein, the terms "prevent," "prevents," or "prevention" and "preventable" refer to delaying the onset of a disease or condition, reducing the risk of developing a condition, delaying the development of symptoms associated with a condition, or some combination thereof. The terms do not imply complete elimination of the disease, but rather encompass any type of preventative treatment that reduces the incidence of a condition or delays the onset and / or progression of a condition.

[0155] In some embodiments, the preventable condition is characterized by a condition that can benefit from a protective effect (e.g., an immune response) that can be induced by an exogenous nucleic acid or its expression product. In some embodiments, the exogenous nucleic acid delivered to the subject can be used to express an immunogen that induces a protective immune response against a pathogen or cancer cell.

[0156] In certain embodiments, the treatment or prevention method comprises: delivering the exogenous nucleic acid to the subject, wherein the subject has been or is concurrently administered an aspirin compound.

[0157] In certain embodiments, the method of treatment or prevention comprises administering an aspirin compound to the subject prior to or concurrently with delivering the exogenous nucleic acid to the cell.

[0158] In certain embodiments, the treatment or prevention method comprises: a) administering an aspirin compound to the subject; and b) delivering the exogenous nucleic acid to the subject, wherein step a) is performed before or simultaneously with step b).

[0159] The exogenous nucleic acid is delivered to the subject in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" with respect to an exogenous nucleic acid means that the amount of the exogenous nucleic acid delivered to the subject is sufficient to produce a therapeutic or preventive benefit in the subject, for example, to provide some relief, alleviation or reduction (for therapeutic purposes) to at least one clinical symptom of the subject, or to delay the onset of a disease or condition or to alleviate symptoms when a disease or condition strikes (for preventive purposes). For example, a therapeutically effective amount of an exogenous nucleic acid can allow delivery to a sufficient number of cells and expression of the exogenous nucleic acid in the subject (for example, expression of a protein of interest from the exogenous nucleic acid) to produce a therapeutic benefit or a preventive benefit.

[0160] In some embodiments, the exogenous nucleic acid comprises or is included in a viral vector (e.g., an AAV vector or an AAV viral particle), and the therapeutically effective amount of the viral vector can be 10 6 vg / kg to 10 14 vg / kg (vector genome / kg), e.g., 10 7 vg / kg to 10 14 vg / kg, 10 8 vg / kg to 10 14 vg / kg, 10 9 vg / kg to 10 14 vg / kg, 10 10 vg / kg to 10 13 vg / kg, 10 10 vg / kg to 10 12.5 vg / kg, 10 10 vg / kg to 1012 vg / kg, 10 10 vg / kg to 10 11.5 vg / kg or 10 10 vg / kg to 10 11 In some embodiments, the therapeutically effective amount of the AAV vector is about or no more than 10 6 vg / kg, 10 7 vg / kg, 10 8 vg / kg, 10 9 vg / kg, 10 10 vg / kg, 10 11 vg / kg, 10 11.5 vg / kg, 10 12 vg / kg, 10 12.5 vg / kg or 10 13 vg / kg. A therapeutically effective amount of a viral vector can vary depending on many factors, such as the type of viral vector, the cells to be transfected, the condition to be treated, the subject being treated (e.g., disease state, age, sex, and weight), the ability of the viral vector to elicit a desired response in the individual, etc. A therapeutically effective amount can be determined by starting with a low but safe dose and gradually increasing to higher doses while monitoring the therapeutic effect (e.g., reduction in cancer cell growth) and the presence of any adverse side effects.

[0161] In certain embodiments, the therapeutically effective amount in the methods of treatment provided herein is a subtherapeutic amount. As used herein, the term "subtherapeutic amount" refers to an amount of exogenous nucleic acid that is less than the conventional amount required to produce a therapeutic benefit in conventional methods of treatment, wherein an aspirin compound is not administered to the subject prior to or concurrently with the delivery of the exogenous nucleic acid ("conventional methods of treatment").

[0162] In certain embodiments, a subtherapeutic amount of an exogenous nucleic acid produces insufficient or no therapeutic effect in conventional treatment methods (where an aspirin compound is not administered).

[0163] In some embodiments, the subtherapeutic amount is no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, no more than 2% or no more than 1% of the conventional amount of the same exogenous nucleic acid, which conventional amount would be required without administration of the aspirin compound.

[0164] In certain embodiments, the therapeutically effective amount in the methods of treatment provided herein is comparable or identical to a conventional amount, but provides a therapeutic effect higher than that of conventional methods of treatment. In certain embodiments, the methods provided herein can achieve a therapeutic effect that is at least 10%, higher than that achievable by the conventional methods of treatment, higher than that of ...

[0165] In some embodiments, an aspirin compound can be administered to the subject prior to or concurrently with administration of the exogenous nucleic acid.

[0166] In some embodiments, the aspirin compound is administered to the subject at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to delivering the exogenous nucleic acid to the subject. In some embodiments, the aspirin compound is administered to the subject once or repeatedly (e.g., twice, three times, four times, etc.) prior to delivering the nucleic acid. In some embodiments, the aspirin compound is administered to the subject simultaneously with the exogenous nucleic acid.

[0167] In some embodiments, the aspirin compound is administered in an amount sufficient to effectively increase the expression of the exogenous nucleic acid in the cell or the subject. In certain embodiments, the aspirin compound is administered in an effective amount that increases the expression of the exogenous nucleic acid in the subject by at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more.

[0168] In some embodiments, the aspirin compound is administered to the subject in an amount of no more than 30 mg / kg, no more than 50 mg / kg, no more than 60 mg / kg, no more than 70 mg / kg, no more than 80 mg / kg, no more than 90 mg / kg, no more than 100 mg / kg, no more than 110 mg / kg, no more than 120 mg / kg, no more than 120 mg / kg, no more than 130 mg / kg, no more than 140 mg / kg, no more than 150 mg / kg, no more than 160 mg / kg, no more than 170 mg / kg, no more than 180 mg / kg, no more than 190 mg / kg or no more than 200 mg / kg. In some embodiments, the aspirin compound is administered to the subject in an amount of 30 mg / kg to 200 mg / kg, 30 mg / kg to 150 mg / kg, 30 mg / kg to 120 mg / kg, 30 mg / kg to 100 mg / kg, 30 mg / kg to 90 mg / kg, 30 mg / kg to 80 mg / kg, 30 mg / kg to 70 mg / kg, 30 mg / kg to 60 mg / kg, 30 mg / kg to 50 mg / kg, 30 mg / kg to 40 mg / kg. In certain embodiments, the aspirin compound is administered to the subject in an amount of 30 mg / kg to 50 mg / kg, for example, about 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg. In certain embodiments, the aspirin compound is administered to the subject in an amount of 50 mg / kg to 100 mg / kg, for example, at about 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, or 100 mg / kg.

[0169] In certain embodiments, the aspirin compound and the exogenous nucleic acid are applied to the experimenter simultaneously. In certain embodiments, the aspirin compound and the exogenous nucleic acid can be applied close enough in time (for example, simultaneously, or in a short period before or after each other) so that it is expected that they work substantially simultaneously in the experimenter. Aspirin compound and exogenous nucleic acid can be considered to be applied simultaneously, as long as two medicaments enter into cells in a short period before or after each other or simultaneously. In certain embodiments, the aspirin compound and the exogenous nucleic acid are applied simultaneously in the form of a single composition. In some other embodiments, the aspirin compound and the exogenous nucleic acid are applied simultaneously with different compositions.

[0170] In some embodiments, the aspirin compound and the exogenous nucleic acid are mixed before being administered to the subject simultaneously. In some embodiments, the aspirin compound and the exogenous nucleic acid are co-administered to the subject intravenously. In some embodiments, the aspirin compound and the exogenous nucleic acid are administered to the subject via different routes of administration. In some embodiments, the aspirin compound is administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intrathecally, intracerebroventricularly (ICV), or rectally to the subject.

[0171] In certain embodiments, the aspirin compound and / or the exogenous nucleic acid are administered to treat central nervous system (CNS) disorders. In certain embodiments, the CNS disorders are treated by systemically administering the aspirin compound and / or the exogenous nucleic acid. In certain embodiments, the exogenous nucleic acid suitable for systemic administration to treat CNS disorders comprises an AAV vector (e.g., AAV viral particles), for example, an AAV9 vector (e.g., AAV9 particles). In certain embodiments, the systemic administration comprises intravenous, subcutaneous, or intramuscular administration. In certain embodiments, the CNS disorders are treated by intraventricular or intrathecal administration of the aspirin compound and / or the exogenous nucleic acid. CNS disorders can be any condition that can be treated by delivering therapeutic nucleic acids to the brain or spinal cord. Examples of CNS disorders include, e.g., Parkinson's disease, Alzheimer's disease, mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, Batten disease, spinocerebellar ataxia, spinal muscular atrophy, Canavan disease, and Friedreich's ataxia.

[0172] Without wishing to be bound by any theory, it is believed that the present invention is particularly advantageous in treating CNS disorders and that this is at least in part due to increased expression of exogenous nucleic acids in target sites in the brain. The inventors surprisingly discovered that by systemically administering AAV in combination with the aspirin compounds provided herein, much lower AAV doses can be used while still achieving the same or even greater efficacy as higher doses. It is known in the art that if AAV9 is administered by a systemic route, a dose of 10 14vg / kg threshold doses are required to achieve transgene expression in the brain, however, such doses are both technically challenging and expensive to manufacture, and this significantly limits the use of AAV9 in treating CNS disorders (for details, see Perez BA et al. Brain Sci. 2020 Feb 22;10(2).pii:E119; Duque S et al. Mol Ther. 2009 Jul;17(7):1187-96; Foust KD et al. Nat Biotechnol. 2009 Jan;27(1):59-65). As disclosed in this application, systemic administration of less than 10 14 The amount of vg / kg (for example, 10 13 vg / kg, 10 12.5 vg / kg, 10 12 vg / kg, 10 11 vg / kg or even lower) in combination with aspirin compounds can achieve transgene expression in the brain and, as with AAV9 delivered at or above 10 14 vg / kg of AAV9 administered without aspirin compounds was equally or even more effective than AAV9 administered with aspirin alone. This could reduce the dose of AAV required to treat CNS disorders and thereby reduce the complexity of the manufacturing process, allowing, for example, the use of lower dose AAV formulations to treat brain disorders.

[0173] On the other hand, the present disclosure provides a method for reducing the side effects of a subject or improving tolerance to exogenous nucleic acids. As used herein, "side effects" caused by the delivery of exogenous nucleic acids can be any type of side effects known in the art regarding drug delivery, including but not limited to nausea, vomiting, dizziness, drowsiness / sedation, allergies, itching, decreased gastrointestinal motility (including constipation), dysuria, peripheral vasodilation (including causing orthostatic hypotension), headache, dry mouth, sweating, fatigue, dependence, mood changes (e.g., irritability, euphoria), dizziness or even respiratory depression, apnea, respiratory arrest, circulatory depression, hypotension or shock.

[0174] In some embodiments of the present disclosure, the exogenous nucleic acid is delivered to the subject in a subtherapeutic amount. In certain embodiments, the subtherapeutic amount is therapeutically effective in the treatment methods provided herein, but causes significantly reduced side effects compared to conventional amounts.

[0175] In some embodiments, the side effects are dose-dependent on the exogenous nucleic acid delivered to the subject. It is believed that by administering the exogenous nucleic acid at subtherapeutic levels, side effects associated with the exogenous nucleic acid can be reduced due to less exposure.

[0176] Composition

[0177] In yet another aspect, the present disclosure provides a composition comprising a combination of an aspirin compound and an exogenous nucleic acid, wherein the exogenous nucleic acid comprises double-stranded DNA or can be converted to double-stranded DNA in the cell after delivery of the exogenous nucleic acid to the cell.

[0178] In some embodiments, the exogenous nucleic acid in the composition comprises a coding sequence that encodes a protein of interest or a portion thereof or encodes a functional RNA or a portion thereof.

[0179] In some embodiments, the therapeutic protein is selected from the group consisting of: SMN1, NAGLU, SGSH, IDS, FVIII, FIX, BTK, ABCD1, ACADVL, AR, HBB, SCN1A, CFTR, CSF2RA, IL2AG, PHA, STK11, PIGA, OTC, NAGS, DMPK, CNBP, ACADM, GNAS, FBN1, LIPA, SLC7A7, HADHA, GHR, IDV, ALPL, SLC25A15, HTT, HCS, NOTCH3, ALDOB, ATP7B, GAA, GCDH, SLC12A3, GBA, MEFV, GLA, CLCN1 NR0B1, ASS1, SLC25A13, SLC22A5, SCN5A, BTD, ACAT1, ARG1, CYP21A2, chimeric antigen receptors (CARs), antibodies (e.g., monoclonal or bispecific or multispecific), insulin, glucagon-like peptide-1, peptide hormones, growth factors, erythropoietin (EPO), cytokines, coagulation factors, antihemophilic factor, interferon, Fc fusion proteins (such as CTLA-4 Fc fusion, VEGFR Fc fusion), and therapeutic enzymes (e.g., lysosomal hydrolases and sulfatases).

[0180] In some embodiments, the immunogenic protein is selected from the group consisting of an immunogenic protein from an orthomyxovirus (e.g., influenza virus), a lentivirus (e.g., HIV, SIV), an arenavirus (Lassa fever virus), a poxvirus (e.g., vaccinia), a flavivirus (e.g., yellow fever virus), a filovirus (Ebola virus), a bunyavirus (RVFV, CCHF, or SFS virus), a coronavirus (e.g., SARS, MERS, or COVID-19), a poliovirus, a herpesvirus (CMV, EBV, HSV), a mumps virus, a measles virus, a rubella virus, a diphtheria toxin, a pertussis virus, and a hepatitis virus (e.g., HAV, HBV, or HCV).

[0181] In some embodiments, the nuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Cas family protein (e.g., Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas 10, Cas 11, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, homologs thereof, or modified versions thereof).

[0182] In some embodiments, the reporter protein is selected from the group consisting of a fluorescent protein (e.g., EGFP, GFP, RFP, BFP, YFP, or dsRED2), an enzyme that produces a detectable product, such as luciferase (e.g., from Gauss, Renilla, or Pho), β-galactosidase, β-glucuronidase, alkaline phosphatase, chloramphenicol acetyltransferase genes, and proteins that can be directly detected.

[0183] In some embodiments, the therapeutic target protein (e.g., CTLA-4, HER2, fibronectin-4, sclerostin, P-selectin, VEGF, RSV F, VEGFR2, CD79, IL23p19, vWF, IFN-γ, C5, PD-1, PD-L1, CGRP, CD3, CD11a, CD20, CD22, CD30, CD33, CD38, CD40, CD52, IgE, KLK, CCR4, FGF-23, IL-6R, IL- 5, IL-23p19, IL-2R, IL-17R, IL-17, CD4, FIX / FX, IL-12, IL-23, IL-1β, IL-5R, IL-6R, IL-4 / IL-13, PDGF-α, dabigatran, SLAMF7, EGFR, PCSK9, GD2, CD3, CD19, α4β7 integrin, α4β1 integrin, PA, BLyS, RANK, TNF-α, EpCAM, GGTA1, endostatin, angiostatin).

[0184] In some embodiments, the functional RNA regulates a biological target selected from the group consisting of: a multiple drug resistance (MDR) protein target, a tumor target (e.g., VEGF, Her2, EGFR, PD-L1, etc.), a pathogen target such as a viral surface antigen (e.g., hepatitis B surface antigen gene), a defective gene product (mutated dystrophin), or a therapeutic target (e.g., myostatin).

[0185] Pharmaceutical composition

[0186] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a subtherapeutic amount of an exogenous nucleic acid provided herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition further comprises an aspirin compound provided herein.

[0187] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the exogenous nucleic acid provided herein, the aspirin compound provided herein, and a pharmaceutically acceptable carrier.

[0188] As used herein, the term "pharmaceutically acceptable carrier" refers to any and all pharmaceutical carriers that can facilitate the storage and administration of the nucleic acids and expression vectors of the present disclosure to a subject and / or to a host cell, such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. In certain embodiments, the host cell to which the pharmaceutical composition has been administered is suitable for administration to a subject. A pharmaceutically acceptable carrier can comprise any suitable component, such as, but not limited to, saline, liposomes, polymeric excipients, colloids, or carrier particles.

[0189] In certain embodiments, the pharmaceutically acceptable carrier is a saline solution that can dissolve or disperse the nucleic acid, expression vector, and / or host cell of the present disclosure. Illustrative examples of saline solutions include, but are not limited to, buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, and polysorbate solution.

[0190] In certain embodiments, the pharmaceutically acceptable carrier is a liposome. Liposome is a single layer or multilayer vesicle with a film formed by a lipophilic material and an internal aqueous portion. The nucleic acid, expression vector and / or host cell of the present disclosure can be encapsulated in the aqueous portion of the liposome. The illustrative examples of liposomes include but are not limited to liposomes based on 3 [N- (N ', N '- dimethylaminoethane) aminoformyl] cholesterol (DC-Chlo), liposomes based on N- (2,3- dioleoyloxy) propyl-N, N, N- trimethylammonium chloride (DOTMA) and liposomes based on 1,2- dioleoyloxy -3- trimethylammonium propane (DOTAP). The method for preparing liposomes and encapsulating expression vectors in liposomes is well known in the art (see, for example, D D Lasic et al., " Liposomes in gene delivery ", published by CRC Press, 1997).

[0191] In certain embodiments, the pharmaceutically acceptable carrier is a polymeric excipient, such as, but not limited to, microspheres, microcapsules, polymeric micelles, and dendrimers. The nucleic acids, expression vectors, and / or host cells disclosed herein can be encapsulated, adhered, or coated on a polymer-based component by methods known in the art (see, for example, W. Heiser, Nonviral gene transfer technologies, published by Humana Press, 2004; U.S. Pat. No. 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0192] In certain embodiments, the pharmaceutically acceptable carrier is a colloid or carrier particle, such as gold colloid, gold nanoparticles, silica nanoparticles, and multi-segment nanorods. The nucleic acid, expression vector, or cell can be coated on, adhered to, or associated with the carrier in any suitable manner known in the art (see, for example, M. Sullivan et al., Gene Therapy, 10:1882–1890 (2003), C. McIntosh et al., Journal of the American Chemical Society (J. Am. Chem. Soc.), 123(31):7626–7629 (2001), D. Luo et al., Nature Biotechnology, 18:893-895 (2000), and A. Salem et al., Nature Materials, 2:668-671 (2003)).

[0193] In certain embodiments, the pharmaceutical composition may further include additives, such as but not limited to stabilizers, preservatives and transfection facilitators that contribute to the cellular uptake of the drug. Suitable stabilizers may include but are not limited to monosodium glutamate, glycine, EDTA and albumin (e.g., human serum albumin). Suitable preservatives may include but are not limited to 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenols, thimerosal and antibiotics. Suitable transfection facilitators may include but are not limited to calcium ions.

[0194] The pharmaceutical composition can be adapted for administration by any suitable route known in the art, including but not limited to parenteral, oral, enteral, intrabuccal, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, cardiac, subcutaneous, intraparenchymal, intracerebroventricular, or intrathecal administration routes.

[0195] The pharmaceutical composition can be administered to a subject in the form of a formulation or preparation suitable for each route of administration. Formulations suitable for administering the pharmaceutical composition may include, but are not limited to, solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposome-based formulations, patches, implants, and suppositories.

[0196] The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Methods for preparing these formulations or compositions comprise the steps of providing the exogenous nucleic acid of the present disclosure to one or more pharmaceutically acceptable carriers and optionally one or more adjuvants. Methods for preparing such formulations can be found, for example, in Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., A.R. Gennaro (ed.), Mack Publishing Co., NJ, 1995; R. Stribling et al., Proceedings of the National Academy of Sciences of the United States of America, 89:11277-11281 (1992); T.W. Kim et al., The Journal of Gene Medicine, 7(6):749-758 (2005); S.F. Jia et al., Clinical Cancer Research, 9:3462 (2003); A. Shahiwala et al., Recent patents on drug delivery and formulation. formulation, 1: 1-9 (2007); A. Barnes et al., Current Opinion in Molecular Therapeutics 2000 2: 87-93 (2000), which are incorporated herein by reference in their entireties).

[0197] In certain embodiments, the pharmaceutical composition of the present disclosure containing exogenous nucleic acid (e.g., AAV vector or AAV viral particles) is suitable for administration to a subject in need of treatment by local delivery to a target tissue or organ at the diseased site. In certain embodiments, the pharmaceutical composition is suitable for direct injection into a diseased site palpable through the skin using a syringe. In certain embodiments, the pharmaceutical composition is suitable for injection using an implantable drug delivery device connected to a catheter line or other medical access device, and can be used in conjunction with an imaging system directed to the diseased site. In certain embodiments, the pharmaceutical composition is suitable for direct injection into a diseased site visible in an exposed surgical area at an effective dose. In certain embodiments, the pharmaceutical composition (e.g., carrier-coated gold particles) is suitable for bombardment of the diseased site using a gene gun, which shoots the particles directly into the diseased site (see, for example, R. Muangmoonchai et al., Molecular Biology, 20(2):145-151 (2002)). In certain embodiments, the pharmaceutical composition is suitable for administration to a subject by intravenous injection. In certain embodiments, the pharmaceutical composition is suitable for oral or transmucosal administration to a subject. For references to methods for introducing therapeutic nucleic acids into cells or animals, see, for example, Yang, NS., Crit. Rev. Biotechnol. 12:335-356 (1992); Anderson, WF, Science 256:808-813 (1992); Miller, AS, Nature 357:455-460 (1992); Crystal, RG, Amer. J. Med. 92(Suppl. 6A):44S-52S (1992); Zwiebel, JA et al., Ann. NY Acad. Sci. 618:394-404 (1991); McLachlin, JR et al., Prog. Nucl. Acids Res. Molecular Biology. Res. Molec. Biol. 38:91-135 (1990); Kohn, DB et al., Cancer Invest. 7:179-192 (1989). These references are incorporated herein by reference in their entirety.

[0198] In some embodiments, the exogenous nucleic acid in the pharmaceutical composition comprises a coding sequence encoding a protein of interest or a portion thereof or encoding a functional RNA or a portion thereof. In some embodiments, the exogenous nucleic acid in the pharmaceutical composition comprises a nucleic acid of interest encoding a therapeutic protein. In some embodiments, the exogenous nucleic acid in the pharmaceutical composition comprises a nucleic acid of interest encoding a therapeutic protein suitable for treating CNS disorders or brain diseases. In some embodiments, the therapeutic protein or therapeutic target of the functional RNA for treating CNS disorders or brain diseases includes, for example, but not limited to, Tau, MeCP2, NGF, APOE, GDNF, SUMF, SGSH, AADC, CD, p53, ARSA arylsulfatase A, ABCD1, SMN1, NAGLU, SOD1, C9ORF72, TARDBP, FUS, HTT, LRRK2, PARIS, PARKIN, GAD, and α-synuclein. These genes are known in the art and have been described in, for example, Maguire CA et al. Neurotherapeutics. 2014 Oct;11(4):817-39; Bowers WJ et al. Hum Mol Genet. 2011 Apr 15;20(R1):R28-41).

[0199] In some embodiments, the exogenous nucleic acid comprises an AAV vector (e.g., an AAV viral particle). In some embodiments, the AAV viral particle in the pharmaceutical composition is suitable for providing an amount of no more than 10 14 vg / kg (e.g. no more than 10 13 vg / kg, 10 12.5 vg / kg, 10 12 vg / kg, 10 12.5 vg / kg, 10 12 vg / kg, 10 11.5 vg / kg, 10 11 vg / kg, 10 10.5 vg / kg, 10 10 vg / kg or 10 9 In some embodiments, the AAV viral particles in the pharmaceutical composition are suitable for providing a subtherapeutic dose.

[0200] In some embodiments, the pharmaceutical composition is in a unit dose and contains no more than 10 10 vg, 10 10.5 vg, 10 11 vg, 10 11.5 vg, 10 12vg, 10 12.5 vg, 10 13 vg, 10 13.5 vg, 10 14 vg, 10 14.5 vg, 10 15 vg, 10 15.5 vg or 10 16 vg of AAV viral particles. As used herein, a "unit dose" is a dose sufficient to provide one treatment. In some embodiments, the unit dose is for human use, for example, for human adults (e.g., an average weight of 60 kg), human adolescents, human children, or human infants. In some embodiments, the pharmaceutical composition is in a formulation suitable for systemic administration, such as for intravenous injection, intravenous infusion, and intramuscular injection.

[0201] In some embodiments, the aspirin compound is packaged with the AAV particles, for example, in a mixture or a combination thereof. In some embodiments, the aspirin compound is packaged separately from the AAV particles, for example, the aspirin compound can be provided in a separate container in any commercially available form.

[0202] In some embodiments, the pharmaceutical composition of the present disclosure further comprises instructions for use, wherein the instructions for use indicate that the aspirin compound is to be administered prior to or concurrently with the administration of the pharmaceutical composition. In some embodiments, the instructions for use indicate that for the treatment of brain diseases, the AAV virus is to be administered systemically, preferably at a dose of less than 10 14 The dosage of vg / kg is administered. The instructions for practicing the method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Therefore, the instructions can be present in the test kit as a package insert, or present in the label of the container of the test kit or its component (that is, relevant to packaging or subpackaging). In certain embodiments, the instructions exist as an electronic storage data file present in a suitable computer-readable storage medium (for example, CD-ROM, floppy disk, etc.). In certain embodiments, there are no actual instructions in the test kit, but there is provided for obtaining instructions from a remote source, for example, by the Internet. The example of this embodiment is a test kit comprising a website, and instructions can be viewed in the website and / or instructions can be downloaded from the website. The same as instructions, the means for obtaining instructions are recorded on a suitable substrate.

[0203] Reagent test kit

[0204] In another aspect, the present disclosure provides a kit comprising: a) a first composition comprising the aspirin compound; and b) a second composition comprising the exogenous nucleic acid provided herein. In some embodiments, the kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure.

[0205] In certain embodiments, the first composition and the second composition are in separate containers. The first composition and the second composition can be combined before use, or can be used separately, for example, at different times or by different administration routes.

[0206] In another aspect, the present disclosure provides a kit comprising a composition comprising the aspirin compound and the exogenous nucleic acid provided herein. In some embodiments, the kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure.

[0207] In some embodiments, the kit further comprises instructions for use, the instructions for use indicating that the second composition is to be administered before or simultaneously with the second composition. In some embodiments, the first composition and the second composition can be easily mixed prior to use to provide a combined composition. In some embodiments, the second composition comprises a subtherapeutic amount of the exogenous nucleic acid.

[0208] In yet another aspect, the present disclosure provides a composition comprising: an aspirin compound and a subtherapeutic amount of the exogenous nucleic acid provided herein.

[0209] The following examples are presented to illustrate the invention and to help those of ordinary skill in the art to make and use the invention. The examples are not intended to limit the scope of the invention in any other way.

[0210] Examples

[0211] Example 1.

[0212] Unless otherwise indicated, the practice of certain examples described herein can employ conventional techniques of molecular biology, cell culture, recombinant nucleic acid (e.g., DNA) technology, immunology, and / or nucleic acid and polypeptide synthesis, detection, manipulation, and quantification, which are all within the ordinary skill of the art. See, for example, Ausubel, F. et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all published by John Wiley & Sons, New York, as of January 2010 or later; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001, or 4th ed., 2012.

[0213] Material

[0214] The vector plasmid AAV-Lxp3.3-Gluc was prepared based on the method disclosed in U.S. Patent Publication No. US20160229904. Briefly, the synthetic promoter Lxp3.3 was synthesized based on the nucleotide sequence disclosed in US20160229904 (also provided herein as SEQ ID NO: 1), and the LXP3.3 promoter was linked at its 3' end to the reporter gene encoding Gaussia luciferase (Gluc) (its sequence can be found in GenBank Accession No. LC006266.1, see also SEQ ID NO: 2) to obtain the Lxp3.3-Gluc expression cassette. The Lxp3.3-Gluc expression cassette is inserted between the AAV2 ITRs in the AAV expression plasmid and co-transfected with a second plasmid expressing the Rep gene and a third plasmid encoding the Cap gene of AAV8, AAV9 or AAV843 into HEK 293 cells to be packaged into AAV8-Lxp3.3-Gluc virus, AAV9-Lxp3.3-Gluc virus or AAV843-Lxp3.3-Gluc virus, respectively. The Cap gene of AAV8 is shown in SEQ ID NO: 4 (obtained from GenBank Accession No. AF513852), and the encoded Cap protein sequence is shown in SEQ ID NO: 8 (see also GenBank Accession No. AAN03857.1). The Cap gene of AAV9 is from GenBank Accession No. AY530579 (see also SEQ ID NO: 5), and the encoded Cap protein sequence is shown in SEQ ID NO: 9. The Cap gene of AAV843 is derived from the synthetic capsid gene sequence of AAVXL32 as disclosed in WO 2019241324A1 (see also SEQ ID NO: 6), and the encoded Cap protein sequence is shown in SEQ ID NO: 10.

[0215] Virus titers were quantified by real-time PCR and dot blot using commercially available kits according to the manufacturer’s instructions. AAV8-Lxp3.3-Gluc and AAV9-Lxp3.3-Gluc were generated, showing titers of 2.67 × 10 13 (vector genomes / mL) vg / mL and 2.99×10 13 vg / mL. AAV843-Lxp3.3-Gluc was produced in-house with a titer of 4 × 10 12 vg / mL.

[0216] Acetylsalicylic acid (aspirin) with CAS number: 50-78-2 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0217] Example 2

[0218] 1.1 Aspirin therapy and viral vector delivery

[0219] Male C57BL / 6J mice (purchased from Shanghai Jiesijie Experimental Animals Co., Ltd.) (n=4) with an average weight of 20 g were divided into six groups. Aspirin was injected intraperitoneally into five groups of mice for 7 consecutive days (day -7 to day -1) at doses of 1 mg / kg, 15 mg / kg, 30 mg / kg, 50 mg / kg, and 100 mg / kg, respectively. The sixth group was injected with 5% castor oil (in PBS buffer (Catalog No.: 02-024-1ACS, BI)) as a control. On day 0, each mouse in each group was intravenously injected with AAV9-Lxp3.3-Gluc virus (5×10 12 vg / kg), and the expression of luciferase was detected from day 1.

[0220] 1.2 Sample collection

[0221] Blood samples of mice were collected by retroorbital bleeding. Half the volume of blood sample was anticoagulated with sodium citrate at a ratio of 1:9, mixed, centrifuged within 30 minutes (6000rpm, 5 minutes), and the supernatant was collected as mouse plasma for detection of Gluc expression. The remaining blood sample was placed at room temperature for 2 hours to separate serum, centrifuged at 6000rpm for 15 minutes, and the supernatant was collected as mouse serum sample. The content of IFN-α and IFN-β was determined by using mouse IFN-β ELISA kit (720131-2, Shanghai Mlbio Co., Ltd.), LEGEND MAX TM The expression of IFN-β was determined by mouse ELISA kit (439407, Biolegend) and IFN-α by mouse ELISA kit (706291-2, Shanghai ELISA Biotechnology Co., Ltd.).

[0222] 1.3 Detection of Gluc

[0223] First prepare coelenterazine h (40906ES02, Shanghai Yeasen Biotech Co., Ltd.) buffer: add 29.72g of sodium ascorbate to 500mL of ultrapure water, and then add 50mL of Tris-HCl (1mol / L, pH 7.4). The concentration of the coelenterazine h stock solution is 200μM. The coelenterazine h working solution is prepared at a ratio of 1: 1 and used in a freshly prepared form. 10μL of the above working solution is added to 10μL of plasma sample, and the relative light units (RLU) are detected with a Synergy H1 multifunctional microplate reader.

[0224] 1.4 Results

[0225] The results showed that the expression levels of luciferase in all groups gradually increased over time, but the expression levels in some groups decreased to a certain extent starting from day 11. The expression level of luciferase in the aspirin pre-treated group showed a dose-dependent increase with aspirin concentration, as revealed by comparison between groups administered with different concentrations of aspirin, with the highest expression level of luciferase reaching 100 mg / kg of aspirin ( Figure 1 ). It was found that the IFN-α levels in the groups treated with 50 mg / kg and 100 mg / kg aspirin on day 3 were significantly lower than those in the control group ( Figure 2 ).

[0226] Example 3.

[0227] Aspirin at a dose of 50 mg / kg was injected for further study of AAV-mediated exogenous gene expression by three different AAV serotypes. The study design was similar to Example 2. Briefly, for each AAV serotype (i.e., AAV8-Lxp3.3-Gluc, AAV9-Lxp3.3-Gluc, or AAV843-Lxp3.3-Gluc), male C57BL / 6J mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) (n=4) with an average weight of 20 g were divided into two groups. One group was injected with 50 mg / kg of aspirin for 7 consecutive days, and the other group was injected with 5% castor oil (in PBS buffer (Catalog No.: 02-024-1ACS, BI)) as a control. On day 8, 5×10 12 vg / kg of AAV8-Lxp3.3-Gluc, AAV9-Lxp3.3-Gluc or AAV843-Lxp3.3-Gluc virus (diluted in 150-200 μl PBS) was delivered to each mouse via tail vein injection. Blood samples were collected and the expression level of luciferase was measured in the same manner as described in Example 2.

[0228] The results showed that aspirin can significantly promote the transduction of all tested virus serotypes, namely AAV8-Lxp3.3-Gluc, AAV9-Lxp3.3-Gluc and AAV843-Lxp3.3-Gluc viruses in vivo. As compared with the control group, the expression of the luciferase gene contained in the three AAV serotypes showed significant differences from the first day after AAV injection. Over time, the expression of luciferase increased rapidly, peaked on the 15th day after AAV injection, and then decreased. Throughout the period, the aspirin-treated group showed significant differences from its corresponding control group ( Figure 3A 、 3B , 3C). The expression of luciferase in the control group also reached a peak on the 15th day.

[0229] AAV and aspirin have been studied for co-administration in one formulation or in separate formulations. Preliminary results suggest that co-administration of AAV and aspirin can also increase transgene expression levels.

[0230] In summary, our results demonstrate that appropriate treatment of mice with aspirin before or concurrently with AAV injection can significantly enhance AAV transduction and increase transgene expression levels in a serotype-independent manner.

[0231] Example 4.

[0232] In the study described in Example 3, the levels of IFN-α and IFN-β were determined by using mouse IFN-β ELISA kit (720131-2, Shanghai ELISA Biotechnology Co., Ltd.), LEGEND MAX TM Mouse IFN-β ELISA kit (439407, Baijin Biotechnology Co., Ltd.) and mouse IFN-α ELISA kit (706291-2, Shanghai ELISA Biotechnology Co., Ltd.) were used for determination according to the user manual. Analysis of IFN-α and IFN-β levels showed that aspirin significantly inhibited the expression of type I interferon in mice after injection of AAV8, AAV9 and AAV843. The IFN-α level of the control group for AAV8 injection increased significantly from the second day and gradually decreased from the 15th day, while the IFN-α in mice treated with aspirin remained at a relatively low level, although it increased from the 3rd to the 11th day and from the 23rd to the 31st day after injection of AAV8 ( Figure 4A). Except for day 31, the IFN-α level in the control group was significantly higher than that in the aspirin-treated group. The IFN-β level in the control group for AAV8 injection was significantly higher than that in the aspirin-treated group. The IFN-β level in the control group gradually decreased over time but remained significantly higher than that in the aspirin-treated group ( Figure 4B ).

[0233] After AAV9 injection, IFN-α levels in the aspirin-treated group increased from day 11 to day 18 but were significantly lower than those in the control group ( Figure 5A In the control group, IFN-α gradually decreased from the 23rd day ( Figure 5A ). Similar to the trend of IFN-α levels in the control group, IFN-β levels in the control group also decreased significantly after day 23, while in the aspirin-treated group, IFN-β remained at a low level from day 1 to day 31 and showed a statistically significant difference from the level of the control group from day 1 to day 23 ( Figure 5B ).

[0234] Measurement of IFN-α and IFN-β levels after AAV843 injection showed that both type I interferons increased gradually over time in the aspirin-treated group, but their levels were significantly lower than those in the control group ( Figure 6A and Figure 6B The level of type I interferon in the control group showed a gradual decline. On day 31 after virus injection, there was no significant difference in the level of type I interferon between the aspirin-treated group and the control group ( Figure 6A and Figure 6B ).

[0235] In summary, our results demonstrate that aspirin significantly inhibits type I IFN activation following transduction of different AAV serotypes, suggesting that such effects are independent of serotype.

[0236] Example 5.

[0237] To confirm the expression of the transgene in the brain, male C57BL / 6J mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) with an average weight of 20 g (n = 6-8 mice per group) were divided into five groups. Four groups were first injected with 50 mg / kg aspirin for 7 consecutive days, and on the 8th day, 10 10 vg / kg, 10 11 vg / kg, 10 12 vg / kg and 10 13vg / kg of AAV virus were delivered to mice in the corresponding groups. The fifth group was the control group, which was injected with 5% castor oil (in PBS buffer (Catalog No.: 02-024-1ACS, BI)) for 7 consecutive days, and only 10 14 vg / kg of AAV was delivered to each mouse in the group. The animals were anesthetized and perfused transcardially 15 to 25 days after injection, and the brain of each mouse was fixed and then sectioned. Transgene expression was analyzed in different regions of the brain. It is expected that intravenous administration of aspirin and AAV effectively produces transgene expression in the brain, with a significant increase in CNS therapeutic effects. Interestingly, the dose of AAV used in the preliminary study was much lower than the dose believed to be able to express in the brain (10 14 vg / kg). Preliminary studies have shown that despite relatively low doses of AAV and low doses of aspirin, transgene expression in the brain is significantly increased.

[0238] Further studies are being designed and conducted to further validate the CNS effects.

[0239] Example 6.

[0240] The expression of the exogenous gene was detected in mice pretreated with aspirin or co-administered with aspirin. AAV9-CB-Gluc was prepared according to the method provided in Example 1, except that the nucleic acid sequence of the CB promoter (see SEQ ID NO: 3) was used. Briefly, one group of mice was pre-administered with aspirin 50 mg / kg by intraperitoneal injection for 7 days, and then AAV9-CB-Gluc (5×10 13 vg / kg). In contrast, a group of mice not pre-administered with aspirin were simultaneously administered with 50 mg / kg of aspirin in combination with AAV9-CB-Gluc. Mice were sacrificed 15 days after AAV9-CB-Gluc administration, and brains and livers were harvested for determination of Gluc expression.

[0241] In the brain, Gluc mRNA levels were significantly higher in mice pre-injected with aspirin compared to the non-treated group (i.e., mice administered AAV9-CB-Gluc without any aspirin treatment). Figure 7A , p<0.01) and enzyme activity levels ( Figure 7B , p<0.01) were upregulated by 5.5-fold and 7.13-fold, respectively. In addition, compared with the non-treatment group, the mRNA level of Gluc in the simultaneous treatment group ( Figure 7A , p<0.01) and enzyme activity levels ( Figure 7B , p<0.01) were also upregulated by 2.95-fold (p<0.05) and 3.55-fold (p<0.01).

[0242] In the liver, the mRNA level and enzyme activity of mice pre-injected with aspirin were upregulated by 1.43-fold ( Figure 7C , p<0.05) and 1.95 times ( Figure 7D , p<0.01). However, compared with the non-treatment group, the treatment group did not increase the mRNA level or enzyme activity level in the liver ( Figure 7C 、 7D , p<0.05). The mRNA level was even lower than that of the non-treatment group ( Figure 7C ), while there was no significant difference in enzyme activity levels ( Figure 7D ).

[0243] In conclusion, aspirin has been shown to significantly improve AAV9 transduction and increase exogenous gene expression in the brain. Increased exogenous gene expression in the liver was also observed in the aspirin pretreatment group.

[0244] Example 7.

[0245] The effect of aspirin on improving expression of a therapeutic transgene in the brain was determined. The AAV9-CB-IDS vector was generated using the methods described in Example 1, except that the iduronate 2-sulfatase (IDS) gene expression cassette was inserted between the AAV ITRs. The gene encoding IDS is provided in SEQ ID NO: 7 and the protein sequence of IDS is provided in SEQ ID NO: 11. To determine the therapeutic effect of the transgene, B6N.Cg-Ids was used. tm1Muen / J mice (n=5, obtained from JAX mice, catalog number: 024744), a model of mucopolysaccharidosis II (MPSII) with inactive iduronate-2-sulfatase (IDS) due to a mutation. A group of wild-type control mice were used as controls. The AAV9-CB-IDS vector for MPSII treatment was administered as described in Example 1 at a concentration of 3×10 13 vg / kg (i.e. 3E13 group) or 1×10 14 vg / kg (i.e., 1E14 group) was administered to mice pretreated with aspirin injection. One month after AAV9-CB-IDS injection, IDS enzyme activity was detected in the brain ( Figure 8 The results showed that in the 3E13 group, aspirin pretreatment significantly increased IDS enzyme activity in MPSII mice, and its level was even higher than that in the wild-type mouse group, and such level was significantly higher than that in the group that did not receive aspirin but only received the same dose (3×10 13 vg / kg)(p<0.05) or even higher doses (10 14These results demonstrate that aspirin can significantly reduce the AAV dose required to treat the disease in the absence of aspirin. Sequence Listing <110> Shanghai Belief-Delivery BIOMED CO., LTD. <120> Novel use of aspirin compounds in increasing nucleic acid expression <130> 075580-8002WO02 <150> PCT / CN2020 / 078843 <151> 2020-03-11 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 287 <212> DNA <213> Artificial Sequence <220> <223> synthesis <400> 1 ctgtttactc tggttaattt ttaaaggagg gtaaacagtg cctgaaagct gacctttgcc 60 cacattcctc cggtagacat taacttatta aattgattct gattacaaat ctgacctttg 120 cccccatctc acccagtaac aatgcaagag ttgatgtcag tctataaaaa gcgaagcgcg 180 cggtgggcgg ggttcgctgc ctgcaggtga gtatctcagg gatccagaca tggggatatg 240 ggaggtgcct ctgatcccag ggctcactgt gggtctctct gttcaca 287 <210> 2 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 2 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgactaa 558 <210> 3 <211> 718 <212> DNA <213> Artificial Sequence <220> <223> Synthesis <400> 3 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240 catgacctta tgggactttc ctacttggca gtacatctac tcgaggccac gttctgcttc 300 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 360 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg 420 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg 480 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc 540 gcgcggcggg cgggagcggg atcagccacc gcggtggcgg cctagagtcg acgaggaact 600 gaaaaaccag aaagttaact ggtaagttta gtctttttgt cttttatttc aggtcccgga 660 tccggtggtg gtgcaaatca aagaactgct cctcagtgga tgttgccttt acttctag 718 <210> 4 <211> 2217 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 4 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctgc aggcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420 ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc 480 ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca 540 gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtggga cctaacaa tggctgcagg cggtggcgca ccaatggcag acaataacga aggcgccgac ggagtgggta gttcctcggg aaattggcat tgcgattcca catggctggg cgacagagtc 720 atcaccacca gcacccgac ctgggccctg cccacctaca acaaccacct ctacaagcaa atctccaacg ggacatcggg aggagccacc aacgacaaca cctacttcgg ctacagcacc ccctgggggt attttgactt taacagattc cactgccact tttcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cccaagagac tcagcttcaa gctcttcaac atccaggtca aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taacctcacc agcaccatcc aggtgtttac ggactcggag taccagctgc cgtacgttct cggctctgcc 1080. caccagggct gcctgcctcc gttcccggcg gacgtgttca tgattcccca gtacggctac 1140 ctaacactca acaacggtag tcaggccgtg ggacgctcct ccttctactg cctggaatac tttccttcgc agatgctgag aaccggcaac aacttccagt ttacttacac cttcgaggac gtgcctttcc acagcagcta cgcccacagc cagagcttgg accggctgat gaatcctctg 1320 attgaccagt acctgtacta cttgtctcgg actcaaacaa caggaggcac ggcaaatacg 1380 cagactctgg gcttcagcca aggtgggcct aatacaatgg ccaatcaggc aaagaactgg 1440 ctgccaggac cctgttaccg ccaacaacgc gtctcaacga caaccgggca aaacaacaat 1500 agcaactttg cctggactgc tgggaccaaa taccatctga atggaagaaa ttcattggct 1560 aatcctggca tcgctatggc aacacacaaa gacgacgagg agcgtttttt tcccagtaac 1620 gggatcctga tttttggcaa acaaaatgct gccagagaca atgcggatta cagcgatgtc 1680 atgctcacca gcgaggaaga aatcaaaacc actaaccctg tggctacaga ggaatacggt 1740 atcgtggcag ataacttgca gcagcaaaac acggctcctc aaattggaac tgtcaacagc 1800 cagggggcct tacccggtat ggtctggcag aaccgggacg tgtacctgca gggtcccatc 1860 tgggccaaga ttcctcacac ggacggcaac ttccacccgt ctccgctgat gggcggcttt 1920 ggcctgaaac atcctccgcc tcagatcctg atcaagaaca cgcctgtacc tgcggatcct 1980 ccgaccacct tcaaccagtc aaagctgaac tctttcatca cgcaatacag caccggacag 2040 gtcagcgtgg aaattgaatg ggagctgcag aaggaaaaca gcaagcgctg gaaccccgag 2100 atccagtaca cctccaacta ctacaaatct acaagtgtgg actttgctgt taatacagaa 2160 ggcgtgtact ctgaaccccg ccccattggc acccgttacc tcacccgtaa tctgtaa 2217 <210> 5 <211> 2211 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60 gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120 aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180 aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240 cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300 caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420 ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actctcccgc gggtattggc 480 aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540 tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600 cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660 gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720 accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780 tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcacccc 840 tggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900 ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960 caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020 acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080 gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140 acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200 ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260 cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320 gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380 ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440 ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500 tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560 ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620 ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680 accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740 gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800 atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860 aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920 aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980 gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040 gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100 tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160 tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta a 2211 <210> 6 <211> 2214 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 6 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctgg agcacgacaa ggcctacgac 240 cagcagctgc aggcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420 ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc 480 ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca 540 gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtggga 600 cctaatacaa tggcttcagg cggtggcgca ccaatggcag acaataacga aggcgccgac 660 ggagtgggta atgcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc 720 atcaccacca gcacccgaac atgggccttg cccacctata acaaccacct ctacaagcaa 780 atctccagtg cttcaacggg ggccagcaac gacaaccact acttcggcta cagcaccccc 840 tgggggtatt ttgatttcaa cagattccac tgccatttct caccacgtga ctggcagcga 900 ctcatcaaca acaattgggg attccggccc aagagactca acttcaagct cttcaacatc 960 caagtcaagg aggtcacgac gaatgatggc gtcacgacca tcgctaataa ccttaccagc 1020 acggttcaag tcttctcgga ctcggagtac cagttgccgt acgtcctcgg ctctgcgcac 1080 cagggctgcc tccctccgtt cccggcggac gtgttcatga ttccgcaata cggctacctg 1140 acgctcaaca atggcagcca agccgtggga cgttcatcct tttactgcct ggaatatttc 1200 ccttctcaga tgctgagaac gggcaacaac tttaccttca gctacacctt tgaggaagtg 1260 cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320 gaccagtacc tgtattacct gaagaact cagaatcagt ccggaagtgc ccaaaaaag 1380 gacttgctgt ttagccgtgg gtctccagct ggcatgtctg ttcagcccaa aaactggcta 1440 cctggaccct gttaccggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500 aactttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560 cctggcactg ctatggcctc acaaagac gacaaagaca agttctttcc catgagcggt 1620 gtcatgattt ttggaaagga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680 atcacagacg aagaggaaat caaagccact aaccccgtgg ccaccgaaag atttgggact 1740 gtggcagtca atctccagag cagcagcaca gaccctgcga ccggagatgt gcatgttatg 1800 ggagccttac ctggaatggt gtggcaagac agagacgtat acctgcaggg tcctatttgg 1860 gccaaaattc ctcacacgga tggacacttt cacccgtctc ctctcatggg cggctttgga 1920 cttaagcacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980 gcagagtttt cggctacaaa gtttgcttca ttcatcaccc agtattccac aggacaagtg 2040 agcgtggaga ttgaatggga gctgcagaaa gaaaacagca aacgctggaa tcccgaagtg 2100 cagtatacat ctaactatgc aaaatctgcc aacgttgatt ttactgtgga caacaatgga 2160 ctttatactg agcctcgccc cattggcacc cgttacctca cccgtcccct gtaa 2214 <210> 7 <211> 1653 <212> DNA <213> Homo sapiens <400> 7 atgccgccac cccggaccgg ccgaggcctt ctctggctgg gtctggttct gagctccgtc tgcgtcgccc tcggatccga aacgcaggcc aactcgacca cagatgctct gaaacgttctt ctcatcatcg tggatgacct gcgcccctcc ctgggctgtt atggggataa gctggtgagg 180 240. tccccaata ttgaccaact ggcatcccac agcctcctct tccagaatgc ctttgcgcag caagcagtgt gcgccccgag ccgcgtttct ttcctcactg gcaggagacc tgacaccacc 300 cgcctgtacg acttcaactc ctactggagg gtgcacgctg gaaacttctc caccatcccc 360 cagtacttca aggagaatgg ctatgtgacc atgtcggtgg gaaaagtctt tcaccctggg 420 atatcttcta accataccga tgattctccg tatagctggt cttttccacc ttatcatcct 480 tcctctgaga actccatgcc tgtcgagggc actccatgcc aacctgcttt gccctgtgga tgtgctggat gttcccgagg gcaccttgcc tgacaaacag 600. agcactgagc aagccataca gttgttggaa aagatgaaaa cgtcagccag tcctttcttc 660. ctggccgttg ggtatcataa gccacacatc cccttcagat accccaagga atttcagaag 720 ttgtatccct tggagaacat caccctggcc cccgatcccg aggtccctga tggcctaccc 780 cctgtggcct acaacccctg gatggacatc aggcaacggg aagacgtcca agccttaaac 840 atcagtgtgc cgtatggtcc aattcctgtg gactttcagc ggaaaatccg ccagagctac 900 tttgcctctg tgtcatattt ggatacacag gtcggccgcc tcttgagtgc tttggacgat 960 cttcagctgg ccaacagcac catcattgca tttacctcgg atcatgggtg ggctctaggt 1020 gaacatggag aatgggccaa atacagcaat tttgatgttg ctacccatgt tcccctgata 1080 ttctatgttc ctggaaggac ggcttcactt ccggaggcag gcgagaagct tttcccttac 1140 ctcgaccctt ttgattccgc ctcacagttg atggagccag gcaggcaatc catggacctt 1200 gtggaacttg tgtctctttt tcccacgctg gctggacttg caggactgca ggttccacct 1260 cgctgccccg ttccttcatt tcacgttgag ctgtgcagag aaggcaagaa ccttctgaag 1320 cattttcgat tccgtgactt ggaagaggat ccgtacctcc ctggtaatcc ccgtgaactg 1380 attgcctata gccagtatcc ccggccttca gacatccctc agtggaattc tgacaagccg 1440 agtttaaaag atataaagat catgggctat tccatacgca ccatagacta taggtatact 1500 gtgtgggttg gcttcaatcc tgatgaattt ctagctaact tttctgacat ccatgcaggg 1560 gaactgtatt ttgtggattc tgacccattg caggatcaca atatgtataa tgattcccaa 1620 ggtggagatc ttttccagtt gttgatgcct tga 1653 <210> 8 <211> 738 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185 190 Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly 195 200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser 210 215 220 Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp 260 265 270 Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285 Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300 Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn 305 310 315 320 Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330 335 Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln 340 345 350 Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe 355 360 365 Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn 370 375 380 Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr 385 390 395 400 Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr 405 410 415 Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430 Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445 Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly 450 455 460 Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp 465 470 475 480 Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly 485 490 495 Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His 500 505 510 Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr 515 520 525 His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile 530 535 540 Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val 545 550 555 560 Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565 570 575 Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala 580 585 590 Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val 595 600 605 Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile 610 615 620 Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe 625 630 635 640 Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655 Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe 660 665 670 Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 675 680 685 Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690 695 700 Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu 705 710 715 720 Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg 725 730 735 Asn Leu <210> 9 <211> 736 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 9 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 10 <211> 737 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185 190 Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ser Gly Gly 195 200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn 210 215 220 Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn 260 265 270 His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr 405 410 415 Phe Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn 435 440 445 Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe 450 455 460 Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu 465 470 475 480 Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp 485 490 495 Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu 500 505 510 Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His 515 520 525 Lys Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe 530 535 540 Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met 545 550 555 560 Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu 565 570 575 Arg Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro 580 585 590 Ala Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp 595 600 605 Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro 610 615 620 His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly 625 630 635 640 Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro 645 650 655 Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile 660 665 670 Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu 675 680 685 Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser 690 695 700 Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly 705 710 715 720 Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro 725 730 735 Leu <210> 11 <211> 550 <212> PRT <213> Homo sapiens <400> 11 Met Pro Pro Pro Arg Thr Gly Arg Gly Leu Leu Trp Leu Gly Leu Val 1 5 10 15 Leu Ser Ser Val Cys Val Ala Leu Gly Ser Glu Thr Gln Ala Asn Ser 20 25 30 Thr Thr Asp Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp Leu Arg 35 40 45 Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro Asn Ile 50 55 60 Asp Gln Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala Gln 65 70 75 80 Gln Ala Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr Gly Arg Arg 85 90 95 Pro Asp Thr Thr Arg Leu Tyr Asp Phe Asn Ser Tyr Trp Arg Val His 100 105 110 Ala Gly Asn Phe Ser Thr Ile Pro Gln Tyr Phe Lys Glu Asn Gly Tyr 115 120 125 Val Thr Met Ser Val Gly Lys Val Phe His Pro Gly Ile Ser Ser Asn 130 135 140 His Thr Asp Asp Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr His Pro 145 150 155 160 Ser Ser Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly 165 170 175 Glu Leu His Ala Asn Leu Leu Cys Pro Val Asp Val Leu Asp Val Pro 180 185 190 Glu Gly Thr Leu Pro Asp Lys Gln Ser Thr Glu Gln Ala Ile Gln Leu 195 200 205 Leu Glu Lys Met Lys Thr Ser Ala Ser Pro Phe Phe Leu Ala Val Gly 210 215 220 Tyr His Lys Pro His Ile Pro Phe Arg Tyr Pro Lys Glu Phe Gln Lys 225 230 235 240 Leu Tyr Pro Leu Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu Val Pro 245 250 255 Asp Gly Leu Pro Pro Val Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln 260 265 270 Arg Glu Asp Val Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly Pro Ile 275 280 285 Pro Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala Ser Val 290 295 300 Ser Tyr Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp Asp 305 310 315 320 Leu Gln Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr Ser Asp His Gly 325 330 335 Trp Ala Leu Gly Glu His Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp 340 345 350 Val Ala Thr His Val Pro Leu Ile Phe Tyr Val Pro Gly Arg Thr Ala 355 360 365 Ser Leu Pro Glu Ala Gly Glu Lys Leu Phe Pro Tyr Leu Asp Pro Phe 370 375 380 Asp Ser Ala Ser Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu 385 390 395 400 Val Glu Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu 405 410 415 Gln Val Pro Pro Arg Cys Pro Val Pro Ser Phe His Val Glu Leu Cys 420 425 430 Arg Glu Gly Lys Asn Leu Leu Lys His Phe Arg Phe Arg Asp Leu Glu 435 440 445 Glu Asp Pro Tyr Leu Pro Gly Asn Pro Arg Glu Leu Ile Ala Tyr Ser 450 455 460 Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln Trp Asn Ser Asp Lys Pro 465 470 475 480 Ser Leu Lys Asp Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr Ile Asp 485 490 495 Tyr Arg Tyr Thr Val Trp Val Gly Phe Asn Pro Asp Glu Phe Leu Ala 500 505 510 Asn Phe Ser Asp Ile His Ala Gly Glu Leu Tyr Phe Val Asp Ser Asp 515 520 525 Pro Leu Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly Asp Leu 530 535 540 Phe Gln Leu Leu Met Pro 545 550

Claims

1. Use of an aspirin compound in the preparation of a medicament for increasing the expression level of an exogenous nucleic acid in a cell, the use comprising: administering an aspirin compound to the cell under conditions suitable for expression prior to or simultaneously with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid comprises double-stranded DNA, or is capable of being converted to double-stranded DNA in the cell after delivery, and The expression level of the exogenous nucleic acid is thereby increased as compared to a control expression level obtained in a control cell not administered the aspirin compound.

2. Use of an aspirin compound in the preparation of a medicament for increasing the expression level of an exogenous nucleic acid in a cell, the use comprising: delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein the cell has been or is being concurrently administered with an aspirin compound, wherein the exogenous nucleic acid comprises double-stranded DNA, or is capable of being converted to double-stranded DNA in the cell after delivery, and The expression level of the exogenous nucleic acid is thereby increased as compared to a control expression level obtained in a control cell not administered the aspirin compound.

3. Use of an aspirin compound in the preparation of a medicament for increasing the expression level of an exogenous nucleic acid in a cell, the use comprising: a) administering an aspirin compound to the cell; as well as b) delivering the exogenous nucleic acid to the cell under conditions suitable for expression, wherein said step a) is performed before or simultaneously with said step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or is capable of being converted to double-stranded DNA in the cell after delivery, and wherein said expression level of said exogenous nucleic acid is increased compared to a control expression level obtained in a control cell not subjected to said step a).

4. The use according to any one of claims 1 to 3, wherein the cell is in vitro, ex vivo or in vivo.

5. The use of any one of claims 1-3, wherein the aspirin compound is administered to the cell at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to delivering the nucleic acid.

6. The use according to any one of claims 1 to 3, wherein the aspirin compound is administered once or repeatedly to the cell prior to the delivering of the nucleic acid.

7. The use according to claim 6, wherein the aspirin compound is repeatedly administered to the cell 7 times, once a day, before the delivering of the nucleic acid.

8. The use according to any one of claims 1 to 3, wherein the aspirin compound is administered in an amount of 30 mg / kg to 120 mg / kg.

9. The use according to claim 8, wherein the aspirin compound is administered in an amount of 50 mg / kg or 100 mg / kg.

10. The use of any one of claims 1-3, wherein the aspirin compound is administered in an amount sufficient to increase expression of the exogenous nucleic acid in the cell by at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more.

11. The use according to any one of claims 1 to 3, wherein the expression level is based on mRNA level or protein level.

12. The method of claim 11, wherein the expression level is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 400%, 500%, 600%, 700%, 800% or 900%.

13. The use according to any one of claims 1 to 3, wherein the expression level is determined over the duration of expression of the exogenous nucleic acid.

14. The use according to any one of claims 1 to 3, wherein the exogenous nucleic acid comprises a viral vector, a plasmid or an exosome, or is contained in a viral vector, a plasmid or an exosome.

15. The use according to claim 14, wherein the viral vector comprises an adeno-associated virus (AAV) vector.

16. The use according to claim 15, wherein the adeno-associated virus AAV vector is an AAV9, AAV8, and / or AAV843 vector.

17. The use according to claim 15, wherein the AAV vector comprises AAV virus particles.

18. The method according to any one of claims 1 to 3, wherein the exogenous nucleic acid comprises a coding sequence encoding a protein of interest or a portion thereof, or encoding a functional RNA or a portion thereof.

19. The method of claim 18, wherein the protein of interest comprises a therapeutic protein, an immunogenic protein, a reporter protein, a nuclease or a therapeutic target protein, and / or the functional RNA comprises an antisense oligonucleotide, a ribozyme, an RNA that affects spliceosome-mediated / primary splicing, an interfering RNA or other non-translated functional RNA.

20. The use according to claim 19, wherein the other non-translated functional RNAs include guide RNAs and single guide RNAs.

21. The use according to claim 19, wherein: The therapeutic protein is selected from the group consisting of: SMN1, NAGLU, SGSH, IDS, FVIII, FIX, BTK, ABCD1, ACADVL, AR, HBB, SCN1A, CFTR, CSF2RA, IL2AG, PHA, STK11, PIGA, OTC, NAGS, DMPK, CNBP, ACADM, GNAS, FBN1, LIPA, SLC7A7, HADHA, GHR, IDV, ALPL, SLC25A15, HTT, HCS, NOTCH3, ALDOB, ATP7B, GAA, GCDH, SLC12A3, GBA, MEFV, GLA, CLCN1 NR0B1, ASS1, SLC25A13, SLC22A5, SCN5A, BTD, ACAT1, ARG1, CYP21A2, chimeric antigen receptor CAR, antibodies, insulin, glucagon-like peptide-1, peptide hormones, growth factors, erythropoietin EPO, cytokines, coagulation factors, antihemophilic factor, interferon, Fc fusion proteins and therapeutic enzymes; The immunogenic protein is selected from the group consisting of: an immunogenic protein from an orthomyxovirus, a lentivirus, an arenavirus, a poxvirus, a flavivirus, a filovirus, a bunyavirus, a coronavirus, a poliovirus, a herpesvirus, a mumps virus, a measles virus, a rubella virus, a diphtheria toxin, a pertussis virus, and a hepatitis virus; The nuclease comprises zinc finger nuclease ZFN, transcription activator-like effector nuclease TALEN or Cas family protein or homolog thereof or a modified version thereof, The reporter protein is selected from the group consisting of a fluorescent protein, an enzyme that produces a detectable product, and a protein that can be directly detected; The therapeutic target proteins include CTLA-4, HER2, fibronectin-4, sclerostin, P-selectin, VEGF, RSVF, VEGFR2, CD79, IL23p19, vWF, IFN-γ, C5, PD-1, PD-L1, CGRP, CD3, CD11a, CD20, CD22, CD30, CD33, CD38, CD40, CD52, IgE, KLK, CCR4, FGF-23, IL-6R, IL- 5, IL-23p19, IL-2R, IL-17R, IL-17, CD4, FIX / FX, IL-12, IL-23, IL-1β, IL-5R, IL-4 / IL-13, PDGF-α, dabigatran, SLAMF7, EGFR, PCSK9, GD2, CD19, α4β7 integrin, α4β1 integrin, PA, BLyS, RANK, TNF-α, EpCAM, GGTA1, endostatin, angiostatin; or The functional RNA regulates a biological target selected from the group consisting of a multiple drug resistance (MDR) protein target, a tumor target, a pathogen target, a defective gene product, or a therapeutic target.

22. The use according to claim 21, wherein the antibody comprises a monoclonal antibody, a bispecific antibody or a multispecific antibody.

23. The use according to claim 21, wherein the Fc fusion protein comprises CTLA-4Fc fusion protein or VEGFRFc fusion protein.

24. The use of claim 21, wherein the therapeutic enzymes comprise lysosomal hydrolases and sulfatases.

25. The use according to claim 21, wherein the orthomyxovirus comprises an influenza virus.

26. The use according to claim 21, wherein the lentivirus comprises HIV or SIV.

27. The use of claim 21, wherein the arenavirus comprises Lassa fever virus.

28. The use according to claim 21, wherein the poxvirus comprises vaccinia.

29. The use of claim 21, wherein the flavivirus comprises yellow fever virus.

30. The use of claim 21, wherein the filovirus comprises an Ebola virus.

31. The use according to claim 21, wherein the bunyavirus comprises RVFV, CCHF or SFS virus.

32. The method of claim 21, wherein the coronavirus comprises SARS, MERS, or COVID-19.

33. The method of claim 21, wherein the herpes virus comprises CMV, EBV, or HSV.

34. The use according to claim 21, wherein the hepatitis virus comprises HAV, HBV or HCV.

35. The method of claim 21, wherein the Cas family protein comprises Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas 10, Cas 11, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1.

36. The use according to claim 21, wherein the fluorescent protein comprises EGFP, GFP, RFP, BFP, YFP or dsRED2.

37. The method of claim 21, wherein the enzyme producing a detectable product comprises luciferase, b-galactosidase, b-glucuronidase, alkaline phosphatase, or chloramphenicol acetyltransferase genes.

38. The use according to claim 21, wherein the tumor targets include VEGF, Her2, EGFR, and PD-L1.

39. The use according to claim 21, wherein the pathogen target comprises a viral surface antigen.

40. The use according to claim 21, wherein the pathogen target comprises the hepatitis B surface antigen gene.

41. The use according to claim 21, wherein the defective gene product comprises a mutant dystrophin protein.

42. The use according to claim 21, wherein the therapeutic target comprises myostatin.

43. The use according to claim 21, wherein the therapeutic protein is iduronate 2-sulfatase IDS. The use according to claim 43 , wherein the iduronate 2-sulfatase IDS comprises the sequence shown in SEQ ID NO:

11.

45. The use of claim 18, wherein the coding sequence is operably linked to one or more regulatory sequences.

46. Use of an aspirin compound in the preparation of a medicament for preparative treatment of a subject suffering from a condition treatable by an exogenous nucleic acid or an expression product thereof, the preparative treatment comprising: administering to the subject an effective amount of an aspirin compound prior to or concurrently with delivering the exogenous nucleic acid to the cell, wherein the exogenous nucleic acid comprises double-stranded DNA, or is capable of being converted to double-stranded DNA in the cell following delivery; The aspirin compound is used to increase the expression level of exogenous nucleic acid in cells.

47. Use of an aspirin compound and an exogenous nucleic acid in the preparation of a medicament for treating or preventing a condition that can be treated or prevented by an exogenous nucleic acid or its expression product, the treatment or prevention comprising: delivering a therapeutically effective amount of the exogenous nucleic acid to a subject, wherein the exogenous nucleic acid comprises double-stranded DNA, or is capable of being converted to double-stranded DNA in the subject after delivery, and wherein the subject has been or is concurrently administered an aspirin compound; The aspirin compound is used to increase the expression level of exogenous nucleic acid in cells.

48. Use of an aspirin compound and an exogenous nucleic acid in the preparation of a medicament for treating or preventing a condition that can be treated or prevented by an exogenous nucleic acid or its expression product, the treatment or prevention comprising: a) administering an effective amount of an aspirin compound to a subject; as well as b) delivering a therapeutically effective amount of the exogenous nucleic acid to the subject, wherein said step a) is performed before or simultaneously with said step b), wherein the exogenous nucleic acid comprises double-stranded DNA, or is capable of being converted to double-stranded DNA in the subject following delivery; The aspirin compound is used to increase the expression level of exogenous nucleic acid in cells.

49. The use according to any one of claims 46 to 48, wherein the exogenous nucleic acid comprises or is included in a viral vector, a plasmid or an exosome.

50. The use according to claim 49, wherein the viral vector comprises an adeno-associated virus (AAV) vector.

51. The use according to claim 50, wherein the adeno-associated virus AAV vector is an AAV9, AAV8, and / or AAV843 vector.

52. The use according to claim 50, wherein the AAV vector comprises an AAV viral particle.

53. The method of any one of claims 46 to 48, wherein the exogenous nucleic acid comprises a coding sequence encoding a protein of interest or a portion thereof, or encoding a functional RNA or a portion thereof.

54. The use according to claim 53, wherein the protein of interest comprises a therapeutic protein, and the therapeutic protein is iduronate 2-sulfatase IDS.

55. The use according to claim 54, wherein the iduronate 2-sulfatase IDS comprises the sequence shown in SEQ ID NO:

11.

56. The use according to any one of claims 54-55, wherein the sequence encoding the protein of interest is operably linked to one or more regulatory sequences.

57. The use according to any one of claims 46-48, wherein the condition is characterized by the absence of one or more functional genes or functional proteins.

58. The use according to claim 57, wherein the condition is a monogenic disorder.

59. The use of claim 58, wherein the monogenic disorder is autosomal dominant, autosomal recessive, X-linked, Y-linked or mitochondrial.

60. The use according to any one of claims 46-48, wherein the condition is a central nervous system (CNS) disorder.

61. The use according to claim 60, wherein the CNS disorder is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, Batten disease, spinocerebellar ataxia, spinal muscular atrophy, Canavan disease and Friedreich's ataxia.

62. The use according to claim 61, wherein the CNS disorder is mucopolysaccharidosis type II.

63. The use according to any one of claims 46-48, wherein the exogenous nucleic acid is administered by a systemic route.

64. The use of claim 60, wherein the exogenous nucleic acid comprises a sequence encoding a protein of interest or a portion thereof, wherein the protein of interest is selected from the group consisting of: Tau, MeCP2, NGF, APOE, GDNF, SUMF, SGSH, AADC, CD, p53, ARSA arylsulfatase A, ABCD1, SMN1, NAGLU, SOD1, C9ORF72, TARDBP, FUS, HTT, LRRK2, PARIS, PARKIN, GAD, and α-synuclein.

65. The use according to any one of claims 46-48, wherein the exogenous nucleic acid comprises an AAV vector of the AAV9 serotype.

66. The use according to claim 65, wherein the AAV vector of the AAV9 serotype comprises an AAV virus particle of the AAV9 serotype.

67. The use according to any one of claims 46-48, wherein the therapeutically effective amount of the exogenous nucleic acid is 10 6 vg / kg to 10 14 vg / kg range.

68. The use according to any one of claims 46-48, wherein the therapeutically effective amount of the exogenous nucleic acid is no more than 10 14 vg / kg.

69. The method of claim 68, wherein the therapeutically effective amount is no more than 10 13 vg / kg, 10 12.5 vg / kg, 10 12 vg / kg, 10 11 vg / kg or even lower.

70. The use according to any one of claims 46-48, wherein the therapeutically effective amount of the exogenous nucleic acid is a subtherapeutic amount.

71. The use of claim 70, wherein the subtherapeutic amount is no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, no more than 2% or no more than 1% of the conventional amount of the same exogenous nucleic acid, which conventional amount would be required without administration of the aspirin compound.

72. The use according to claim 71, wherein the subtherapeutic amount is no more than 10 7 vg / kg, not more than 10 8 vg / kg, not more than 10 9 vg / kg, not more than 10 10 vg / kg, not more than 10 11 vg / kg, not more than 10 12 vg / kg, not more than 10 13 vg / kg or not more than 10 14 vg / kg.

73. The use of any one of claims 46-48, wherein the aspirin compound is administered to the subject at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to delivering the exogenous nucleic acid to the subject, and / or is administered once or repeatedly prior to delivering the nucleic acid.

74. The use of claim 73, wherein repeated administration comprises administering twice, administering three times, or administering four times.

75. The use of any one of claims 46-48, wherein the aspirin compound and / or the exogenous nucleic acid is administered to the subject by a parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, cardiac, subcutaneous, intraparenchymal, intracerebroventricular, or intrathecal route of administration.

76. The use of any one of claims 46-48, wherein the aspirin compound is administered to the subject in an amount of no more than 30 mg / kg, no more than 50 mg / kg, no more than 100 mg / kg, no more than 110 mg / kg, no more than 120 mg / kg, no more than 130 mg / kg, no more than 140 mg / kg, no more than 150 mg / kg, no more than 160 mg / kg, no more than 170 mg / kg, no more than 180 mg / kg, no more than 190 mg / kg, or no more than 200 mg / kg.

77. The use of claim 76, wherein the aspirin compound is administered to the subject in an amount of 30 mg / kg to 120 mg / kg.

78. The use according to claim 77, wherein the aspirin compound is administered to the subject in an amount of 50 mg / kg or 100 mg / kg.

79. The use according to any one of claims 46-48, wherein the subject is repeatedly administered an aspirin compound seven times, once daily, prior to delivering the exogenous nucleic acid.

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