CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application relates to and claims the benefit of U.S. Provisional Application No. 63/143,035, filed January 29, 2021; the content of the application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. FIELD OF THE INVENTION

[0003] The present disclosure in general relates to the field of nucleic acid delivery. More particularly, the present disclosure relates to kits and uses thereof for delivering nucleic acids into a host.

[0004] 2. DESCRIPTION OF RELATED ART

[0005] Transfection is a process of introducing nucleic acids, such as deoxyribonucleic acids (DNAs) or ribonucleic acids (RNAs), into eukaryotic cells that allows the synthesis of proteins of interest, or the regulation (e.g., upregulation or downregulation) of target genes in the cells. As an important tool widely used in studies investigating gene function, modification and regulation, transfection contributes to the advancement of basic cellular research, medical development, and target validation.

[0006] At present, transfection may be done via various approaches, such as virally (e.g., adenoviral vector-based or lentiviral vector-based transfection), chemically (e.g., calcium phosphate, liposome, dendrimer or FUGENE® transfection), and physically (e.g., electroporation, microinjection, cell squeezing, sonoporation or impalefection). However, each method has its own disadvantages. For examples, viral transfection is usually limited by its instability, cytotoxicity, immunogenicity, and possible insertional mutagenesis caused by random integration into the host genome. The efficacy of chemical transfection is largely determined by factors such as the ratio of nucleic acid and chemical reagent, pH value and cell conditions that results in low transfection efficacy, especially in vivo, as compared to other methods. Also, the efficacy of chemical transfection varies with cell types, and the chemical reagents may be toxic to some types of cells. The major drawbacks of physical transfection are low efficacy, labor-intensive, technical demanding, potential cell damage, and nonspecific transportation of molecules into and out of cells. Further, a method suitable for transfecting DNA may not provide a satisfactory result for RNA transfection due to the structural difference between the DNA and RNA molecules.

[0007] In view of the foregoing, there remains a continued interest in developing a novel method for transfecting DNA or RNA in a safer and more efficient manner.

SUMMARY

[0008] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0009] The first aspect of the present disclosure is directed to a kit comprising the nucleocapsid (N) protein of SARS-CoV-2, and a nucleic acid. According to some embodiments of the present disclosure, the N protein and the nucleic acid are provided in the kit in a weight ratio from 1 : 1 to 1: 10; preferably, in a weight ratio from 1 : 1 to 1 :4.

[0010] According to some embodiments, the N protein comprises an amino acid sequence at least 85% identical to SEQ ID NO: 1; preferably, at least 90% identical to SEQ ID NO: 1; more preferably, at least 95% identical to SEQ ID NO: 1. In one specific example, the N protein comprises an amino acid sequence 100% identical to SEQ ID NO: 1.

[0011] According to some embodiments of the present disclosure, the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Depending on desired purposes, the nucleic acid may encode a therapeutic peptide, or an antigen (e.g., a viral protein or a tumor-associated antigen). In some exemplary embodiments, the nucleic acid encodes the spike (S) protein of SARS-CoV-2 (z.e., SARS-CoV-2 S protein). In some exemplary embodiments, the nucleic acid encodes a fragment of the SARS-CoV-2 S protein. In alternative embodiments, the nucleic acid is a small interfering RNA (siRNA).

[0012] Optionally, the present kit may further comprise an alpha-hydroxy acid (a-hydroxy acid, AHA) or a salt thereof. According to one exemplary embodiment, the kit further comprises lactate.

[0013] Also disclosed herein is a method of preventing and/or treating a disease in a subject by using the present kit. The method comprises,

(a) mixing the N protein of SARS-CoV-2 and a nucleic acid in a weight ratio of 1 : 1 to 1 : 10 to form a mixture; and

(b) administering to the subject an effective amount of the mixture of step (a). [0014] According to some preferred embodiments, in the step (a), the N protein and the nucleic acid are mixed in a weight ratio of 1 : 1 to 1 :4.

[0015] Depending on desired purposes, the nucleic acid may be a DNA or an RNA, in which the RNA may have a single-stranded or double-stranded structure.

[0016] According to some embodiments, the present method is useful in preventing a disease (e.g., SARS-CoV-2 infection). In these embodiments, the nucleic acid encodes an antigen associated with the disease (e.g., the S protein of SARS-CoV-2) so as to stimulate an immune response (e.g., producing anti-S antibodies) in the subject against the occurrence or progression of the disease.

[0017] According to certain embodiments, the present method is useful in treating a disease (e.g., SARS-CoV-2 infection). In these embodiments, the nucleic acid may encode a therapeutic peptide (i.e., a peptide exhibiting an inhibitory effect on the activity or function of SARS-CoV-2), or being an siRNA specific to a peptide associated with the disease (e.g., an siRNA against the structural protein of SARS-CoV-2) so as to ameliorate or alleviate the symptoms associated with the disease.

[0018] Optionally, the method further comprises administering to the subject an AHA or the salt thereof (e.g., lactate), prior to the administration of the mixture of the N protein the nucleic acid.

[0019] In general, the subject is a mammal; preferably, a human.

[0020] Another aspect of the present disclosure pertains to a method of in vitro delivering a nucleic acid into a host cell. The method comprises,

(a) mixing the N protein of SARS-CoV-2 and the nucleic acid in a weight ratio of 1 : 1 to 1 : 10 to form a mixture; and

(b) exposing the host cell to the mixture of step (a) so as to deliver the nucleic acid into the host cell

[0021] According to some preferred embodiments, in the step (a), the N protein and the nucleic acid are mixed in a weight ratio of 1 : 1 to 1 :4.

[0022] The nucleic acid may be a DNA or an RNA, in which the RNA may have a single-stranded or double-stranded structure.

[0023] Optionally, the method further comprises exposing the host cell to an AHA or the salt thereof (e.g., lactate), prior to the step (b).

[0024] Also disclosed herein is the use of the N protein of SARS-CoV-2 for the manufacture of a medicament in combination with a nucleic acid for the treatment of a disease. According to some embodiments, the N protein and the nucleic acid are combined in a weight ratio from 1 : 1 to 1 : 10; preferably, 1 : 1 to 1:4.

[0025] As discussed above, the nucleic acid may be a DNA or an RNA, in which the RNA may have a single-stranded or double-stranded structure. Depending on desired purposes, the nucleic acid may encode a therapeutic peptide, or an antigen associated with the disease (e.g., a viral protein or a tumor-associated antigen). Alternatively, the nucleic acid may be an siRNA specific to a peptide associated with the disease.

[0026] Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:

[0028] Figs. 1A to IE respectively depict the binding of SARS-CoV-2 nucleocapsid (SARS2-N) protein to mammalian cells according to Example 1 of the present disclosure. Fig. 1 A: A549 lung cancer cells were incubated with His tag-crotonase (Crt, as a control protein) or specified His tag-N proteins followed by the analysis of flow cytometry using anti-His-APC antibody. Fig. IB: mammalian cells, including HEK293T, HCT-8, HPAEpiC, A549, HeLa, 4T1 and LL2 cells, were respectively administered with specified treatments followed by the analysis of flow cytometry using anti-His-APC antibody. Crt: His tag-crotonase treatment; Ccr: His-tag control protein treatment; SARS2-N: His tag-SARS2-N treatment. The level of protein binding was presented by mean fluorescence intensity (MFI). Figs. 1C and ID: the treatment of anti-SARS2-N sera inhibited the binding of SARS2-N protein to A549 cells (Fig. 1C) and HPAEpiC cells (Fig. ID). Fig. IE: the treatment of anti-SARS2-N monoclonal antibody (NP-m Ab-40) neutralized SARS2-N protein binding to cell surface. SARS1-N: N protein of severe acute respiratory syndrome coronavirus (SARS-CoV); SARS2-N: N protein of SARS-CoV-2; MERS-N: N protein of middle east respiratory syndrome coronavirus (MERS); NL63-N: N protein of human coronavirus NL63 (HCoV-NL63); OC43-N: N protein of human coronavirus OC43 (HCoV-OC43); HKU1-N: N protein of human coronavirus HKU1 (HCoV-HKUl); 229E-N: N protein of human coronavirus 229E (CoV-229E). **, p < 0.01; ***, p < 0.001. [0029] Figs. 2A and 2B are histograms respectively depicting that membrane protein STEAP2 serves as a receptor for SARS2-N surface binding according to Example 2 of the present disclosure. Fig. 2A: knockdown of STEAP2 significantly inhibited the binding of SARS2-N protein to HPAEpiC cells. Fig. 2B: STEAP2 knockout significantly inhibited the binding of SARS2-N protein to A549 cells (Fig. 2B). *, p < 0.05; **, p < 0.01.

[0030] Figs. 3 A to 3C are histograms respectively depicting the effect of SARS2-N protein on transfecting RNA molecules into host cells according to Example 3 of the present disclosure. Figs. 3 A and 3B: the binding of specified SARS2-N-RNA complex to A549 cells (Fig. 3 A) or HPAEpiC cells (Fig. 3B). Crt: mock control; SARS2-N: SARS2-N only; SARS2-N+siRNA: SARS2-N mixed with an siRNA; SARS2-N+PS RNA#1: SARS2-N mixed with a double-stranded RNA having 21 nucleotides; SARS2-N+PS RNA#12: SARS2-N mixed with a single-stranded RNA having 50 nucleotides; SARS2-N+RBD: SARS2-N mixed with an mRNA encoding the receptor binding domain (RBD) of SARS-CoV-2; SARS2-N+RBD: SARS2-N mixed with an mRNA encoding the spike protein of SARS-CoV-2. SARS2-N+Luc2AGFP mRNA: SARS2-N mixed with an mRNA encoding luciferase-P2A-EGFP. Fig. 3C: expression levels of c-JUN and IFNAR1 genes in the cells administered with specified treatment. N protein: SARS2-N only; siJUN: c-JUN siRNA only; silFNARl : IFNAR1 siRNA only; N protein+siJUN: SARS2-N mixed with c-JUN siRNA; N protein+ silFNARl : SARS2-N mixed with IFNARJ siRNA. *,p < 0.05; **,p < 0.01; ***, p < 0.001.

[0031] Fig. 4 is the data depicting the effect of SARS2-N protein on transfecting DNA molecules into host cells according to Example 4 of the present disclosure, in which the cells were administered with specified treatment and the cell images were taken.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0033] I. DEFINITION

[0034] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Also, unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more.

[0035] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0036] The term “nucleic acid” is known in the art, and refers to a single-stranded and/or double-stranded polynucleotide, such as a DNA, a single-stranded RNA, a double-stranded RNA, an RNA/DNA hybrid, or the analog or derivative thereof. As used herein, the nucleic acid may be naturally isolated or chemically synthesized (in vitro synthesized) with or without modification (e.g., having one or more modifications in the nucleobase structure, phosphodiester backbone, and/or ribofuranosyl sugar). A preferred method for chemically synthesizing nucleic acids may be one that is automated and involves solid-state support synthetic schemes, although other schemes such as enzymatic methods could also be used. Within the practice of the present invention, a “nucleic acid” may be of any length.

[0037] As used herein, the term “protein” is synonymous with “polypeptide”, and as is generally understood in the art, refers to at least one chain of amino acids liked via sequential peptide bonds. This term also does not specify or exclude chemical or post-expression modifications of the protein/polypeptide of the invention, although chemical or post-expression modifications of the protein/polypeptide may be included or excluded as specific embodiments. Therefore, for example, modifications to proteins/polypeptides that include the covalent attachment of glycosyl group, acetyl group, phosphate group, lipid group and the like are expressly encompassed by the term “protein” or “polypeptide”. Further, proteins or polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention. Throughout the present disclosure, the positions of any specified amino acid residues within a protein/polypeptide are numbered starting from the N terminus of the protein/polypeptide. When amino acids are not designated as either D-or L-amino acids, the amino acid is either an L-amino acid or could be either a D- or L- amino acid, unless the context requires a particular isomer. Further, the notation used herein for the polypeptide amino acid residues are those abbreviations commonly used in the art.

[0038] As used herein, the term “synthetic polypeptide” refers to a polypeptide which does not comprise an entire naturally occurring protein molecule. The polypeptide is “synthetic” in that it may be produced by human intervention using such techniques as chemical synthesis, recombinant genetic techniques, or fragmentation of whole antigen or the like.

[0039] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are “generally regarded as safe,” e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0040] As used herein, the term “carrier” has its conventional meaning and refers to substances used to formulate the ingredients (i.e., the N protein of SARS-CoV-2 and the nucleic acid of the present disclosure) into a composition. Carriers (e.g., mannitol, CAPTISOL®, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like) help to achieve the desired product profile including but not limited to an aid in the manufacture process, and/or modifying the ingredient’s stability and efficacy. Acceptable carriers are non-toxic and do not adversely affect the benefit of at least one chemical entity described herein. Such carrier may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous carrier, that is generally available to one of skill in the art. Preferably, carriers are approved for or considered to be safe for human and animal administration. [0041] The term “adjuvant” has its conventional meaning, i.e., the ability to enhance the immune response to a particular antigen. Such ability is manifested by a significant increase in immune-mediated protection. Enhancement of humoral immunity is typically manifested by a significant increase (usually >10%) in the titer of antibody raised to the antigen.

[0042] The term “administered”, “administering” or “administration” are used interchangeably herein to refer a mode of delivery, including, without limitation, intranasal, intramuscular, intradermal, subcutaneous and intravenous application of an agent (e.g., the mixture of the N protein of SARS-CoV-2 and nucleic acid) of the present invention to a subject.

[0043] The term “prevent” or “preventing” as used herein are interchangeable, and refers to the prophylactic treatment of a subject who is at risk of developing a symptom, a secondary disorder or a condition associated with a disease (e.g., SARS-CoV-2 infection), in which the probability of the subject developing the symptom, secondary disorder or condition of the disease is decreased. Specifically, the term “prevent” or “preventing” refers to inhibit the occurrence of a symptom, a secondary disorder or a condition associated with the disease (e.g., SARS-CoV-2 infection), that is to reduce the incidence or the frequency of occurrence of the symptom, secondary disorder or condition. The term “prevent” or “preventing” as used herein referring to a mixture of the N protein of SARS-CoV-2 and nucleic acid and/or a method, does not mean or imply that use of the mixture and/or the method will provide a guarantee that the symptom, secondary disorder or condition will never occur, but rather that the mixture and/or the method will inhibit the occurrence of the symptom, secondary disorder or condition, and that the incidence and/or frequency of the symptom, secondary disorder or condition will be reduced.

[0044] As used herein, the term “treat,” “treating” or “treatment” are interchangeable, and encompasses partially or completely ameliorating, mitigating and/or managing a symptom, a secondary disorder or a condition associated with a disease (e.g., SARS-CoV-2 infection). The term “treating” as used herein refers to application or administration of the mixture of the N protein of SARS-CoV-2 and nucleic acid of the present disclosure to a subject, who has a symptom, a secondary disorder or a condition associated with a disease (e.g., SARS-CoV-2 infection), with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms, secondary disorders or features associated with the disease (e.g., SARS-CoV-2 infection). Treatment may be administered to a subject who exhibits only early signs of such symptoms, disorder, and/or condition for the purpose of decreasing the risk of developing the symptoms, secondary disorders, and/or conditions associated with the disease (e.g., SARS-CoV-2 infection). Treatment is generally “effective” if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is “effective” if the progression of a symptom, disorder or condition is reduced or halted.

[0045] The term “subject” refers to a mammal including the human species that is treatable with the mixture of the N protein of SARS-CoV-2 and nucleic acid and/or the method of the present invention. The term “subject” is intended to refer to both the male and female gender unless one gender is specifically indicated.

[0046] II. DESCRIPTION OF THE INVENTION

[0047] The present invention is based, at least in part, on the discovery that the N protein of SARS-CoV-2 (hereinafter as ’’SARS2-N protein”) can bind to cellular surface and assist the entry of nucleic acids (e.g., DNA or RNA) into cells. Accordingly, the SARS2-N protein may serve as a carrier to deliver nucleic acids into cells thereby generating proteins of interest and/or modifying gene expression in the host cells. Based on this discovery, the present disclosure provides a kit comprising the SARS2-N protein as a transfection carrier, and uses thereof in preventing and/or treating diseases (e.g., SARS-CoV-2 infection).

[0048] H-(l) Kits

[0049] The first aspect of the present disclosure is directed to a kit for transfecting host cells with nucleic acids. The kit comprises a polypeptide and a nucleic acid respectively housed in a first and a second containers (e.g., glass or plastic vials, ampoules or bottles). According to certain embodiments of the present disclosure, the polypeptide comprises an amino acid sequence at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1. Preferably, the polypeptide of the present disclosure comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. More preferably, the polypeptide of the present disclosure comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some working examples of the present disclosure, the polypeptide is the SARS2-N protein, and has the amino acid sequence of SEQ ID NO: 1.

[0050] According to some embodiments of the present disclosure, the polypeptide and the nucleic acid are provided in the kit in a weight ratio about 1 : 1 to 1 : 10; for example, 1 : 1, 1 : 1.5, 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, 1 :5, 1 :5.5, 1 :6, 1:6.5, 1 :7, 1 :7.5, 1 :8, 1 :8.5, 1 :9, 1 :9.5 or 1 : 10. Preferably, the polypeptide and the nucleic acid are provided in the kit in a weight ratio about 1 : 1 to 1 :4; more preferably, 1 :3 to 1 :4. In one embodiment, the polypeptide and the nucleic acid are provided in the kit in a weight ratio of 1: 1.3. In another embodiment, the polypeptide and the nucleic acid are provided in the kit in a weight ratio of 1 :2. In still another embodiment, the polypeptide and the nucleic acid are provided in the kit in a weight ratio of 1 :3.3. In further another embodiment, the polypeptide and the nucleic acid are provided in the kit in a weight ratio of 1 :4.

[0051] The polypeptide of the present disclosure can be isolated from a natural source or artificially produced by commonly used methods, such as being synthesized by host cells (e.g., bacterial cells, yeast cells or mammalian cells), which are transfected with a nucleic acid encoding the polypeptide. Alternatively, the present polypeptide may be synthesized by t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide. Peptides of the invention can also be synthesized by the well-known solid phase peptide synthesis methods. According to some embodiments, the polypeptide of the present kit is a synthetic polypeptide. Optionally, the N-terminus of the synthetic polypeptide is acetylated. Additionally or alternatively, the C-terminus of the synthetic polypeptide is amidated.

[0052] The nucleic acid may be a DNA or an RNA isolated from nature sources or artificially synthesized. According to some embodiments, the nucleic acid is a single-stranded RNA. According to alternative embodiments, the nucleic acid is a double-stranded RNA.

[0053] According to certain embodiments, the nucleic acid is a synthetic nucleic acid. The methods for synthesizing nucleic acids are known in the art, such as column-based synthesis and microarray-based synthesis. In general, DNA synthesis begins with solid-phase phosphoramidite chemistry, in which single-stranded DNA molecules about 10 and 100 base pairs in length are constructed. These single stranded molecules are then enzymatically assembled into larger molecules. The process is repeated until the synthesized molecule reaches the desired length. Compared to naturally occurring nucleic acids, in vitro synthesized nucleic acids may comprise one or more structural features generated during the synthesis procedure, such as non-phosphorylated 5’-end, overhang structure (i.e., 3’- and/or 5’-overhangs) and/or cloning sequence. Further, the synthetic nucleic acid may comprise one or more chemical modifications for intended purposes, such as the incorporation of triethylene glycol (TEG) moiety, 2’-O-methylation, thymidine (dT), 2’ -< -m ethyl -uridine (OMe), phosphorothioate thymidine (PS), L-threoninol-acridine (ACR), 2-deoxyribitol (RIB), thymine glycol nucleic acid (GNA), P-L-thymidine (MIR) or L-threoninol-thymine (THR).

[0054] The nucleic acid may encode a desired protein or polypeptide. For example, in the case when the present kit is used as a vaccine to prevent a disease, the nucleic acid may comprise a polynucleotide encoding an antigen associated with the disease, for example, a viral antigen (c.g, the S protein of SARS-CoV-2, the surface protein of hepatitis B virus, the early protein of human papilloma virus, or the hemagglutinin or neuraminidase of influenza virus), or a tumor-associated antigen (e.g., alpha-fetoprotein, carcinoembryonic antigen, CA-125, melanoma-associated antigen or human epidermal growth factor receptor 2). After administering to a subject, the polypeptide (e.g., the SARS2-N protein) would interact with and transport the nucleic acid into cells, e.g., lung cells or immune cells, thereby allowing the nucleic acid to be expressed and induce an immune response (i.e., an active immunity) against the disease in the vaccinated subject. According to some exemplary embodiments, the nucleic acid encodes the S protein of SARS-CoV-2. According to alternative embodiments, the nucleic acid encodes the receptor binding domain (RBD) of SARS-CoV-2 S protein.

[0055] Optionally, the kit for preventing a disease may further comprise a pharmaceutically acceptable adjuvant to enhance the immune response induced by the nucleic acid. The pharmaceutically acceptable adjuvant may be any substance known to enhance the immune response to an antigen. Examples of pharmaceutically acceptable adjuvant suitable to be used in the present disclosure include, but are not limited to Emulsigen-D, aluminum hydroxide, incomplete Fruend's adjuvant (IFA), complete Fruend's adjuvant (CFA), endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminium salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexe, Gebru adjuvant, super carrier, elvax 40w, L-tyrosine, montanide, Adju prime, Squalene, sodium phthalyl lipopolysaccharide (SPLPS), calcium phosphate, saponin, and muramyl dipeptide (MDP).

[0056] According to some embodiment, the present kit is used to treat a disease. In these embodiments, the nucleic acid may comprise a polynucleotide encoding a therapeutic peptide (i.e., a peptide exhibiting an inhibitory activity to the disease) so as to ameliorate or alleviate symptoms of the disease. Alternatively, the nucleic acid may be a small interfering RNA (siRNA) specific to a protein associated with the disease so as to alleviate or ameliorate the symptoms of the disease via suppressing the expression of the peptide. According to certain exemplary embodiments, the nucleic acid is an siRNA specific to the S protein of SARS-CoV-2. [0057] Optionally, the kit for treating a disease may further comprise a pharmaceutically acceptable carrier to integrate the polypeptide (e.g., the SARS2-N protein) and nucleic acid into a formulation, and/or modifying the ingredient’s stability and efficacy. Non-limiting examples of pharmaceutically acceptable carrier suitable to be used in the present disclosure include, but are not limited to, a solubilizing agent, stabilizer, diluent, bulking agent, pH buffering agent, tonicifying agent, antimicrobial agent, wetting agent, and emulsifying agent (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like). [0058] Optionally, the nucleic acid is conjugated to a reporter, e.g., a fluorophore. Non-limiting examples of reporter suitable to be conjugated with the nucleic acid include polyhistidine-tag (His-tag), FLAG-tag, myc-tag, ALFA-tag, polyglutamate tag, polyarginine tag, AVITAG™, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), orange fluorescent protein (OFP), fluorescein amidite (FAM™), tetrachlorofluorescein (TET™), hexachlorofluorescein (HEX™), carboxy-X-rhodamine (ROX™), TEXAS RED®, carboxytetramethylrhodamine (TAMRA™), ALEXA FLUOR® 488, ALEXA FLUOR®568, ALEXA FLUOR® 596, ALEXA FLUOR® 594, allophycocyanin (APC), cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5. According to one working example, the nucleic acid is conjugated to FAM™.

[0059] According to some optional embodiments, the present kit may further comprise an AHA (z.e., a class of chemical compounds distinguished by one or more hydroxyl groups ovalently bonded to the a-carbon of a carboxylic acid) or a salt thereof. Examples of AHA suitable for use in the present disclosure include, but are not limited to, citric acid, glycolic acid, 2-hydroxycaproic acid, 2-hydroxycaprylic acid, lactic acid, malic acid, tartaric acid and the like. According to some embodiments, the present kit further comprises lactate. According to the embodiments, the presence of lactate enhances the entry and expression of the nucleic acid in host cells.

[0060] Optionally, the present kit may further comprise an instruction indicating how the materials (z.e., the polypeptide and nucleic acid) within the kit are employed.

[0061 ] U-(2) Methods of delivering nucleic acids into host cells

[0062] The second aspect of the present disclosure pertains to a method of in vitro introducing a nucleic acid (e.g., DNA or RNA) into a host cell. The method comprises, (a) mixing the present polypeptide (e.g., SARS2-N protein) and the nucleic acid to form a mixture; and (b) exposing the host cells to the mixture of step (a) so as to deliver the nucleic acid into the host cell.

[0063] In general, the host cells may be any types of cells, for example, epithelial cells, endothelial cells, fibroblasts, immune cells (such as T cells, B cells, dendritic cells, macrophages and neutrophils), adipocytes, keratinocytes, melanocytes, smooth muscle cells, and neural cells. According to some exemplary embodiments, the host cells are epithelial cells derived from lung, kidney, colon, pulmonary alveoli, cervix or mammary gland. [0064] In the step (a), the present polypeptide and nucleic acid are mixed in a weight ratio from 1 : 1 to 1 : 10; preferably, from 1 : 1 to 1 :4; more preferably, from 1 :3 to 1 :4. As described above, the nucleic acid may be a DNA or RNA, in which the RNA may have a single-stranded or double-stranded structure. In one exemplary example, the nucleic acid encodes the S protein of SARS-CoV-2 (1,255 amino acid residues). In another example, the nucleic acid encodes the receptor binding domain (RBD, 222 amino acid residues) of the S protein of SARS-CoV-2. In still another example, the nucleic acid is an siRNA of the S protein of SARS-CoV-2.

[0065] Then, in the step (b), the mixture of the present polypeptide and nucleic acid are added to the host cell and incubated for a sufficient period of time (e.g., 24 or 48 hours) until the nucleic acid is transported into the host cell via endocytosis.

[0066] Optionally, the present method further comprises exposing the host cell to an AHA (e.g., citric acid, glycolic acid, 2-hydroxycaproic acid, 2-hydroxycaprylic acid, lactic acid, malic acid or tartaric acid) or a salt thereof, prior to, concurrently with or after the step (b), so as to enhance the entry and expression of the nucleic acid. According to some embodiments, the AHA is added to the host cells prior to the step (b). In one specific example, the AHA is lactate.

[0067] II-/3) Methods of preventing and/or treating diseases

[0068] The third aspect of the present disclosure is directed to a method of preventing or treating a disease in a subject. The method comprises, (a) mixing the present polypeptide (e.g., SARS2-N protein) and a nucleic acid in a weight ratio of 1 : 1 to 1 : 10 to form a mixture; and (b) administering to the subject an effective amount of the mixture of step (a) so as to prevent, alleviate and/or ameliorate the symptoms associated with the disease.

[0069] The effective amount may vary with different factors, such as the particular condition being treated, the severity of the condition, the individual patient parameters (including age, physical condition, size, gender and weight), the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of a skilled artisan or a health practitioner.

[0070] In general, the mixture of the present polypeptide and nucleic acid is administered to the subject at intervals of several days to several months, preferably one to four weeks, for 2-10 times, until the desired antibody is produced. For example, the mixture of the present polypeptide and nucleic acid may be administered to the subject once every week, every 2 weeks, every 3 weeks, every month, every 2 months, every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the active agent used) can vary over time.

[0071] The subject vaccinated by or treatable with the present method is a mammal, for example, a human, mouse, monkey, rat, cat, dog, sheep, goat, or rabbit. Preferably, the subject is a human.

[0072] Depending on the intended routes of administration, the kit may comprise different types of adjuvant or carriers for preparing a formulation containing the active agents (i.e., the SARS2-N and nucleic acid) of the present kit. The thus-prepared formulation (e.g., vaccine or pharmaceutical) may be administered intramuscularly, intranasally, intradermally, subcutaneously, intraveneously, intraarterially, intraperitoneally, intralesionally, intrapleurally or intratracheally, in any suitable forms such as liquids, aerosols and etc. In some embodiments, the formulation is prepared in a form suitable for parenteral administration, such as administration by intramuscular or intranasal injection. The formulation for parenteral administration is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred formulation for parenteral injection should contain, in addition to the present polypeptide (e.g., the SARS2-N protein) and nucleic acid, an isotonic vehicle such as sodium chloride injection, Ringer’s injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer’s injection, or other vehicle as known in the art. The formulation of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

[0073] According to some preferred embodiments, the method further comprises administering to the subject an AHA (e.g., citric acid, glycolic acid, 2-hydroxycaproic acid, 2-hydroxycaprylic acid, lactic acid, malic acid or tartaric acid) or the salt thereof, prior to, concurrently with or after the administration of the mixture of the present polypeptide and nucleic acid. According to some embodiments, the AHA is administered to the subject prior to the administration of the mixture of the present polypeptide and nucleic acid. In one specific example, the AHA is lactate.

[0074] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. EXAMPLE

[0075] Materials and Methods

[0076] Cell Culture

[0077] HEK293T cells (human embryonic kidney cells), HCT-8 cells (human colon cells), A549 cells (human lung cells), HeLa cells (human cervical cells), 4T1 cells (mouse mammary gland cells) and LL2 (mouse lung cells) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HPAEpiC (Human pulmonary alveolar epithelial cells) were cultured in alveolar epithelial cell medium. All cells were maintained at 37°C in humid air with 5% CO2 condition and are free from mycoplasma contamination. For Endocytosis inhibition observation, cells were blocked by pretreatment of hydroxychloroquine (HCQ).

[0078] Plasmid

[0079] All-in-one CRISPR vector, pAll-Cas9.Ppuro, was digested with BsmPA and ligated with annealed oligonucleotides (SEQ ID NOs: 2 and 3) for expression of STEAP2 sgRNAs, which could bind to the targeting regions of STEAP2 gene. The CRISPR activity of each sgRNA was validated by surrogate reporter assay.

[0080] Protein purification and analysis

[0081] SARS2-N protein sequence from the original Wu-han strain was constructed and codon optimized into pET-15b plasmid by using Nhel and Xhol sites. The constructed plasmid were then transformed into Escherichia coli (E. coll) cultured in terrific broth (TB) medium and inducted by isopropyl [3-D- l -thiogalactopyranoside (IPTG) at 30°C for 8 hours upon the medium OD value reached 0.4 to 0.6. The bacteria pellet were collected and rinsed with binding buffer (500 mM NaCI, 10 mM sodium phosphate buffer, 80 mM imidazole, pH7.6), followed by lysing with continuous high pressure cell disrupter. The lysates were then passed through 0.45 um filter and purified by column. Next, 20 mL wash buffer (500 mM NaCI, 10 mM sodium phosphate buffer, 10 mM imidazole, pH7.6) was added to the column thereby removing the unbound proteins; the purified SARS2-N protein were eluted by elution buffer (500 mM NaCI, 10 mM sodium phosphate buffer, 250 mM imidazole, pH7.6). The final SARS2-N protein were then further dialyzed two times with dialysis buffer (50 mM Tris, 500 mM NaCI, pH7.6) at 4°C and concentrated by concentrate tube. The purification of SARS2-N protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions and stained with IMPERIAL™ protein stain.

[0082] Cell surface binding and blocking assays [0083] Cells were detached by cell dissociation buffer and suspended in cultured medium. After transferring into 96-well plate ( I N O ⁵ cells in 100 pL cultured medium/well), cells were washed with ice-cold phosphate-buffered saline (PBS) once, and re-suspended in 100 pL of ice-cold FACS buffer (PBS supplemented with 2% FBS) containing indicated concentrations of His-tag control protein (Crt), or His-tag N protein derived from human coronavirus (including SARS-CoV, SARS-CoV-2, MERS, HCoV-NL63, HCoV-OC43, HCoV-HKUl and HCoV-229E) followed by incubation at 4°C for 1 hour. Cells were washed with ice-cold PBS once, stained by anti-His-APC antibody (1 :300 dilution) at 4°C for 30 minutes. After staining, cells were washed with ice-cold PBS once, and re-suspended in 200 pL of ice-cold FACS buffer for flow cytometry analysis.

[0084] DNA and siRNA delivery by SARS2-N protein

[0085] HEK293T were seeded onto chamber slides (50,000 cell/well) in 200 uL medium. For the purpose of evaluating the efficacy of SARS2-N protein on delivering DNA molecule, the SARS2-N protein was mixed with a GFP plasmid at a weight ratio of 1 : 1 to 1 :4 (Table 1). After incubating at 4°C for 1 hour, the thus-formed SARS2-N-DNA complex was added to cell condition medium with gently pipetting. After 24 hours, the expression of GFP were detected by an imaging system.

[0086] Table 1 Weight ratio of SARS2-N protein and DNA plasmid

[0087] For the purpose of evaluating the efficacy of SARS2-N protein on delivering RNA molecule, 10 ug SARS2-N protein was mixed with 40, 80, 100 uL siRNA specific to c-JUN and IFNAR1 genes at 4°C for 1 hour. The thus-formed SARS2-N-siRNA complex was added to cell condition medium with gently pipetting. After 72 hours, total RNA was harvested from the cells, and the gene expression was detected by quantitative PCR (qPCR). [0088] Alternatively, 5* 10 ⁴ HEK293T were seeded onto chamber slides (80,000 cells/well) in 200 uL medium. FAM™-labeled siRNA (SEQ ID NO: 4) was mixed with the SARS2-N protein at 4°C for 1 hour. The thus-formed SARS2-N-siRNA-FAM complex was added to cell condition medium with gently pipetting. After 24 hours, the localization of siRNA-FAM was observed by confocal microscopy.

[0089] Immunofluorescent staining

[0090] Cells were seeded onto chamber slides. FAM™-labeled siRNA was premixed with SARS2-N protein for 1 hour on ice. The thus-formed SARS2-N-siRNA-FAM complex was added into each chamber for overnight incubation. After treatment, cells were washed PBS once, fixed by 4% paraformaldehyde (PF A) for 10 minutes, and permeabilized by 0.1% TRITON™ X-100 for 5 minutes. Nonspecific binding sites on the cells were blocked with blocking buffer (1% BSA in PBS). For the purpose of detecting GFP, cells were stained with antibodies against GFP (1 : 100). After washing with PBS, the slides were incubated with goat anti-rabbit antibody conjugated with ALEXA FLUOR® 568 (1 :250) for 1 hour. After washing, the nuclear were stained with 4’,6-diamidino-2-phenylindole (DAPI) (1 : 1,000) and observed under confocal microscopy. To detect His-tag SARS2-N protein, cells were stained with anti-His monoclonal antibody (1 :5,000) for 90 minutes. After incubation, cells were washed by PBS and treated by goat anti-mouse-ALEXA FLUOR® 568 or anti-rabbit APC (1 :400) for 1 hour. The localization of SARS2-N protein and siRNA-FAM were assessed by confocal microscopy.

[0091] Time lapse analysis

[0092] 5x l0 ⁵ HPAEpiC cells were seeded on 6-well dish. 20 ug siRNA-FAM was then mixed with 20 ug SARS2-N protein at 4°C for 1 hour. The thus-formed SARS2-N-siRNA-FAM complex was added to cells and quickly washed one time by PBS. The live cell image was observed by lattice light sheet microscopy.

[0093] Isolation and digestion of SARS2-N protein-interacted membrane proteins

[0094] For isolating cell membrane proteins, cells were detached by cell dissociation buffer, followed by washing with cultured medium and PBS once. Cells were resuspended in hypotonic buffer (10 mM HEPES, 1.5 mM MgCL, 10 mM KC1, and lx protease inhibitor mixture, pH7.4) and incubated on ice for 15 minutes. After homogenizing by dounce homogenizer, the membrane fraction was purified by two-step centrifugation. First, the nuclei were pelleted by centrifugation at 1,000g at 4°C for 10 minutes. The supernatant was mixed with 1.8 M sucrose to a final concentration of 0.25 M sucrose, and centrifuged by 13,000 rpm at 4°C for 1 hour. The pellet remaining membrane proteins was dissolved in 90% (v/v) formic acid (FA) and the concentration of the membrane protein was determined by protein assay. Membrane protein samples were further vacuum-dried for -80°C storage.

[0095] For the preparation of control and SARS2-N-beads, 10 pg of His-tag control or SARS2-N protein was mixed with 1 pL of anti-His antibody and 50 pL of protein G magnetic beads in 500 pL RIPA buffer, and then rotated at 4°C overnight. The control or SARS2-N-beads were washed with RIPA buffer three times. In the meantime, the freeze-dried membrane proteins were re-suspended in 500 pL of 1% NP40 buffer (150 mM NaCl, 50 mM Tris-HCl, 1% NP40) containing protease inhibitor, and sonicated to clear. Resolved membrane proteins were mixed with prepared control or SARS2-N-beads, and rotated at 4°C for 3 hours. Membrane proteins pull-downed-beads were washed with 500 pL of RIPA buffer three times and stored at -80°C.

[0096] For DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) crosslinking, cells were detached by cell dissociation buffer and re-suspended in ice-cold PBS containing 2% FBS at l * 10 ⁷ cells/ml density. 100 pg of control or SARS2-N protein was mixed with cell suspension and rotated at 4°C for 1 hour surface binding. After 1 hour surface binding, cells were washed with ice-cold PBS twice and re-suspended in 1.2 mL of fresh prepared DTSSP linker (3 mM in PBS), and rotated at 4°C for 1.5 hours. 133.3 pL of 10x TBS buffer (0.5 M Tris, 1.5 M NaCl, pH7.6) was added into cell solution and rotated at 4°C for 15 minutes to stop crosslinking. Cells were washed with ice-cold PBS once and lyzed by RIPA buffer containing protease inhibitor. His-tag control or SARS2-N protein bound surface proteins were immunopreci ptaed by using anti-His antibody.

[0097] To elute pull-downed membrane proteins from the beads, magnetic beads were incubated with elution buffer (1% SDS, 20 mM Tris-HCl and 150 mM NaCl) at 4°C for 20 minutes. The eluent was collected and dried by vacuum concentrator.

[0098] For protein digestion, the protein pellet was resuspended in 0.1 M triethylammonium bicarbonate (TEABC) buffer containing 6M urea, 5 mM EDTA and 2% (w/v) SDS. Proteins were chemically reduced by adding 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 37°C for 30 minutes, and alkylated by 2 mM methyl methanethiosulfonate (MMTS) at room temperature for 30 minutes. To incorporate proteins into a gel directly in the eppendorf vial, acrylamide/bisacrylamide solution (40%, v/v, 29: 1), 10% (w/v) ammonium persulfate (APS), and N,N,N',N'-tetramethylethylenediamine (TEMED) was then applied to the protein solution. The gel was cut into small pieces and washed with 25 mM TEABC and 25 mM TEABC containing 50% (v/v) acetonitrile (ACN). The gel samples were further dehydrated with 100% ACN and then completely dried by vacuum concentrator. Proteolytic digestion was then performed by sequencing grade modified trypsin (proteimtrypsin = 10: 1, g/g) in 25 mM TEABC at 37°C overnight. Peptides were extracted from the gel using sequential extraction with 25 mM TEABC, 0.1% (v/v) trifluoroacetic acid (TFA) in water, 0.1% (v/v) TFA in 50% ACN, and 100% ACN. The solutions were dried in vacuum concentrator and resuspended in 0.1% (v/v) TFA for further desalting.

[0099] Generation of STEAP2 Knockout Mutant

[0100] To generate STEAP2 knockout A549 cells, adequate all-in-one CRISPR plasmid was transfected by LIPOFECT AMINE® reagent. After 24 hours, the medium was replaced with fresh culture medium with 2 pg/mL of puromycin and was selected for additional 3 days. Before establishing single cell clones by limiting dilution method, the edited cells in the cell pool were analyzed to verify knockout effects. Briefly, genomic DNA was extracted from cell pool by kits, and was used as a template for polymerase chain reaction (PCR) amplification. The PCR reaction was carried out in a 25 pL of reaction mixture of DNA polymerase, 100 ng of genomic DNA and 0.3 pM of forward and reverse primers (SEQ ID NOs: 5 and 6). A wild-type (1,513 bp) or knockout (1,076 bp) PCR amplicon spanning the target site was generated at an initial denaturation at 95°C for 3 minutes, 30 cycles with DNA denaturation at 98°C for 20 seconds, annealing at 60°C for 15 seconds, DNA extension at 72°C for 25 seconds, and final extension at 72°C for 25 seconds. Finally, 24 single cell clones were selected.

[0101] Statistics

[0102] All statistical significance was analyzed by two-tailed unpaired t-tests. P<0.05 was considered significant. The data of the present study was presented as mean ± SEM from at least three independent replicates

[0103] Example 1 SARS2-N protein binds to and enters into cells through endocytosis

[0104] Since N protein is the most abundant viral protein during infection and is known to be expressed in patient blood, whether the N protein would bind to cell surface was examined in this example. The N proteins derived from SARS-CoV, SARS-CoV-2, MERS, HCoV-NL63, HCoV-OC43, HCoV-HKUl and HCoV-229E (hereinafter, as the “SARS1-N”, “SARS2-N”, “MERS-N”, “NL63-N”, “OC43-N”, “HKU1-N” and “229E-N” proteins; each had a His-tag attached thereto) were respectively expressed in E. coli and incubated with A549 lung cancer cells. The binding between the N proteins and A549 cells was detected via flow cytometry analysis using anti-His antibody. The data of Fig. 1A indicated that among seven coronavirus N proteins, the SARS2-N protein exhibited the highest binding affinity to A549 cells at concentrations less than 10 ug/ml, followed by NL63-N and OC43-N proteins, in which OC43-N only exhibited binding affinity at 100 ug/ml. The SARS-CoVl-N and HKU1-N proteins exhibited less binding affinity as compared to the SARS2-N protein, and the MERS-N protein exhibited no binding affinity to the surface of A549 cells.

[0105] To further examine whether the binding of the SARS2-N protein to cell surface is cell type-dependent, five human cell lines (including HEK293T kidney cells, HCT-8 colon cells, HPAEpiC and A549 pulmonary alveolar cells, and HeLa cervical cells) and two mouse cell lines (including 4T1 breast cells and LL2 lung cells) were respectively treated with purified SARS2-N protein, and the binding of the SARS2-N protein and cells was assessed by flow cytometry analysis. As the data of Fig. IB depicted, the SARS2-N protein bound to all cell lines, suggesting that the SARS2-N surface binding was not cell-type dependent.

[0106] In addition, five mice were immunized by the SARS2-N protein, in which the titers of anti-SARS2-N protein sera were similar in all immunized mice (data not shown). The sera were collected from the immunized mice so as to test whether polyclonal anti-SARS2-N sera would block the SARS2-N protein binding to cell surface. Surprisingly, the data of antibody blocking assay indicated that 2 out of 5 anti-SARS2-N sera efficiently suppressed the binding of the SARS2-N protein to the surface of A549 and HPAEpiC cells (Figs. 1C and ID). Moreover, it was found that an anti-SARS2-N monoclonal antibody (NP-mAb-40) existed about 94.32% neutralization effect on SARS2-N protein surface binding at an antibody /N protein ratio of 10 (Fig. IE). Since not all the polyclonal anti-SARS2-N sera exhibited blocking effect, this result indicated that the cell binding was dependent on particular SARS2-N epitopes. Interestingly, NP-mAb-40 didn’t recognize the RBD and dimerization domain (DD) of SARS2-N protein, suggesting that the N-terminal domain (NTD), SR or CTD may contribute to the SARS2-N protein surface binding. Indeed, it was found that NTD, SR or CTD deletion abrogated the binding of SARS2-N protein to cell surface (data not shown), suggesting that the SARS2-N protein binds to cell surface through the NTD, SR or CTD domain. The binding activity data confirmed that the NTD, SR or CTD domain can be recognized by NP-mAb-40 (data not shown), suggesting that NP-mAb-40 neutralizes the SARS2-N protein binding to cell surface through blocking its NTD, SR or CTD domain.

[0107] Since the SARS2-N protein bound to cell surface, whether it can enter the cells was further investigated in this example. Three N proteins (including SARS1-N, SARS2-N and MERS-N) were respectively added to HPAEpiC cells, followed by incubating at 37°C for 1 hour. The N proteins were detected by confocal microscopy using anti-N antibodies. The results indicated that the signals of cells (surface and cytosol) treated with SARS2-N protein were significantly higher than that of cells treated with SARS1-N or MERS-N protein (data not shown). The image results were consistent with the cell surface binding results.

[0108] Next, whether the SARS2-N protein enters the cells through endocytosis was also examined. The HPAEpiC cells were treated with two endocytosis inhibitors, dynasore (a cell permeable dynamin inhibitor) and hydroxychloroquine (HCQ), for 1 hour to block cellular endocytosis. The results of immunofluorescent staining indicated that the SARS2-N signal can be detected in the group of SARS2-N treatment, while the pretreatment of dynasore or HCQ significantly reduced the signal of subcellular localization of SARS2-N protein (data not shown). These results suggested that the SARS2-N protein enters the cell through endocytosis.

[0109] Example 2 STEAP2 is involved in SARS2-N protein endocytosis

[0110] Protein pull-down experiment was carried out to identify the cellular receptor mediating the entry of SARS2-N protein. As described in Materials and Methods, the HPAEpiC cell membrane fraction was extracted and immunoprecipitated by SARS2-N-bound beads; in the meantime, DTSSP-crosslinked SARS2-N-treated cells were also extracted, and the SARS2-N protein interacted surface proteins were further immunoprecipitated by SARS2-N-bound beads. The pulled down proteins were analyzed by LC-MS/MS, and the putative membrane proteins were further sorted.

[0111] Proteomic analysis data indicated that 16 and 19 significantly enriched SARS2-N-interacted membrane proteins were respectively identified in A549 and HPAEpiC membrane fraction (data not shown). Moreover, 10 and 1 (STEAP2) significantly enriched SARS2-N-interacted membrane proteins were respectively identified in A549 and HPAEpiC DTSSP crosslinking cells (data not shown). Interestingly, among these SARS2-N-interacted membrane proteins, STEAP2 (a metalloreductase localized in the plasma membrane and Golgi complex) was the common receptor protein significantly enriched in pull-downed experiments (data not shown). Accordingly, lentivirus-mediated shRNA knockdown and CRISPR/Cas9 knockout were used to confirm whether STEAP2 serves as a receptor for SARS2-N surface binding. The data of Figs. 2A and 2B indicated that the STEAP2 knockdown in HPAEpiC cells (Fig. 2A) or knockout in A549 cells (Fig. 2B) significantly inhibited SARS2-N protein binding to cell surface. The control protein (Ccr) exhibited no binding affinity as expected. These data suggested that STEAP2 acts as a surface receptor for SARS2-N protein. However, since STEAP2 knockout cells did not abolish the SARS2-N protein binding to cell surface, these data suggested that other surface receptors might exist for the SARS2-N protein to bind.

[0112] Example 3 Effect of SARS2-N protein on transfecting RNA molecules [0113] In this example, two short RNAs (including an FAM™-labeled double-stranded siRNA having 20 nucleotides, and a single-stranded RNA fragment derived from the packaging sequence of SARS-CoV-2 and having 50 nucleotides), and three long RNA (>500 nucleotides; including a single-stranded RBD mRNA having 1,007 nucleotides, a single-stranded full-length spike mRNA having 4,111 nucleotides, and a luciferase-P2A-EGFP mRNA having 2,708 nucleotides) were used to test whether RNA binding to the SARS2-N protein would affect its surface binding. The results indicated that the short RNAs did not affect the binding of SARS2-N protein to cell surface, while long RNA-bound SARS2-N proteins dramatically increased the surface binding ability as compared to short RNA-bound SARS2-N proteins (Figs. 3A and 3B).

[0114] Whether the RNA molecules bound with SARS2-N protein would be delivered into cells was further examined. The SARS2-N protein was premixed with FAM™-labeled control siRNA (RNA-FAM) for 1 hour to form SARS2-N-RNA complex. The SARS2-N-RNA complex was then added to cells followed by detecting via confocal microscopy. The data demonstrated the presence of RNA-FAM signal in the cell, while there were no signals in the mock and RNA-FAM only groups (data not shown). To clearly detect the dynamics of SARS2-N-mediated RNA-FAM entry to the cells, lattice light sheet microscopy was used and time-lapse study was carried out to visually trace the biological process of SARS2-N-RNA entering. The results indicated that the RNA signal was initially widely dispersed, and as the time increased, the RNA-FAM signal transferred from outside to the inner part of cells (data not shown), that clearly demonstrated that the SARS2-N protein was capable of carrying and transferring RNA into the cells.

[0115] The efficiency of SARS2-N protein in transferring double-strand and single-strand RNAs into cells was also determined. The SARS2-N protein was mixed with a double-strand siRNA-FAM, or its positive or negative single-strand sequence, followed by analyzing with cell entering assay. The immunofluorescence results indicated that in comparison to the positive or negative siRNA sequence, the mixture of SARS2-N protein and double-strand siRNA have dramatically higher signals in cells (data not shown). This data demonstrated that the SARS2-N protein prefers carrying double-strand nucleic acid, even though it can bind to both single- and double-stranded RNA.

[0116] For the purpose of determining whether the RNA carried by the SARS2-N protein exhibits desired bio-activity in cells, siRNAs respectively specific to c-JUN and IFNAR1 genes were synthesized. After mixing with the SARS2-N protein, the SARS2-N-siRNA complex was added to cells. The knockdown efficacy of the siRNA on c-JUN and IFNAR1 gene expression was determined by qPCR. It was found that the expression levels of both c-JUN and IFNAR1 genes were significantly inhibited by SARS2-N-siRNA treatment as compared to that of control group (Fig. 3C). These results indicated that the RNA carried by the SARS2-N protein exhibited desired bio-activity in cells.

[0117] Example 4 Effect of SARS2-N protein on transfecting DNA molecules

[0118] In this example, the SARS2-N protein was mixed with a DNA plasmid (encoding GFP, i.e., GFP plasmid) at different ratio as described in Materials and Methods. The thus-formed SARS2-N-DNA complex was then added to cells. Strong GFP signals were detected in all testing groups, while the best ratio of the SARS2-N protein and GFP plasmid was about 1 :3 to 1 :4 (w/w) (Fig. 4). The mock and control group (DNA only) showed no expression signals as expected. The size distribution of the SARS2-N-DNA complex (SARS2-N: DNA= 1 :4) was determined by particle size analyzers (PSA). The analytic results showed a bimodal distribution, in which 30% of the complex had a particle size (diameter) ranging from about 180 nm to about 200 nm, and 70% of the complex had a particle size (diameter) ranging from about 1,200 nm to about l,400nm (data not shown), suggesting that there might be an optimal size of SARS2-N-DNA complex for DNA delivery.

[0119] The pretreatment of lactate (10, 25 or 50 mM) significantly enhanced the expression level of the GFP plasmid in cells, in which the numbers of GFP-positive cells administered with 0, 10, 25 and 50 mM lactate were 14.4±6, 21.2±6, 21.7±2 and 41.5±4, respectively. This result suggested that the DNA delivery effect might be amplified in vivo under hypoxic conditions where lactate is produced.

[0120] To further examine whether the SARS2-N protein would promote nucleic acid transfer in vivo, a trans-well assay was carried out, in which HEK293T and A549 cells were respectively seeded in the upper and bottom wells. The HEK293T cells were co-transfected with a SARS2-N expressing plasmid and a GFP expressing plasmid. Three days later, the GFP signals in A549 cells were detected by fluorescent microscopy. The data indicated that the GFP signals were higher in the SARS2-N and GFP co-transfected cells as compared to the control group (cells co-transfected with empty vector and GFP expressing plasmid) (data not shown). These data suggested that the SARS2-N protein secreted from the transfected cell would be able to bind and deliver intracellular DNA into neighboring cells.

[0121] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.