Antiviral nucleic acids and compositions

Abstract

The present invention relates generally to compositions and methods for inhibiting coronavirus replication and treating diseases caused by coronavirus infection. More specifically, the present invention relates to nucleic acids capable of inhibiting coronavirus (e.g., SARS-CoV-2) replication and the use of such nucleic acids in treating patients infected by this virus.

Description

[Technical Field]

[0001] The present invention relates generally to compositions and methods for inhibiting coronavirus replication and treating diseases caused by coronavirus infection. More specifically, the present invention relates to nucleic acids capable of inhibiting coronavirus (e.g., SARS-CoV-2) replication and the use of such nucleic acids in treating patients infected by this virus. [Background technology]

[0002] SARS-CoV-2 is a positive-sense, single-stranded RNA virus that is infectious in humans and other animals. It is the causative agent of coronavirus disease 2019 (COVID-19), which has had a significant impact on people worldwide. As of September 2022, conservative estimates suggest that the number of SARS-CoV-2 human infections since the start of the current pandemic has exceeded 610 million, and more than 6.5 million people have died from COVID-19 disease (see the WHO Coronavirus (COVID-19) Dashboard).

[0003] In humans, SARS-CoV-2 infection results in a range of outcomes, with many people experiencing very mild or barely perceptible symptoms and others presenting with moderate to severe disease, with an estimated case fatality rate of approximately 1% worldwide. While a significant amount of funding and effort has been devoted to protective measures through vaccine development, it is well recognized that therapeutic measures are crucial to reducing morbidity and mortality. Antiviral agents suitable for administration to patients, particularly during the early stages of disease, offer the opportunity to reduce viral replication and thereby inhibit the onset of severe disease, particularly in people in high-risk categories (e.g., immunocompromised individuals, the elderly, etc.).

[0004] There is a need for effective therapeutic treatments to combat COVID-19 disease, including treatments that can inhibit viral replication during the early stages of COVID-19 disease. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention addresses a need in the field of therapeutic treatments for COVID-19 disease by providing nucleic acids capable of inhibiting SARS-CoV-2 expression. [Means for solving the problem]

[0006] Various embodiments of the present invention relate to nucleic acids capable of inhibiting the replication of coronaviruses, such as SARS-CoV-2. These nucleic acids, and compositions containing such nucleic acids, may be used to treat diseases resulting from coronavirus infection, including COVID-19.

[0007] In some embodiments, nucleic acids are provided that comprise 15 to 30 nucleotides and are capable of specifically hybridizing to a SARS-CoV-2 sequence as defined in SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or to a fragment of the above SARS-CoV-2 sequence. The nucleic acids may comprise or consist of: 15 to 25 nucleotides; 15 to 23 nucleotides; 15 to 22 nucleotides; 15 to 21 nucleotides; 15 to 20 nucleotides; 15 to 19 nucleotides; 19 nucleotides; 20 nucleotides; 21 nucleotides, 22 nucleotides, or 23 nucleotides.

[0008] In other embodiments: a sequence as defined in SEQ ID NO: 2 or 24, wherein the nucleic acid is capable of specifically hybridizing to the SARS-CoV-2 sequence as defined in SEQ ID NO: 23, or a sequence as defined in SEQ ID NO: 4 or 26, wherein the nucleic acid is capable of specifically hybridizing to the SARS-CoV-2 sequence as defined in SEQ ID NO: 25, or A sequence as defined in SEQ ID NO: 6 or 28, wherein the nucleic acid is capable of specifically hybridizing to the SARS-CoV-2 sequence as defined in SEQ ID NO: 27. Nucleic acids having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the

[0009] In further embodiments, there are provided fragments of the nucleic acids of the invention that are 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length.

[0010] In yet another embodiment, there is provided a double-stranded nucleic acid for inhibiting expression of SARS-CoV-2, comprising a sense strand and an antisense strand: the sense strand comprises a sequence as defined in SEQ ID NO: 1 or 23, or a variant or fragment thereof, and the antisense strand comprises a sequence as defined in SEQ ID NO: 2 or 24, or a variant or fragment thereof; or the sense strand comprises a sequence as defined in SEQ ID NO: 3 or 25, or a variant or fragment thereof, and the antisense strand comprises a sequence as defined in SEQ ID NO: 4 or 26, or a variant or fragment thereof; or The sense strand comprises a sequence as defined in SEQ ID NO: 5 or 27, or a variant or fragment thereof, and the antisense strand comprises a sequence as defined in SEQ ID NO: 6 or 28, or a variant or fragment thereof. A double-stranded nucleic acid is provided.

[0011] In further embodiments, nucleic acids of the invention may comprise: 2'-deoxy-2'-fluoro modified nucleotides; 2'-deoxy modified nucleotides; locked nucleic acids; abasic nucleotides; 2'-amino modified nucleotides; 2'-alkyl modified nucleotides; morpholino nucleotides; nucleotides containing unnatural bases; 2'-O-methyl modified nucleotides; 2'O-methoxyethoxy modified nucleotides; 2'fluoro modified nucleotides; 5-methyl modified cytidines; pseudouridines; nucleotides containing 5'-phosphorothioate groups and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group; nucleotides containing phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methyl phosphoramidite; or nucleotides containing deoxyribose.

[0012] In other embodiments, the nucleic acids of the invention may be conjugated to a ligand.

[0013] In other embodiments, the nucleic acids of the invention may be RNA, antisense RNA, or siRNA.

[0014] Other aspects of the invention provide cells comprising the nucleic acids described herein, vectors comprising nucleic acid sequences encoding the RNA, antisense RNA, or siRNA described herein, lipid nanoparticles comprising the nucleic acids and / or vectors described herein, and pharmaceutical compositions comprising the nucleic acids, vectors, and / or lipid nanoparticles described herein.

[0015] In some embodiments, the lipid nanoparticles of the present invention may comprise any one or more of: a non-cationic liquid, a cationic lipid, a conjugated lipid to prevent aggregation of the nanoparticles.

[0016] In other embodiments, the pharmaceutical composition may be a liquid for intravenous administration or an aerosol for intranasal administration.

[0017] Further aspects of the invention provide methods for inhibiting coronavirus replication in a cell, methods for treating a coronavirus infection in a subject, and methods for treating COVID-19 disease in a subject.

[0018] In some embodiments, provided are methods for inhibiting coronavirus replication in a cell, comprising administering to the cell a nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition described herein, thereby causing degradation of coronavirus mRNA molecules in the cell and inhibiting said replication of coronavirus.

[0019] In other embodiments, provided are methods for treating a coronavirus infection in a subject, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition described herein, thereby inhibiting coronavirus replication and treating the infection.

[0020] In a further embodiment, there is provided a method for treating COVID-19 disease in a subject, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, lipid nanoparticle or pharmaceutical composition described herein, thereby inhibiting coronavirus replication and treating said COVID-19 disease.

[0021] In some embodiments, the coronavirus is SARS-CoV-2.

[0022] Yet another aspect of the present invention provides methods for preparing medicaments relating to the various nucleic acids, vectors, lipid nanoparticles and pharmaceutical compositions described herein, and medical uses thereof.

[0023] In some embodiments, there is provided a use of a nucleic acid, vector, lipid nanoparticle or pharmaceutical composition described herein in the preparation of a medicament for inhibiting coronavirus replication in a cell, treating a coronavirus infection in a subject, or treating COVID-19 disease in a subject.

[0024] In other embodiments, there is provided a nucleic acid, vector, lipid nanoparticle or pharmaceutical composition described herein for use in inhibiting coronavirus replication in a cell, treating a coronavirus infection in a subject, or treating COVID-19 disease in a subject.

[0025] In some embodiments, the coronavirus is SARS-CoV-2.

[0026] definition As used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" also includes plural cells unless otherwise stated.

[0027] As used herein, the term "comprising" means "including" in a non-inclusive sense. Variations of the word "comprise," such as "comprise" and "comprises," have altered meanings, as appropriate. Thus, for example, a composition "comprising" a given component A may consist solely of component A, or may include one or more additional components, such as component B. Similarly, a pharmaceutical composition "comprising" a given nucleic acid may include one or more additional components, such as pharmaceutically acceptable excipients, diluents, and / or carriers.

[0028] As used herein, the term "about," when used in reference to a recited value, includes the recited value and values ​​within plus or minus ten percent of the recited value.

[0029] As used herein, the term "SARS-CoV-2" refers to severe acute respiratory syndrome-associated coronavirus 2.

[0030] As used herein, the term "nucleic acid" refers to nucleotides and nucleotide polymers. Contemplated nucleotides include ribonucleotides, deoxyribonucleotides, modified forms thereof, and combinations thereof. Nucleotide polymers may be single-stranded or double-stranded, including single-stranded and double-stranded RNA, single-stranded and double-stranded DNA, and double-stranded RNA / DNA hybrids. Non-limiting examples of nucleic acids include all types of RNA, such as siRNA, mRNA, miRNA, shRNA, antisense RNA, guide RNA, and dicer substrate RNA and siRNA. All of these types of RNA can functionally target and inhibit viruses. Other non-limiting examples of "nucleic acid" include all types of DNA, such as genomic DNA, complementary DNA (cDNA), minicircle DNA, and plasmid DNA. Also included within the scope of the term "nucleic acid" are those containing nucleotide analogs, modified backbone linkages or residues, etc., those that have a physical structure related to DNA or RNA molecules or residues, and those that can form hydrogen bonds with DNA or RNA residues or their analogs (i.e., can hybridize and form base pairs with DNA or RNA residues or their analogs) and therefore have similar binding properties to the structurally related ribonucleotide or deoxyribonucleotide residues (i.e., base nucleotides) and can be metabolized in a manner similar to base nucleotides. Non-limiting examples of nucleic acid analogs include methylated, iodinated, brominated, or biotinylated residues, and those with modified sugar moieties or phosphodiester backbones. Contemplated nucleic acid analogs also include, but are not limited to, those containing modifications to nucleotide bases, such as pseudouridine, 2'-fluoro, 2'-O-methyl, 5-methylcytidine, 2'-O-methoxyethoxy, and those containing peptide nucleic acid backbones and linkages. Other analog nucleic acids include those containing modified sugars (e.g., deoxyribose), non-ionic backbones, positive backbones, and non-ribose backbones (e.g., locked nucleic acids or phosphorodiamidate morpholino oligos).

[0031] As used herein, the term "RNA interference" refers to a mechanism of action imbued in mammalian cells that can specifically silence the production of proteins in cells in a sequence-specific and potent manner. In this process, RNA molecules are involved in the sequence-specific suppression of gene expression by double-stranded RNA through translational or transcriptional repression. For example, RNA interference can be initiated through the introduction of small interfering RNA (siRNA, a 15-30 bp double-stranded RNA) that specifically targets mRNAs through sequence complementarity and causes their subsequent degradation. Additionally, small hairpin RNA (shRNA) can also suppress transcripts and gene expression.

[0032] As used herein, an "antisense" nucleic acid or sequence is one whose complementarity to at least a component of a specific target nucleic acid promotes hybridization with the target (e.g., under physiological conditions), thereby inhibiting a biological activity associated with the target, non-limiting examples of which include inhibition of translation of the target nucleic acid, alteration of transcription splicing, etc. Antisense nucleic acids specifically hybridize to their nucleic acid target, meaning that they have a greater tendency to hybridize to their nucleic acid target, e.g., under physiological conditions, compared to other non-target nucleic acids. Antisense nucleic acids can include those containing nucleotide analogs, modified backbone linkages or residues, etc.

[0033] As used herein, the terms "siRNA" and "small interfering RNA" refer to single- or double-stranded RNA (ribonucleic acid) capable of hybridizing to a target nucleic acid to inhibit the expression of a given target nucleic acid present in a cell containing the target nucleic acid, thereby inhibiting its normal biological activity (e.g., its translation into protein). The target nucleic acid may be single- or double-stranded RNA or single- or double-stranded DNA, non-limiting examples of which include messenger RNA (mRNA) and promoter sequences. Single- or double-stranded siRNAs may typically be 15 to 50 nucleotides in length. siRNAs are capable of functionally targeting and inhibiting viruses.

[0034] As used herein, the terms "shRNA" and "small hairpin RNA" refer to artificial RNA molecules that contain a tight hairpin turn that can be used to silence target gene expression through RNA interference (RNAi). Similar to endogenous microRNAs, shRNAs contain paired antisense and sense stem regions linked to unpaired nucleotides that form a loop. Those skilled in the art will appreciate that once delivered to a cell, shRNAs: RNA-induced silencing complex It will be recognized that the passenger sense strand can be loaded into RISC, which degrades the passenger sense strand. The antisense (guide) strand guides RISC to degrade mRNA with a complementary sequence. Thus, shRNAs can functionally target and inhibit viruses and are functionally equivalent to siRNAs.

[0035] As used herein, the term "loop" or "loop region" refers to a sequence that connects two complementary strands of nucleic acid. In certain embodiments, the loop region is 4 to 20 nucleotides in length, e.g., 15 to 19 nucleotides in length. 0 to 50% of the loop region may be complementary to another region of the loop region. The nucleotide sequence of the loop region may be, for example, (5'-GCAA-3'), (5'-GCGC-3'), or (5'-TTGC-3'), or other sequences as will be appreciated by those skilled in the art. As used herein, the terms "hybridize" and "hybridization" refer to the binding of nucleic acid strands through complementary base pairing, including binding scenarios based on partial or complete complementarity. As known to those of ordinary skill in the art, the degree of hybridization between two nucleic acid strands can be affected by parameters such as temperature, salt concentration, etc., and conditions under which two complementary or partially complementary nucleic acid strands hybridize in a specific manner and avoid hybridization with other potential binding partners can be readily determined using standard optimization.

[0036] As used herein, the term "variant" refers to a substantially similar nucleic acid or polypeptide sequence. Generally, sequence variants possess a common qualitative biological activity. Furthermore, such sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length of the reference sequence or over a specified region of the reference sequence. Also included within the meaning of the term "variant" are homologs, which are polypeptides or nucleic acids that are typically derived from different species but that share substantially the same biological function or activity as the corresponding polypeptide or nucleic acid disclosed herein.

[0037] As used herein, percent "sequence identity" will be understood to result from a comparison of two sequences when they are aligned to maximize correlation between the sequences. This may include inserting "gaps" in either one or both sequences to enhance the degree of alignment. The percent sequence identity may then be determined across the length of each of the sequences or portions thereof being compared. For example, a nucleotide sequence ("subject sequence") that has at least 95% "sequence identity" with another nucleotide sequence ("query sequence") means that the subject sequence is identical to the query sequence, except that the subject sequence may contain up to five nucleotide alterations per 100 nucleotides of the query sequence. In other words, to obtain a nucleotide sequence that is at least 95% sequence identical to the query sequence, up to 5% (i.e., 5 out of 100) of the nucleotides in the subject sequence may be inserted or substituted with other nucleotides or deleted. The percent sequence identity between two sequences may be determined by comparing two optimally aligned sequences across a comparison window. To optimally align two sequences, the portion of the sequence in the comparison window may contain deletions or additions (i.e., gaps), for example, compared to a reference sequence (e.g., obtained from another species) that does not contain deletions or additions, or vice versa. The percent sequence identity may then be calculated by determining the number of positions where identical nucleotides exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. In the context of two or more nucleic acid sequences, percent sequence identity refers to a specified percentage of nucleotides that are the same across a specified region (or across the entire sequence, if not specified) when compared and aligned for maximum correspondence across a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared.When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are known in the art. Optimal alignment of sequences for determining sequence identity can be suitably achieved using known computer programs, including, but not limited to: CLUSTAL in the PC / gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (version 2.0) in the GCG Wisconsin Genetics Software Package, version 10; and GAP, BESTFIT, BLAST, FASTA, and TFASTA (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignment using these programs can be performed using default parameters. Another method for determining the best overall match between a query sequence and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)).

[0038] As used herein, the term "fragment" in reference to a nucleic acid refers to a component of that nucleic acid. Typically, a fragment possesses a qualitative biological activity in common with the nucleic acid, including, for example, hybridization with another target nucleic acid, capable of reducing, inhibiting, etc., expression of the target nucleic acid. Fragments may be derived from the nucleic acids of the invention or may be synthesized by some other means, for example, chemical synthesis.

[0039] As used herein, the term "isolated" in the context of a nucleic acid or other biological entity will be understood to mean that the isolated nucleic acid or other biological entity is at least partially free, and in some cases free or substantially free, from nucleic acids, proteins, lipids, carbohydrates, or other materials that normally accompany it as found in the natural / natural state. An "isolated" nucleic acid or other biological entity may be purified to facilitate partial or complete separation from other biological entities that normally accompany it as found in the natural / natural state.

[0040] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or alleviating a disorder / disease and / or its associated symptoms. Although not excluded, it will be recognized that treating a disorder or condition does not necessarily require that the disorder, condition, or its associated symptoms be completely eliminated. However, it is intended that the disorder, condition, or its associated symptoms improve when compared to before treatment begins.

[0041] As used herein, the term "subject" includes any animal of economic, social, or research importance, including bovine, equine, ovine, primate, avian, and rodent species. Thus, a "subject" may be a mammal, for example, a human or non-human mammal.

[0042] As used herein, a "therapeutically effective amount" of a given composition or the like upon administration to a subject is understood to be an amount of therapeutic agent sufficient to partially or completely alleviate the condition or disease being treated, including a reduction in the symptom(s) of the condition or disease, e.g., by reducing the severity or frequency of the symptom(s), or by eliminating the symptom(s). For example, with respect to a given symptom, a therapeutically effective amount may result in at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or 100% reduction in the symptom. The actual "therapeutically effective amount" will depend on the particular composition administered, the condition or disease being treated, the age and health of the subject being treated, etc. Evaluation of such parameters is routine and can be readily determined by one of ordinary skill in the art. [Brief explanation of the drawings]

[0043] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] Figure 1 shows the results of an interferon screen of novel candidate siRNAs targeting SARS-COV-2. Error bars represent SEM of triplicate treatments. [Figure 2] We provide the results of a plaque assay screen and evaluation of novel candidate siRNAs for in vitro inhibition of SARS-CoV-2. Data are presented with standard errors of the mean for triplicate treatments; *p<0.05, ***p<0.001, and ****p<0.001 were considered statistically significant when compared against N3675 (control), as determined by one-way ANOVA analysis (Dunnett's post-hoc test). [Figure 3] 1 shows the results of a qRTPCR screen of candidate novel siRNAs. [Figure 4]

[0013] Figure 1 provides the results of a plaque assay screen and evaluation of novel candidate siRNAs for inhibition of SARS-CoV-2 in vitro. Data shown are % plaque inhibition of mean plaque counts for each treatment compared to virus alone. [Figure 5] We provide the results of another plaque assay screen and evaluation of selected candidate siRNAs for inhibition of alpha, beta, kappa, delta, or omicron mutant SARS-CoV-2 VOCs in vitro. Data were collected from triplicate treatments and are shown with the standard error of the mean for triplicate treatments; *p<0.05, ***p<0.001, and ****p<0.001 were considered statistically significant as determined by one-way ANOVA analysis (Dunnett's post-hoc test) when compared against N3675 (control). [Figure 6] Figure 1 shows the results of an additional plaque assay screen and evaluation of novel candidate minus-strand targeting siRNAs for inhibition of SARS-CoV-2 delta mutants in vitro. Data were collected from triplicate treatments and are shown with the standard error of the mean for triplicate treatments; *p<0.05, ***p<0.001, and ****p<0.001 were considered statistically significant as determined by one-way ANOVA analysis (Dunnett's post-hoc test) when compared against N3675 (control). [Figure 7] We provide the results of an additional plaque assay screen and evaluation of novel candidate minus- and plus-strand-targeting siRNA combinations for in vitro inhibition of SARS-CoV-2 Wuhan and Delta mutants. Data were collected from triplicate treatments and are shown with the standard error of the mean for triplicate treatments; *p<0.05, ***p<0.001, and ****p<0.001 were considered statistically significant when compared to N3675 (control), as determined by one-way ANOVA analysis (Dunnett's post-hoc test). Percent viral plaque inhibition by siRNA is also shown. [Figure 8] 1 provides the results of a dose-response evaluation of the top three candidate siRNAs for the inhibition of delta mutants in vitro. Data are collected from triplicate treatments and are shown with the standard error of the mean of the triplicate treatments reflected on each bar graph. [Figure 9]A) Timeline of treatment after viral infection from day -1 to day 3. A daily dose of 1 mg / kg siRNA was administered daily from day 0, and all mice were sacrificed on day 3. B) In vivo evaluation of HelUP2 siRNA delivered by stealth LNPs. Lung virus tissue counts / g at 3 dpi are shown. Each dot represents data from one mouse. **p<0.005, one-way ANOVA. [Figure 10] Results are shown for an experiment in which Vero E6 cells were transfected with siControl and siCoV_1 (30 nM) complexed with Lipofectamine 2000 for 24 hours before infection with delta SARS-CoV-2 Virus Clones (VOC) at 250 plaque-forming units (PFU). Infectious viral plaques were counted 4 days post-infection (dpi). Data are expressed as the mean percent plaque inhibition compared to virus alone (control) and are representative of the standard error of the mean (SEM) for triplicate treatments. One-way ANOVA (Dunnett's post-hoc test) was performed and compared against siControl. [Figure 11] Results are shown for an experiment in which Vero E6 cells were transfected with increasing concentrations (0.1–30 nM) of siControl and siCoV_1 complexed with Lipofectamine 2000 for 24 h before infection with delta SARS-CoV-2 VOCV at 250 PFU. Infectious viral plaques were counted 4 dpi. Data are presented as the mean plaque count and standard error of the mean (SEM) for triplicate treatments. One-way ANOVA (Dunnett's post-hoc test) was performed and compared against siControl. [Figure 12]A) Schematic summary of the in vivo study approach and siRNA treatment regimen, and B) K18h-ACE2 mice infected with 1 x 10 live delta SARS-CoV-2 VOCV and received either daily intravenous (IV) (retroorbital, sLNP) or intranasal (IN, dmLNP) treatment with siRNA-LNP (1 mg / kg) daily (-1 to 2 dpi). The control siRNA used here is siN367 (siControl). Virus counts / g of tissue at 3 dpi are shown. Each dot represents data from one mouse, and bars represent the mean. One-way ANOVA (Dunnett's post-hoc test) was performed against siControl. Mice were weighed daily, and data points represent the mean percent weight gain. Error bars represent the SEM. Two-way ANOVA against siControl was performed at each time point. [Figure 13] Schematic diagram showing the dual shRNA expression cassette used to express SARS-CoV-2 shRNAs packaged into EVs. Both the H1 and U6 promoters are shown here, but either the U6 or H1 promoter and shRNA can be expressed alone, or in combination, as in this case. DETAILED DESCRIPTION OF THE INVENTION

[0044] The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable those of ordinary skill in the art to practice the present invention. Features or limitations of the various described embodiments do not necessarily limit other embodiments of the present invention or the present invention as a whole. Accordingly, the following detailed description does not limit the scope of the present invention, which is defined only by the claims.

[0045] It will be appreciated by those of ordinary skill in the art that many variations and / or modifications may be made to the invention as disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0046] inhibitory nucleic acid The present invention provides nucleic acids that are designed to inhibit the expression of a target gene and / or inhibit the biological activity of an untranslated target sequence (eg, a regulatory sequence).

[0047] Without limitation, the genes and regulatory sequences targeted by the inhibitory nucleic acids described herein can be derived from pathogens that cause a disease or condition upon infection of a host organism. For example, the pathogen can be a virus capable of infecting mammalian subjects, including humans.

[0048] The virus may be an RNA virus, such as a coronavirus. Non-limiting examples include alphacoronaviruses, including the human coronaviruses HCoV-229E and HCoV-NL63, and betacoronaviruses, including the human coronaviruses SARS-CoV-1 (the cause of severe acute respiratory syndrome), SARS-CoV-2 (the cause of coronavirus disease 2019 / COVID-19), MERS-CoV (the cause of Middle East respiratory syndrome), HCoV-HKU1, and HCoV-OC43.

[0049] The inhibitory nucleic acids described herein can inhibit the expression of a viral protein(s), including coronavirus proteins. As a non-limiting example, the inhibitory nucleic acid can inhibit the expression of a structural coronavirus protein (e.g., spike, membrane, envelope, or nucleocapsid protein). The protein, such as a helicase protein, can be essential for viral replication. Additionally or alternatively, the inhibitory nucleic acid can inhibit the expression of a non-structural coronavirus protein. Additionally or alternatively, the inhibitory nucleic acid can inhibit the expression of ORF1ab and / or the polymerase gene.

[0050] The inhibitory nucleic acids described herein can inhibit the biological activity of an untranslated segment of a viral genome, for example, they can inhibit the biological activity of a stem-loop structure in the 5' or 3' untranslated region (i.e., 5'UTR or 3'UTR) of a coronavirus (e.g., stem-loop 5 (SL5) of the coronavirus 5'UTR).

[0051] The inhibitory nucleic acids described herein can be single-stranded (including but not limited to hairpin structures) or double-stranded.

[0052] The inhibitory nucleic acids described herein may have a length of between: 15-30 nucleotides, 15-25 nucleotides, 15-20 nucleotides, 18-25 nucleotides, or 25-30 nucleotides, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0053] The double-stranded inhibitory nucleic acid may be provided in a form in which each strand of the duplex is the same length or different lengths. The double-stranded inhibitory nucleic acid may therefore include one or more single-stranded overhangs of at least 1, 2, 2, or 4 nucleotides, 1 to 2 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 2 to 5 nucleotides, 1 to 10 nucleotides, 2 to 10 nucleotides, or 5 to 10 nucleotides. The overhang(s) may be provided at the 5' and / or 3' end of the sense strand, the 5' and / or 3' end of the antisense strand, the 5' end of the sense strand and the 5' end of the antisense strand, or the 3' end of the sense strand and the 3' end of the antisense strand.

[0054] The inhibitory nucleic acids (e.g., variants of inhibitory nucleic acids) described herein may contain one or more mismatches (e.g., 1, 2, 3, 4, or 5 mismatched nucleotides, or fewer than 5, 4, or 3 mismatched nucleotides) to a target sequence. The mismatches may be located in the 5' and / or 3' to central portions of the nucleic acid, including within 6, 5, 4, 3, or 2 nucleotides of its 3' and / or 5' end. For example, for a nucleic acid sequence defined by any of SEQ ID NOS: 1-20 or 23-42, a mismatch with the respective target sequence may occur within the central 9, 10, 11, 14, 14, 15, 16, 17, or 18 nucleotides of SEQ ID NOS: 1-20 or 23-42. Based on the methods described herein and general knowledge common to the art, one of ordinary skill in the art can readily determine whether a given variant of an inhibitory nucleic acid described herein containing mismatched nucleotide(s) to its target sequence is still effective in inhibiting expression of a target gene and / or biological activity of an untranslated target sequence (e.g., a regulatory sequence). Furthermore, those skilled in the art will recognize that there is substantially no difference in the strength of siRNAs made with different overhangs, and the composition of the overhang does not appear to play a very important role in target mRNA recognition and cleavage. siRNAs may have dTdT overhangs, but may also have UU or AA overhangs or other overhangs, such as overhangs complementary to the mRNA sequence, or no overhangs at all.

[0055] The inhibitory nucleic acids described herein may contain one or more modifications, such as modified backbones, substituted internucleoside linkages, substituted sugar moieties, nucleobases, etc. Typically, but not necessarily, modifications may improve one or more performance parameters of the nucleic acid (e.g., stability, strength of hybridization to target sequences, provide simpler / more cost-effective manufacture). One of ordinary skill in the art is familiar with the wide variety of nucleic acid modifications available, their advantages, and how to introduce them into a reference sequence.

[0056] The inhibitory nucleic acids described herein can be produced using standard methods known in the art, including, for example, those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, the entire contents of which are incorporated herein by cross-reference. Suitable, non-limiting chemical synthesis methods for producing unmodified nucleic acids include those that utilize common nucleic acid groups such as phosphoramidites at the 3' end and dimethoxytrityl at the 5' end (e.g., as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433). For the specific example of inhibitory nucleic acids (e.g., siRNA), non-limiting methods include those involving synthesis, deprotection, and analysis, as set forth in, for example, U.S. Patent Nos. 6,649,751, 6,673,918, 6,686,463, 6,989,442, and 6,995,259. Alternatively, they can be synthesized separately and then combined after synthesis, for example, by ligation (see Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204; International PCT Publication No. WO 93 / 23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Moore et al., 1992, Science 256, 9923), or by hybridization after synthesis and / or deprotection.They can also be synthesized as described in U.S. Patent Nos. 5,889,136; 6,008,400 and 6,111,086.

[0057] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 1 or 23, or a variant or fragment of the sequence set forth in SEQ ID NO: 1 or 23. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 1. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 1 or 23.

[0058] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 2 or 24, or a variant or fragment of the sequence set forth in SEQ ID NO: 2 or 24. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 2 or 24. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 2 or 24.

[0059] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 3 or 25, or a variant or fragment of the sequence set forth in SEQ ID NO: 3 or 25. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 3 or 25. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 3 or 25.

[0060] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 4 or 26, or a variant or fragment of the sequence set forth in SEQ ID NO: 4 or 26. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 4 or 26. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 4 or 26.

[0061] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 5 or 27, or a variant or fragment of the sequence set forth in SEQ ID NO: 5 or 27. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 5 or 27. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 5 or 27.

[0062] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 6 or 28, or a variant or fragment of the sequence set forth in SEQ ID NO: 6 or 28. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 6 or 28. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 6 or 28.

[0063] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 7, or a variant or fragment of the sequence set forth in SEQ ID NO: 7 or 29. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 7 or 29. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 7 or 29.

[0064] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 8 or 30, or a variant or fragment of the sequence set forth in SEQ ID NO: 8 or 30. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 8 or 30. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 8 or 30.

[0065] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 9 or 31, or a variant or fragment of the sequence set forth in SEQ ID NO: 9 or 31. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 9 or 31. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 9 or 31.

[0066] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 10 or 32, or a variant or fragment of the sequence set forth in SEQ ID NO: 10 or 32. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 10 or 32. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 10 or 32.

[0067] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 11 or 33, or a variant or fragment of the sequence set forth in SEQ ID NO: 11 or 33. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 11 or 33. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 11 or 33.

[0068] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 12 or 34, or a variant or fragment of the sequence set forth in SEQ ID NO: 12 or 34. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 12 or 34. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 12 or 34.

[0069] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 13 or 35, or a variant or fragment of the sequence set forth in SEQ ID NO: 13 or 35. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 13 or 35. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 13 or 35.

[0070] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 14 or 36, or a variant or fragment of the sequence set forth in SEQ ID NO: 14 or 36. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 14 or 36. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 14 or 36.

[0071] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 15 or 37, or a variant or fragment of the sequence set forth in SEQ ID NO: 15 or 37. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 15 or 37. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 15 or 37.

[0072] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 16 or 38, or a variant or fragment of the sequence set forth in SEQ ID NO: 16 or 38. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 16 or 38. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 16 or 38.

[0073] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 17 or 39, or a variant or fragment of the sequence set forth in SEQ ID NO: 17 or 39. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 17 or 39. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 17 or 39.

[0074] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 18 or 40, or a variant or fragment of the sequence set forth in SEQ ID NO: 18 or 40. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 18 or 40. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 18 or 40.

[0075] In certain embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 19 or 41, or a variant or fragment of the sequence set forth in SEQ ID NO: 19 or 41. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 19 or 41. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 19 or 41.

[0076] In other embodiments of the present invention, inhibitory nucleic acids are provided that comprise or consist of the sequence set forth in SEQ ID NO: 20 or 42, or a variant or fragment of the sequence set forth in SEQ ID NO: 20 or 42. Variants may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 20 or 42. Where overhangs are specified, variants may include different overhangs. Fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of the sequence set forth in SEQ ID NO: 20 or 42.

[0077] In a further aspect of the present invention, an inhibitory nucleic acid is provided that comprises or consists of a first nucleic acid strand hybridized by complementary base pairing to a second nucleic acid strand. In some embodiments, the first and second strands may be provided in a hairpin structure. In other embodiments, the first and second strands are not provided in a hairpin structure (i.e., they each terminate separately at their 5' and 3' ends). Either or both strands may have an overhang of non-hybridizing nucleotides at their 3' ends (e.g., 1, 2, 3, 4, or 5 non-hybridizing nucleotides). Alternatively, there may be no overhanging sequence on either strand. The 5' ends of either or both strands may be phosphorylated, and / or the 3' ends of either or both strands may be hydroxylated.

[0078] The first strand may comprise or consist of the sequence defined in SEQ ID NO: 1 or 23, or a variant or fragment of the sequence defined in SEQ ID NO: 1 or 23. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 1 or 23. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 1 or 23. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 2 or 24, or a variant or fragment of the sequence defined in SEQ ID NO: 2 or 24. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 2 or 24. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 2 or 24.

[0079] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 3, or a variant or fragment of the sequence defined in SEQ ID NO: 3 or 25. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 3 or 25. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 3 or 25. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 4 or 26, or a variant or fragment of the sequence defined in SEQ ID NO: 4 or 26. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 4 or 26. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 4 or 26.

[0080] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 5, or a variant or fragment of the sequence defined in SEQ ID NO: 5 or 27. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 5 or 27. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 5 or 27. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 6 or 28, or a variant or fragment of the sequence defined in SEQ ID NO: 6 or 28. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 6 or 28. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 6 or 28.

[0081] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 7 or 29, or a variant or fragment of the sequence defined in SEQ ID NO: 7 or 29. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 7 or 29. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 7 or 29. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 8 or 30, or a variant or fragment of the sequence defined in SEQ ID NO: 8 or 30. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 8 or 30. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 8 or 30.

[0082] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 9 or 31, or a variant or fragment of the sequence defined in SEQ ID NO: 9 or 31. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 9 or 31. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 9 or 31. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 10 or 32, or a variant or fragment of the sequence defined in SEQ ID NO: 10 or 32. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 10 or 32. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 10 or 32.

[0083] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 11 or 33, or a variant or fragment of the sequence defined in SEQ ID NO: 11 or 33. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 11 or 33. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 11 or 33. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 12 or 34, or a variant or fragment of the sequence defined in SEQ ID NO: 12 or 34. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 12 or 34. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence set forth in SEQ ID NO: 12 or 34.

[0084] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 13 or 35, or a variant or fragment of the sequence defined in SEQ ID NO: 13 or 35. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 13 or 35. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 13 or 35. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 14 or 36, or a variant or fragment of the sequence defined in SEQ ID NO: 14 or 36. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 14 or 36. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence set forth in SEQ ID NO: 14 or 36.

[0085] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 15 or 37, or a variant or fragment of the sequence defined in SEQ ID NO: 15 or 37. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 15 or 37. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 15 or 37. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 16 or 38, or a variant or fragment of the sequence defined in SEQ ID NO: 16 or 38. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 16 or 38. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence set forth in SEQ ID NO: 16 or 38.

[0086] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 17 or 39, or a variant or fragment of the sequence defined in SEQ ID NO: 17 or 39. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 17 or 39. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 17 or 39. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 18 or 40, or a variant or fragment of the sequence defined in SEQ ID NO: 18 or 40. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 18 or 40. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence set forth in SEQ ID NO: 18 or 40.

[0087] Alternatively, the first strand may comprise or consist of the sequence defined in SEQ ID NO: 19 or 41, or a variant or fragment of the sequence defined in SEQ ID NO: 19 or 41. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 19 or 41. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 19 or 41. The second strand may comprise or consist of the sequence defined in SEQ ID NO: 20 or 42, or a variant or fragment of the sequence defined in SEQ ID NO: 20 or 42. A variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence set forth in SEQ ID NO: 20 or 42. Where overhangs are specified, variants may include different overhangs. A fragment may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence set forth in SEQ ID NO: 20 or 42.

[0088] Pharmaceutical Compositions, Dosages and Routes of Administration The inhibitory nucleic acids of the present invention may be incorporated into pharmaceutical compositions. These can be prepared using methods known to those skilled in the art. Non-limiting examples of suitable methods are described in Gennaro et al. (Eds), (1990), "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pennsylvania, USA.

[0089] Pharmaceutical compositions may include pharmaceutically acceptable carriers, excipients, and / or diluents. As intended herein, a "pharmaceutically acceptable" carrier, excipient, and / or diluent is a substance that does not produce any adverse reaction(s) when administered to a specific recipient, such as a human or non-human animal. Pharmaceutically acceptable carriers, excipients, and diluents are also generally compatible with the other components of the composition. Non-limiting examples of suitable excipients, diluents, and carriers can be found in "Handbook of Pharmaceutical Excipients" 4th Edition, (2003) Rowe et al. (Eds), The Pharmaceutical Press, London, American Pharmaceutical Association, Washington. Non-limiting examples of pharmaceutically acceptable carriers, excipients and diluents include demineralized or distilled water; saline solution; vegetable-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil or coconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenylpolysiloxane and methylphenylpolysiloxane; volatile silicones; mineral oils, such as liquid paraffin, soft paraffin or squalene; cellulose derivatives, such as methylcellulose, ethylcellulose, carboxymethyl ... Examples of suitable carriers include sodium cellulose or hydroxypropyl methylcellulose; lower alkanols such as ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or acacia, and petrolatum. Typically, the carrier or carriers will form 10% to 99.9% by weight of the composition.

[0090] Pharmaceutical compositions may be provided in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (e.g., capsules, tablets, caplets, elixirs), in the form of an ointment, cream, or lotion suitable for topical administration, in a form suitable for delivery as eye drops, in an aerosol form suitable for administration by inhalation, e.g., intranasal or oral inhalation, or in a form suitable for parenteral administration, i.e., intradermal, subcutaneous, intramuscular, or intravenous injection. Injectable solutions or suspensions may use non-toxic parenterally acceptable diluents or carriers, such as Ringer's solution, isotonic saline, phosphate buffered saline, ethanol, and 1,2 propylene glycol.

[0091] The pharmaceutical compositions may include any suitable surfactant, such as an anionic, cationic, or nonionic surfactant, such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents, such as natural gums, cellulose derivatives, or inorganic materials, such as silicaceous silicas, and other ingredients, such as lanolin, may also be included.

[0092] In one embodiment, the inhibitory nucleic acids described herein are administered without a delivery system that associates the molecule, either covalently or non-covalently, ie, as naked siRNA.

[0093] In one embodiment, the inhibitory nucleic acids described herein are protected in pharmaceutical compositions, for example, by encapsulation or conjugation to a ligand. The pharmaceutical composition may be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multilamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes may be used. Liposomal pharmaceutical compositions may contain stabilizers, preservatives, excipients, etc. Preferred lipids are both natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for forming liposomes are known in the art; in this regard, see, inter alia, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), p. 33 et seq.

[0094] The pharmaceutical composition may be formulated as lipid nanoparticles. Any suitable liquid nanoparticle delivery system known to those skilled in the art may be used. For example, the inhibitory nucleic acid described herein may be formulated with a lipid nanoparticle composition comprising, for example, a cationic lipid / cholesterol / PEG-DMG / DSPC in a ratio of 40 / 48 / 2 / 10, or a cationic lipid / cholesterol / PEG-C-DMA / DSPC in a ratio of 40 / 48 / 2 / 10. The cationic lipid may be, for example, CLinDMA or DLinDMA. The PEG may be, for example, PEG-DMG. Other suitable, non-limiting lipid nanoparticle delivery systems include those described in the Examples of the present application; Idris et al. 2021, "A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19", Molecular Therapy Vol. 29 No. 7, 2219-2226; Wu et al. 2008, "Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use", Pharmaceutical Research, Vol. 26, No. 3, 512-522; and those described in U.S. Patent Nos. 7,514,099, 9,061,063, 10,369,226, 11,071,784, and 11,382,979.

[0095] Pharmaceutical compositions may be administered in the form of exosomes. As used herein, the term "exosome" refers to small, cell-derived (20-300 nm diameter, more preferably 40-200 nm diameter) membrane-containing vesicles encapsulating an internal space and derived from cells by direct plasma membrane budding or by fusion of the plasma membrane with late endosomes. Exosomes contain lipids or fatty acids and polypeptides, and may further contain an inhibitory nucleic acid as described herein as a payload. Exosomes may be obtained from producer cells or isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes may be directly loaded with exogenous nucleic acids or drugs by electroporation, lipofection, ultrasound, and contact with calcium chloride. Alternatively, purified exosomes may be loaded ex vivo, for example, by electroporation.

[0096] The exosomes of the present invention can be produced from cells grown in vitro or in the body fluids of a subject. When exosomes are produced from in vitro cell culture, a variety of producer cells can be used, such as HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs).

[0097] Pharmaceutical compositions may also be formulated by incorporation of the inhibitory nucleic acids described herein into adenovirus or adeno-associated virus (AAV), formulated with cell-penetrating peptides, lentiviral vectors, polymers, dendrimers, or prepared as siRNA bioconjugates, such as GalNAc-siRNA conjugate delivery platforms.

[0098] If exosome or vector is used as a vehicle for delivering siRNA, candidate siRNA is delivered as shRNA. Both siRNA and shRNA can target and inhibit viruses, and are functionally equivalent. When candidate siRNA is delivered as shRNA, it is derived from cell system and packaged in exosome or vector (AAV or lentivirus vector) as described above.

[0099] The shRNA may be provided in an expression cassette containing a promoter contiguously linked to the siRNA as described herein. In some embodiments, the promoter is a polII or polIII promoter, for example, a U6 promoter (e.g., a mouse U6 promoter) or an H1 promoter. In some embodiments, the expression cassette further contains a marker gene. In some embodiments, the promoter is a polII promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a polIII promoter. In some embodiments, the promoter is a U6 or H1 promoter.

[0100] Also provided is a vector containing the expression cassette described herein.Examples of suitable vectors include adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, herpes simplex virus (HSV) or murine Maloney virus vector.In one embodiment, the vector is an adeno-associated virus (AAV) vector.

[0101] The shRNA molecule comprises a pair of RNA sequences and a loop portion disposed between the pair of RNA sequences to form a hairpin. The length of the loop may vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In certain embodiments, the loop is 18 nucleotides in length. The hairpin structure may also contain a 3' and / or 5' overhang portion. In some embodiments, the overhang is 0, 1, 2, 3, 4, or 5 nucleotides in length. The nucleotide sequence of the loop region may vary, for example, (5'-GCAA-3'), (5'-GCGC-3'), or (5'-TTGC-3'), or other sequences as would be well understood by one skilled in the art.

[0102] The pharmaceutical compositions described herein may be administered in dosages sufficient to inhibit the expression of a target gene or the biological activity of an untranslated target sequence (e.g., a regulatory sequence) in a cell, tissue, or organism under treatment. The specific dosage of an inhibitory nucleic acid described herein administered to a given subject will depend on factors such as the route of administration and the subject's physical characteristics (including health status). For example, the appropriate dosage of a given pharmaceutical composition comprising an inhibitory nucleic acid described herein may depend on, but is not limited to, the subject's physical characteristics (e.g., age, weight, sex), the progression of a given coronavirus infection (i.e., pathological state), and other factors that will be readily recognized by those skilled in the art. Various general considerations that may be taken into account when determining appropriate dosages are described, for example, in Gennaro et al. (Eds), (1990), "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al. (Eds), (1990), "Goodman and Gilman's: The Pharmacological Bases of Therapeutics", Pergamon Press.Non-limiting examples of suitable dosages of the inhibitory nucleic acids described herein include 0.01 to 200 milligrams per kilogram body weight of the recipient per day, 1 to 50 mg / kg body weight per day, 1 to 40 mg / kg body weight per day, 1 to 30 mg / kg body weight per day, 1 to 30 mg / kg body weight per day, 1 to 10 mg / kg body weight per day, 1 to 5 mg / kg body weight per day, 1 to 3 mg / kg body weight per day, 1 to 2 mg / kg body weight per day, 0.1 to 1 mg / kg body weight per day, 0.1 to 0.9 mg / kg body weight per day, 1 to 2 mg / kg body weight per day, 0.1 to 1 mg / kg body weight per day, 0.1 to 0.9 mg / kg body weight per day, 1 to 2 mg / kg body weight per day, 0.1 to 1 mg / kg body weight per day, 0.1 to 0.9 mg / kg body weight per day, 0.1 to 1 ... These ranges include 0.1-0.8 mg / kg body weight per day, 0.1-0.7 mg / kg body weight per day, 0.1-0.6 mg / kg body weight per day, 0.1-0.5 mg / kg body weight per day, 0.1-0.4 mg / kg body weight per day, 0.1-0.3 mg / kg body weight per day, 0.1-0.2 mg / kg body weight per day, 0.01-0.1 mg / kg body weight per day, 0.01-0.05 mg / kg body weight per day, 0.01-0.02 mg / kg body weight per day, and 0.005-0.01 mg / kg body weight per day.

[0103] One of ordinary skill in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of the pharmaceutical compositions and / or inhibitory nucleic acids described herein to include in a dosage or series of dosages to achieve a desired therapeutic outcome.

[0104] Typically, in therapeutic applications, treatment will refer to the duration of an infection, disease state, or condition. Furthermore, it will be clear to one of ordinary skill in the art that the optimal amount and interval of individual dosages will be determined by the nature and severity of the infection, disease state, or condition being treated, the type, route, and site of administration, and the nature of the particular individual being treated. Such optimal conditions may also be determined using routine techniques.

[0105] In many cases, it will be desirable to administer several or multiple doses of the pharmaceutical compositions described herein. For example, they may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Administration may be at intervals of about 1 to about 12 weeks, and in certain embodiments, at intervals of about 1 to about 4 weeks. Periodic re-administration may also be desirable in cases of repeated exposure to the particular pathogen targeted by the pharmaceutical compositions described herein.

[0106] It will also be apparent to one of ordinary skill in the art that a conventional course of treatment determination testing may be used to ascertain the optimal course of treatment.

[0107] Suitable technologies for introducing the inhibitory nucleic acids described herein into cells, tissues, and organisms include a variety of carrier systems, vectors, and reagents, including, but not limited to, lipid nanoparticles (LNPs), micelles, nucleic acid-lipid particles, lipoplexes, liposomes, nucleic acid polymers, single chemical entity conjugates, virosomes, virus-like particles (VLPs), and mixtures thereof.

[0108] The pharmaceutical compositions of the present invention may be administered by any suitable method, for example, intravenously, bucally, parenterally, intranasally, orally, sublingually, or topically. Thus, administration may be topical, pulmonary (e.g., inhalation or insufflation of an aerosol or powder, including with a nebulizer), intranasal, intratracheal, epithelial, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (e.g., intraparenchymal, intrathecal, or intraventricular) administration.

[0109] In one embodiment, the pharmaceutical composition is adapted for intranasal administration. When the pharmaceutical composition of the present invention is delivered intranasally, significant antiviral targeting can be achieved in both the lungs and nasal cavity of SARS-COV-2 infected mice.

[0110] In one embodiment, the pharmaceutical compositions of the present invention are formulated as a direct acting nasal spray, hi one embodiment, the nasal spray may be self-administered at the point of care.

[0111] therapeutic method Generally, the inhibitory nucleic acids described herein are demonstrated to inhibit, reduce, or prevent coronavirus replication.

[0112] Accordingly, various aspects of the present invention provide methods for inhibiting coronavirus replication, methods for treating coronavirus infection, and methods for treating conditions and diseases resulting from coronavirus infection in a subject in need of treatment.

[0113] In some embodiments, provided are methods for inhibiting coronavirus replication in a cell, comprising administering to the cell an inhibitory nucleic acid described herein, optionally contained in a vector, lipid nanoparticle, or pharmaceutical composition, thereby causing degradation of coronavirus mRNA molecules in the cell and inhibiting coronavirus replication.

[0114] In other embodiments, provided are methods for treating a coronavirus infection in a subject, comprising administering an inhibitory nucleic acid described herein, optionally contained in a vector, lipid nanoparticle, or pharmaceutical composition, thereby inhibiting coronavirus replication and treating the infection.

[0115] In other embodiments, provided are methods for treating COVID-19 disease in a subject, comprising administering to the subject a therapeutically effective amount of an inhibitory nucleic acid described herein, optionally contained in a vector, lipid nanoparticle, or pharmaceutical composition, thereby inhibiting coronavirus replication and treating said COVID-19 disease.

[0116] In yet other embodiments, there is provided a use of an inhibitory nucleic acid described herein, optionally included in a vector, lipid nanoparticle, or pharmaceutical composition, in the preparation of a medicament for inhibiting coronavirus replication in a cell, treating a coronavirus infection in a subject, or treating COVID-19 disease in a subject.

[0117] In further embodiments, there are provided inhibitory nucleic acids described herein, which may be comprised in a vector, lipid nanoparticle, or pharmaceutical composition for use in inhibiting coronavirus replication in a cell, treating a coronavirus infection in a subject, or treating COVID-19 disease in a subject.

[0118] In one embodiment, administration is intranasal. Significant antiviral targeting of SARS-CoV-2 in both the lungs and nasal cavity is achieved in infected mice when the pharmaceutical composition of the present invention is delivered intranasally. Without wishing to be bound by theory, it is believed that intranasally delivered anti-SARS-CoV-2 siRNA antiviral agents can reduce viral replication in the nasal cavity and prevent aerosol transmission of respiratory viruses. In one embodiment, encapsulated siRNA is delivered intranasally.

[0119] In one embodiment, administration is by direct action nasal spray, hi one embodiment, the nasal spray may be self-administered at the point of care.

[0120] For many respiratory viruses, including SARS-CoV-2, the nasal cavity is a primary target for respiratory viral replication in the early stages of disease and a critical source of viral transmission through aerosols. RNAi is cost-effective, scalable, and easily programmable to target any viral RNA. This approach could result in the introduction of an entirely novel, cost-effective, programmable RNA platform technology and a self-administerable IN delivery platform, but could also deliver first-class RNA drugs suitable for any emerging novel RNA respiratory virus of concern.

[0121] The subject may be any animal of economic, social, or research importance, including bovine, equine, ovine, primate, avian, and rodent species. Thus, the subject may be a mammal, such as a human or non-human mammal (e.g., a pig, cat, dog, cow, horse, or sheep). The subject may be a laboratory animal (e.g., a rodent, such as a mouse, rat, or guinea pig; rabbit, etc.), a bird (e.g., poultry), a fish, or a crustacean.

[0122] The treatment may be used in subjects infected with an alphacoronavirus (e.g., HCoV-229E and HCoV-NL63), or a betacoronavirus (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-HKU1, or HCoV-OC43).

[0123] The therapeutic methods may be used to treat subjects diagnosed with a condition or disease resulting from a coronavirus infection, including, but not limited to, severe acute respiratory syndrome, coronavirus disease 2019 (COVID-19), and Middle East respiratory syndrome.

[0124] Identification of subjects in need of treatment by the methods described herein can include those identified by testing for coronavirus infection and / or by symptom assessment.

[0125] Standard techniques known in the art may be used to identify subjects who are positive for coronavirus infection, including, for example, CT scans, PCR-based methods (e.g., cycle threshold (CT), qRT-PCR), sequencing, CRISPR, ELISA, LFA, RT-LAMP, colloidal gold immunolateral flow chromatography.

[0126] The symptoms of coronavirus infection are diverse and often difficult to detect. Furthermore, there is overlap with symptoms resulting from other independent sources. Therefore, in some embodiments, after evaluating a subject for symptoms consistent with coronavirus infection, the symptoms may be confirmed or denied to be caused by coronavirus infection. Non-limiting examples of symptoms resulting from coronavirus infection include, but are not limited to, fever, cough, fatigue, loss of taste or smell, sore throat, headache, muscle or joint pain, nausea or loss of appetite, diarrhea, vomiting, difficulty breathing or shortness of breath, speech or movement disorders, confusion, and / or chest pain.

[0127] The effectiveness of the therapeutic methods described herein may be evaluated using standard methods known to those of ordinary skill in the art. For example, PCR-based techniques (e.g., qRTPCR) for assessing coronavirus load during or after therapy may be used to evaluate the effectiveness of the therapeutic methods described herein. Common techniques for evaluating the effectiveness of the therapeutic methods described herein include in vitro plaque assays and the use of in vivo mouse models. Additionally or alternatively, therapeutic effectiveness may be evaluated using techniques used to diagnose coronavirus infection, including CRISPR, ELISA, LFA, RT-LAMP, and colloidal gold immunolateral flow chromatography, particularly if adapted to provide quantitative data. [Example]

[0128] The present invention is described herein with reference to specific embodiment(s), which should not be construed as limiting in any way.

[0129] Example 1: siRNA targeting of SARS-CoV-2 material and method (a) siRNA design Using an algorithm that selectively discovers siRNA seed sequences based on the print tract in the target RNA (https: / / weinbergmorrislab.wixsite.com / weinbergmorrislab / tgs-algorithm-format; see also Ackley et al. 2013, "An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells", Molecular Therapy-Nucleic Acids (2013) 2, e104), we developed several candidate siRNAs. Regions of the viral genome that are highly conserved and amenable to siRNA targeting and contain a 4- to 6-bp print tract were selected. These non-chemically modified siRNAs were designed against diverse conserved sites in the SARS-CoV-2 genome, including the conserved stem of the 5' untranslated region (5'UTR), the helicase region, and the conserved region in the RNA-dependent RNA polymerase (RdRp) gene.

[0130] (b) Interferon screening THP-1 DUAL cells, a well-recognized standard for measuring immune stimulation, were transfected with the indicated siRNAs using Fugene 6 for 24 hours before quantification for (A) IRF and (B) NFκB gene reporter expression levels. 2'3'-cGAMP (20 μg / ml) and LPS (100 ng / ml) were used as positive controls for IRF and NFκB pathway stimulation, respectively.

[0131] (c) Plaque assay Plaque assays were used to evaluate the ability of various siRNAs to inhibit SARS-CoV-2 (Vic-1; Wuhan). VeroE6 cells were pretreated for 24 hours with either no treatment (virus), no target siRNA complexed with Lipofectamine 2000 (Lipo + virus), or 30 nM target siRNA prior to infection with 250 PFU of Wuhan (ancestral) SARS-CoV-2. Top novel candidate siRNAs were also infected with 250 PFU of alpha, beta, kappa, delta, or omicron mutant SARS-CoV-2. Four days after treatment, viral plaques were counted. Data were collected from triplicate treatments.

[0132] (d) qRTPCR screening VeroE6 cells were pretreated for 24 hours with either no siRNA (Lipo only) or with siRNA complexed with Lipofectamine 2000 prior to infection with 250 PFU of SARS-CoV-2. Selected siRNA combinations were mixed in equimolar ratios to a final concentration of 30 nM, and viral copy numbers were determined by digital droplet PCR against the N gene at 4 dpi. Data are representative of the mean range of duplicate treatments.

[0133] (e) Dose-response assessment of delta mutant suppression Candidate siRNAs targeting viral genes and phylogenetically conserved regions were designed and selected for screening against SARS-CoV-2-infected cells. Prior to infection with 250 PFU of delta mutant SARS-CoV-2, VeroE6 cells were pretreated for 24 hours with either no target siRNA (Lipo + virus) or seven dilutions (30, 20, 10, 5, 2.5, 1, or 0.1 nM) of target siRNA complexed with Lipofectamine 2000. Four days after treatment, viral plaques were counted. Data were collected from triplicate treatments.

[0134] (f) Liponanoparticle (LNP) delivery of siRNA 5x104 K18hACE2 mice infected with the live delta SARS-CoV-2 mutant received daily (-1 to 2 dpi) prophylactic intravenous (retro-orbital) treatment with siRNA-LNP (1 mg / kg). Lung virus tissue counts / g at 3 dpi are shown. Each dot represents data from one mouse. The siRNA Hel2, previously found to inhibit SARS-CoV-2, was contrasted with the novel siRNA siHelUP2.

[0135] result There are several methods for preparing siRNA, including chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes. For chemical synthesis, vendors provide siRNAs with dTdT 3' overhangs. Alternatively, the overhangs may be UU or other overhangs. Those skilled in the art will recognize that there is no substantial difference in the strength of siRNAs made with dTdT or UU overhangs, and the composition of the overhang does not appear to play a significant role in target mRNA recognition and cleavage. The overhangs are not part of the target site in the mRNA, but rather are useful for other purposes, such as Ago-2 and slicer activity and loading into the complex.

[0136] The candidate siRNAs developed are shown in Table 1. The siRNAs in Table 1 have dTdT overhangs and are loaded into the LNPs in this form, but may equally have UU overhangs or other overhangs, such as overhangs complementary to the mRNA sequence, or no overhangs at all. [Table 1] [Table 2] For comparison purposes, the sequences referred to are shown in Tables 3 and 4. [Table 3] [Table 4]

[0137] Regions of the viral genome containing 4-6 bp purine tracts that are highly conserved and highly amenable to siRNA targeting were selected and screened for any interferon activation (Figure 1A-B). None of the siRNAs induced significant interferon activation, suggesting that they were not immunogenic. Next, to determine the ability of various siRNAs to inhibit SARS-CoV-2, Vero cells were transfected and then infected with SARS-CoV-2. Several candidates, including siHelUP2ps, siHELdwn3s, and SL5-si1S, appeared to robustly inhibit SARS-CoV-2, as determined by plaque assay (Figure 2) and qRT-PCR (Figure 3).

[0138] The efficacy of these novel algorithm candidate siRNAs was then contrasted with previously published siRNAs (Idris et al. 2021, ibid.) that were found to potently suppress the Wuhan SARS-COV-2 mutant (Figure 4). The top candidate siRNAs appeared to be conserved for specific regions where previous studies had also found strong siRNA candidates, including helicase loci, e.g., siHel2, RNA-dependent RNA polymerases, e.g., siUC7, and 5'UTRs, e.g., siUTR3 (Idris et al. 2021, ibid.) (Figure 4).

[0139] There are several variants of SARS-COV-2, including alpha, beta, kappa, and delta. We screened the top candidate siRNAs, including those previously published, for their efficacy against various viral variants. All tested siRNAs, including algorithmically generated siRNAs, were found to be effective in suppressing various viral variants (Figure 5). The SARS-COV-2 virus undergoes negative-strand RNA synthesis during its viral life cycle. We examined the efficacy of siRNAs targeting this step in the viral life cycle and observed that some degree of suppression of viral expression is imprinted after negative-strand targeting of delta mutants (Figure 6). To determine the ability of combinations of siRNAs, sense-strand and negative-strand targeting siRNAs, to suppress SARS-COV-2 and whether such combinations offer additional benefits, we contrasted single- and double-treated cells exposed to the virus and observed that the siRNA combination neither significantly enhanced nor diminished the observed suppression (Figure 7). We then performed a dose-response analysis of the top three candidate siRNAs for in vitro suppression of delta mutants (Figure 8). The siRNA target sites of the top three siRNAs in the novel omicron variant of SARS-COV-2 were also evaluated, and all siRNAs were observed to contain 100% sequence targeting homology to the novel omicron variant. Finally, the top candidate siRNA, siHelUP2, was selected and compared with siHEL2, previously reported for the inhibition of delta SARS-COV-2 virus in vivo (Idris et al. 2021, ibid.). Notably, both siHelUP2 and siHel2 inhibited SARS-COV-2 in vivo (Figure 9), suggesting that siHelUP2 is also an excellent candidate siRNA for broad-spectrum inhibition of SARS-COV-2.

[0140] siHELUp2 (siCoV_1), which targets a conserved site in the helicase gene region of SARS-CoV-2, was then screened for efficacy against diverse SARS-CoV-2 VOCs and found to be equally potent across all tested VOCs, including the Omicron variant (Figure 10). Upon bioinformatics evaluation of the siHELUp2 siRNA target site in the Omicron variant, we confirmed that this siRNA exhibited 100% sequence targeting homology to this variant, further highlighting the highly conserved design of this siRNA. The dose response of siHELUp2 against SARS-CoV-2 was significantly higher than that of siControl (IC 50 =1.3x10 12 nM) compared to an IC of 3.99 nM 50 We revealed that SARS-CoV-2 undergoes negative RNA strand synthesis during its viral life cycle (Figure 11).

[0141] Taken together, these data highlight that the algorithmically defined siRNAs are functional in suppressing all variants of SARS-COV-2, and the top three candidate siRNAs all perform equally well and could be used in LNP-based delivery approaches as therapeutics for COVID-19.

[0142] Example 2: Intranasal Administration material and method Under isoflurane anesthesia, 10 μg of live delta SARS-CoV-2 VOCs were administered. 5Mice were infected intranasally (IN) with plaque-forming units (PFU) (20 μL total volume) using the IN injection technique. Subsequently, under isoflurane anesthesia, mice were treated with sLNPs or dmLNPs complexed with siRNA administered retroorbitally (IV) (100 μL total volume) or IN (20 μL total volume). For siRNA dosing on the day of infection (day 0 post-infection (dpi)), siRNA was administered 2 hours before infecting the mice with SARS-CoV-2. Mice were monitored daily for weight measurement and clinical scoring. We used the same prophylactic treatment strategy previously performed in SARS-CoV-2-infected K18-hACE2 mice (Supramaniam et al.), using LNP-based delivery of siRNA (Figure 12A).

[0143] result We observed that IV-delivered siHELUp2 (siCoV_1) suppressed SARS-CoV-2 in the lungs of infected mice when formulated in "stealth LNP" (sLNP) (Figure 12B). We then formulated siHELUp2 in dmLNP, a lung-targeting LNP (Supramaniam et al.) that has been shown to biodistribute and retain siRNA in the nasal cavity. Notably, IN-delivered siHELUp2-LNP significantly reduced not only pulmonary but also nasal viral load. This is the first demonstration that anti-SARS-CoV-2 siRNA targets both the lower and upper respiratory tract. Taken together, these data highlight that the algorithmically defined siRNA, siHELUp2, functions in suppressing all variants of SARS-CoV-2 and can be used in LNP-based delivery to reduce viral load in the lungs and nasal cavity of mice.

[0144] Example 3 - shRNA When using exosomes or vectors as a vehicle to deliver siRNA, candidate siRNAs are delivered as shRNAs. Both siRNAs and shRNAs are capable of targeting viruses, suppressing them, and are functionally equivalent. When candidate siRNAs are delivered as shRNAs, they are derived from cell lines and packaged into exosomes or vectors (AAV or lentiviral vectors). These do not have dTdT overhangs; rather, they contain only the sequences shown in Table 2.

[0145] The shRNA is provided in an expression cassette containing a promoter linked adjacent to the siRNA. A dual shRNA expression cassette (Figure 13) may be used to express siHelUP2 and siHelUP1 for packaging into EVs. While both the H1 and U6 promoters are shown in Figure 13, each promoter and shRNA may be expressed alone or in combination, as in this case. The nucleotide sequence of the loop region is (5'-GCAA-3'), but may also be (5'-GCGC-3') or (5'-TTGC-3'), or other sequences, as would be well understood by one skilled in the art. The sequence of the insert shown in Figure 13 is as follows: JPEG2025534045000005.jpg82170

[0146] References The contents of the following documents are incorporated herein by reference: Wang H, Paulson KR, Pease SA, Watson S, Comfort H, Zheng P, et al. Estimating excess mortality due to the COVID-19 pandemic: a systematic analysis of COVID-19-related mortality, 2020–21. The Lancet. 2022;399(10334):1513-36. Meganck RM, Baric RS. Developing therapeutic approaches for twenty-first century emerging infectious viral diseases. Nature Medicine. 2021;27(3):401-1 Cao Z, Gao W, Bao H, Feng H, Mei S, Chen P, et al. VV116 versus Nirmatrelvir-Ritonavir for Oral Treatment of Covid-1 New England Journal of Medicine. 2022;388(5):406-17. Fischer W, Eron JJ, Holman W, Cohen MS, Fang L, Szewczyk LJ, et al. Molnupiravir, an Oral Antiviral Treatment for COVID-1 withRxiv. 2021. Takashita E, Kinoshita N, Yamayoshi S, Sakai-Tagawa Y, Fujisaki S, Ito M, et al. Efficacy of Antibodies and Antiviral Drugs against Covid-19 Omicron Variant. N Engl J Med. 2022;386(10):995-8 Idris A , Davis A , Supramaniam A , Acharya D , Kelly G , Tayyar Y , et al. A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19. Mol Ther. Vol. 29 No. 7, 2219–2226. 2021. Zhang X, Goel V, Robbie GJ. Pharmacokinetics of Patisiran, the First Approved RNA Interference Therapy in Patients With Hereditary Transthyretin-Mediated Amyloidosis. The Journal of Clinical Pharmacology. 2020;60(5):573-85. To KK, Tsang OT, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020;20(5):565-74. Chang Y-C, Yang C-F, Chen Y-F, Yang C-C, Chou Y-L, Chou H-W, et al. A siRNA targets and inhibits a broad range of SARS-CoV-2 infections including Delta variant. EMBO Molecular Medicine.n / a(n / a):e15298. Supramaniam A, Tayyar Y, Clarke DTW, Kelly G, Acharya D, Morris KV, et al. Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection. Journal of Microbiology, Immunology and Infection. 2023. Amarilla AA, Modhiran N, Setoh YX, Peng NYG, Sng JDJ, Liang B, et al. An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2. Front Microbiol. 2021;12:625136. Khaitov M, Nikonova A, Shilovskiy I, Kozhikhova K, Kofiadi I, Vishnyakova L, et al. Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation. Allergy. 2021;76(9):2840-54. Bowden-Reid E, Ledger S, Zhang Y, Di Giallonardo F, Aggarwal A, Stella AO, et al. Novel siRNA therapeutics demonstrate multi-variant efficacy against SARS-CoV-2. Antiviral Research. 2023;217:105677. Ackley A, Lenox A, Stapleton K, Knowling S, Lu T, Sabir KS, et al. An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells. Molecular Therapy - Nucleic Acids. 2013;2. McCaskill JL, Marsh GA, Monaghan P, Wang LF, Doran T, McMillan NA. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation. PLoS One. 2013;8(5):e64360. Brutlag et al Comp. App. Biosci. 6:237-245 (1990). Usman et al., 1987, J. Am. Chem. Soc., 109, 7845. Scaringe et al., 1990, Nucleic Acids Res., 18, 5433. Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951. Bellon et al., 1997, Bioconjugate Chem. 8, 204. Shabarova et al., 1991, Nucleic Acids Research 19, 4247. Moore et al., 1992, Science 256, 9923. Wu et al. 2008, “Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use”, Pharmaceutical Research, Vol. 26, No. 3, 512-522.

Claims

1. 1. An isolated nucleic acid comprising 15 to 30 nucleotides and capable of specifically hybridizing to a SARS-CoV-2 sequence as defined in SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27, or to a fragment of said SARS-CoV-2 sequence.

2. 20 nucleotides; 21 nucleotides; 22 nucleotides or 23 nucleotides.

3. 1. An isolated nucleic acid comprising: a sequence as defined in SEQ ID NO: 2 or 24, wherein said nucleic acid is capable of specifically hybridizing to a SARS-CoV-2 sequence as defined in SEQ ID NO: 23, or a sequence as defined in SEQ ID NO: 4 or 26, wherein said nucleic acid is capable of specifically hybridizing to a SARS-CoV-2 sequence as defined in SEQ ID NO: 25; or A sequence as defined in SEQ ID NO: 6 or 28, wherein said nucleic acid is capable of specifically hybridizing to a SARS-CoV-2 sequence as defined in SEQ ID NO:

27. The isolated nucleic acid has at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to

4. 4. An isolated fragment of the isolated nucleic acid of any one of claims 1 to 3, wherein the fragment is 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length.

5. 1. An isolated double-stranded nucleic acid for inhibiting expression of SARS-CoV-2, comprising a sense strand and an antisense strand: the sense strand comprises a sequence as defined in SEQ ID NO: 1 or 23, or a variant or fragment thereof, and the antisense strand comprises a sequence as defined in SEQ ID NO: 2 or 24, or a variant or fragment thereof; or the sense strand comprises a sequence as defined in SEQ ID NO: 3 or 25, or a variant or fragment thereof, and the antisense strand comprises a sequence as defined in SEQ ID NO: 4 or 26, or a variant or fragment thereof; or The sense strand comprises a sequence as defined in SEQ ID NO: 5 or 27, or a variant or fragment thereof, and the antisense strand comprises a sequence as defined in SEQ ID NO: 6 or 28, or a variant or fragment thereof. The isolated double-stranded nucleic acid.

6. 6. The isolated nucleic acid of any one of claims 1 to 3, the isolated fragment of claim 4, or the isolated double-stranded nucleic acid of claim 5, comprising: a 2'-deoxy-2'-fluoro modified nucleotide; a 2'-deoxy modified nucleotide; a locked nucleic acid; an abasic nucleotide; a 2'-amino modified nucleotide; a 2'-alkyl modified nucleotide; a morpholino nucleotide; a nucleotide comprising an unnatural base; a 2'-O-methyl modified nucleotide; a 2'O-methoxyethoxy modified nucleotide; a 2'fluoro modified nucleotide; a 5-methyl modified cytidine; a pseudouridine; a nucleotide comprising a 5'-phosphorothioate group and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group; a nucleotide comprising a phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methyl phosphoramidite; or a nucleotide comprising deoxyribose.

7. 10. The isolated nucleic acid of any one of claims 1 to 3 or 6, the isolated fragment of claim 4 or claim 6, or the isolated double-stranded nucleic acid of claim 5 or claim 6, which is conjugated to a ligand.

8. A cell comprising the isolated nucleic acid of any one of claims 1 to 3, 6 or 7, the isolated fragment of any one of claims 4, 6 or 7, or the isolated double-stranded nucleic acid of any one of claims 5 to 7.

9. 9. The isolated nucleic acid of any one of claims 1 to 3 or 6 to 8, the isolated fragment of any one of claims 4, 6, 7 or 8, or the isolated double-stranded nucleic acid of any one of claims 5 to 8, which is RNA, antisense RNA or siRNA.

10. A vector comprising a nucleotide sequence encoding the RNA, antisense RNA, or siRNA of claim 9.

11. An isolated nucleic acid according to any one of claims 1 to 3, 6, 7 or 9; 10. An isolated fragment according to claim 4, or any one of claims 6, 7 or 9; An isolated double-stranded nucleic acid according to any one of claims 5 to 7 or 9; The vector of claim 10 Lipid nanoparticles comprising one or more of the following:

12. The lipid nanoparticle of claim 11, wherein the lipid comprises one or more of: a non-cationic liquid, a cationic lipid, and a conjugated lipid to prevent aggregation of the nanoparticles.

13. An isolated nucleic acid according to any one of claims 1 to 3, 6, 7 or 9; 10. The isolated fragment of any one of claims 4, 6, 7 or 9; An isolated double-stranded nucleic acid according to any one of claims 5 to 7 or 9; The vector of claim 10. Lipid nanoparticles according to claim 11 or claim 12; One or more of the following: a pharmaceutically acceptable excipient, carrier or diluent; A pharmaceutical composition comprising:

14. 14. The pharmaceutical composition of claim 13, wherein the isolated nucleic acid of any one of claims 1 to 3, 6, 7 or 9, the isolated fragment of any one of claims 4, 6, 7 or 9, or the isolated double-stranded nucleic acid of any one of claims 5 to 7 or 9 is protected.

15. 15. The pharmaceutical composition of claim 14, comprising a liposome, an AAV, or an exosome.

16. 15. The pharmaceutical composition of claim 14, comprising the lipid nanoparticles of claim 11 or claim 12.

17. The pharmaceutical composition of claim 13, comprising naked siRNA.

18. The pharmaceutical composition of claim 13, comprising an shRNA.

19. 19. The pharmaceutical composition according to any one of claims 13 to 18, which is a liquid for intravenous administration.

20. The pharmaceutical composition according to any one of claims 13 to 18, which is an aerosol for intranasal administration.

21. 20. A method for inhibiting coronavirus replication in a cell, comprising administering to the cell a nucleic acid described in any one of claims 1 to 3, 6, 7 or 9, a fragment described in any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid described in any one of claims 5 to 7 or 9, a vector described in claim 10, a lipid nanoparticle described in claim 11 or claim 12, or a pharmaceutical composition described in any one of claims 13 to 20, thereby causing degradation of coronavirus mRNA molecules in the cell and inhibiting the replication of coronavirus.

22. 21. A method for treating a coronavirus infection in a subject, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of claims 1 to 3, 6, 7 or 9, the fragment of any one of claims 4, 6, 7 or 9, the double-stranded nucleic acid of any one of claims 5 to 7 or 9, the vector of claim 10, the lipid nanoparticle of claim 11 or claim 12, or the pharmaceutical composition of any one of claims 13 to 20, thereby inhibiting replication of the coronavirus and treating the infection.

23. 21. A method for treating COVID-19 disease in a subject, comprising administering to the subject a therapeutically effective amount of the nucleic acid of any one of claims 1 to 3, 6, 7 or 9, the fragment of any one of claims 4, 6, 7 or 9, the double-stranded nucleic acid of any one of claims 5 to 7 or 9, the vector of claim 10, the lipid nanoparticle of claim 11 or claim 12, or the pharmaceutical composition of any one of claims 13 to 20, thereby inhibiting replication of the coronavirus and treating the COVID-19 disease.

24. Use of a nucleic acid according to any one of claims 1 to 3, 6, 7 or 9, a fragment according to any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid according to any one of claims 5 to 7 or 9, a vector according to claim 10, a lipid nanoparticle according to claim 11 or claim 12, or a pharmaceutical composition according to any one of claims 13 to 20 in the preparation of a medicament for inhibiting coronavirus replication in a cell, treating a coronavirus infection in a subject, or treating COVID-19 disease in a subject.

25. 21. The nucleic acid of any one of claims 1 to 3, 6, 7 or 9, the fragment of any one of claims 4, 6, 7 or 9, the double-stranded nucleic acid of any one of claims 5 to 7 or 9, the vector of claim 10, the lipid nanoparticle of claim 11 or claim 12, or the pharmaceutical composition of any one of claims 13 to 20, for use in inhibiting coronavirus replication in a cell, treating a coronavirus infection in a subject, or treating COVID-19 disease in a subject.

26. The method of any one of claims 21 to 23, the use of claim 24, or the nucleic acid, double-stranded nucleic acid, vector, lipid nanoparticle or pharmaceutical composition of claim 25, wherein the coronavirus is SARS-CoV-2.

Images