Angiotensin-converting enzyme 2 (ACE2) mutant with inactivated catalytic reaction and its applications

Abstract

Angiotensin-converting enzyme 2 (ACE2) has been identified as a specific receptor for severe acute respiratory syndrome coronavirus (SARS-CoV-1) and several beta-group coronaviruses, including SARS-CoV-2, the causative agent of the recent global pandemic COVID-19, and the low-pathogenic coronavirus HCoV-NL63, a member of the alpha-coronavirus group. The viral spike protein (S) of the viral envelope has been identified as a viral receptor that binds to ACE2 and initiates the viral replication cycle. The present invention provides ACE2 and its mutant or variant forms, as well as viral or non-viral vectors thereof. Methods for treating viral infections in human subjects using such mutant or variant forms are also provided.

Description

[Background technology]

[0001] Human angiotensin-converting enzyme 2 (ACE2) is expressed on the cell surface of various tissues, with the highest levels detected in digestive tissues such as the small intestine, large intestine, duodenum, gallbladder, myocardium, airways, and lungs, and lower levels detected in other tissues. ACE2 is a peptidase that catalyzes the removal of a residue (Phe8) from the C-terminal network of angiotensin II, converting it to angiotensin I-7 and maintaining the balance between angiotensin II and angiotensin I-7. It has diverse and complex physiological roles, centering on several types of functions, namely, it functions as a negative regulator of the renin-angiotensin system and a facilitator of amino acid transport.

[0002] Another biological role of ACE2 has been identified as a specific receptor for several β-group coronaviruses. These include the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV-1) (Hofmann et al, 2004, TRENDS in Microbiology, 12 (10), 2004; Jia, HP et al, 2005, J. Virol. 79(23), 14614-14621; Wang et al, 2008, Cell Research, 18:290-301) and the less pathogenic coronavirus HCoV-NL63, a member of the α-coronavirus group (Hofmann et al, 2005, PNAS, 102, 7988-7993). Very recently, human ACE2 was confirmed to be a specific receptor for SARS-CoV-2, the pathogen that caused the global COVID-19 pandemic (Wang et al., 2020, Cell, 181, 894-904; Zhao et al., 2020, Cell Host & Microbe, 28, 1-16). When the spike protein (S) of an enveloped virus binds to the viral receptor ACE2, the viral replication cycle begins, leading to host cell damage and viral transmission. SARS-CoV-2 has severely affected and killed millions of people worldwide. Controlling the binding of the virus to its receptor is a crucial strategy in ending the COVID-19 epidemic.

[0003] SARS-CoV 1 and 2 virions bind to the extracellular domain of the host cell receptor, ACE2, via the enveloped virus spike protein (S1). Resulting entry into the cytosol occurs via acid-dependent proteolytic cleavage of the S protein by cathepsins, TMPRRS2, or other proteases, followed by fusion of the virus with the cell membrane. Viral genomic RNA (gRNA) is released from the nucleocapsid. Replicase synthesis occurs using the gRNA template. This is a crucial step in the replication of replicases, which catalyze the synthesis of genomic and subgenomic RNA fragments. Subgenomic RNA (sgRNA) is used for the synthesis of structural proteins packaged with the gRNA template, which is replicated in the intermediate compartment using minus-strand RNA (-RNA). Following viral gRNA replication, structural proteins S, E, and M are translated and transported to the ER-Golgi intermediate compartment (ERGIC), where mature virions are formed. Release of the newly formed viral particle occurs after maturation is complete. Throughout the entire process, angiotensin-converting enzyme 2 (ACE2) plays a crucial role in the replication cycles of SARS-CoV-1, SARS-CoV-2, and HCoV-NL63, respectively. Circulating ACE-soluble receptors, wild-type or mutant, block the binding of SARS-CoV-1 and SARS-CoV-2 to their receptors on the host cell surface, whether fused or not. Therefore, viral infection and disease are prevented and treated. Furthermore, ACE2 is important in regulating the normal biological functions of many types of tissues and organs. It has been confirmed to be important for cardiovascular disease, enterotoxemia, inflammation, lung disease, diabetic cardiovascular complications, and kidney disease. More detailed information on ACE2 is described in the following review (Gheblawi et al, 2020, Circulation Research, 126: 1457-1475).

[0004] Several approaches have been taken to control COVID-19, including a. vaccine development using inactivated viral particles (inactivated vaccines), b. recombinant spike protein or messenger RNA (mRNA), c. recombinant viral receptor-binding domains (RBDs) of the viral spike protein, and d. recombinant human antibody cocktails. The challenge with these approaches is that they offer little to no protection when spontaneous mutations in the viral spike protein occur during spread, human-to-human transmission, human-to-animal transmission, or vice versa.

[0005] Since its discovery as a receptor for SARS-CoV, no mutations affecting viral binding have been detected, indicating that it is a stable and specific target for the manifestation and treatment of viral diseases. Initial efforts were directed towards using it as a viral decoy receptor for COVID-19. However, if ACE2 is administered directly to a target as a viral receptor blocker, other functions of ACE2 may also be introduced, potentially leading to unwanted activity related to the renin-angiotensin system (RAS). [Overview of the project]

[0006] The present invention provides an isolated extracellular domain (ECD) polypeptide of angiotensin-converting enzyme 2 (ACE2) that has one or more mutations resulting in the loss of ACE2 catalytic activity (referred to herein as ACE2-vECD) but retains binding activity to a viral spike protein, wherein the viral protein is the coronavirus spike protein. In some embodiments, the present invention provides the use of wild-type ACE2 (referred to herein as ACE2-ECD).

[0007] In one embodiment, the mutation that causes loss of ACE2 enzyme activity is located near the N-terminal region covering the amino acid sequence 361-410, where the region contains a catalytic center.

[0008] In one embodiment, the N-terminal catalytic center contains the motif HEXXH..….E, located at H374E375XXH378……E402. The catalytic center contains one or more mutations that terminate the catalytic activity of the enzyme. The mutations of the present invention continue to bind to viral proteins, including, but are not limited to, those derived from SARS-CoV 1, SARS-CoV 2, MERS-CoV-1, and HCoV-NL63.

[0009] This invention provides isolated extracellular domain polypeptides of angiotensin-converting enzyme 2 (ACE2) having one or more mutations that cause loss of ACE2 enzyme catalytic activity, where the loss of enzymatic activity is caused by loss of binding to a divalent metal ion. The divalent metal ion is Zn 2+ Co 2+ and Mn 2+ Selected from a group consisting of the following.

[0010] In one embodiment, the mutation is selected from a group consisting of positions H374, E375, H378, E402 and one or more combinations thereof. These amino acid residues constitute the catalytic center of ACE2. The mutation is a divalent metal ion, namely Zn 2+ Co 2+ and Mn 2+ This would result in the loss of ACE2 binding to the target. The loss of metal ion binding activity would render ACE2 an apoenzyme, and its catalytic activity would be lost.

[0011] In another embodiment, mutations in the R273, H345, H505, H515, and P346 amino acid residues in the N-terminal half of the extracellular domain of ACE2 also result in a loss of enzyme activity, but may retain the ability to bind to the coronavirus spike protein.

[0012] This invention provides an ACE-vECD mutation or variant that enhances the binding affinity of ACE-vECD to the viral S1 protein.

[0013] In one embodiment, the ACE2-ECD or ACE2-vECD mutant binds to the human IgG1 Fc region. Therefore, the ACE2-ECD and ACE2-vECD mutants become the ACE2-ECD-Fc or ACE2-vECD-Fc mutant. The present invention provides at least one extra-catalyzed mutation, along with mutations in the catalytic region. One or more mutations that result in loss of enzymatic activity while enhancing the binding affinity of the ACE2-vECD mutant / mutation to the S1 protein can occur by those skilled in the art. Examples of peptide sequences include, but are not limited to, those in Table 1.

[0014] In yet another embodiment, ACE2-ECD includes SEQ IDs: 1, 2, and 29, or ACE2-vECD includes polypeptides selected from the group consisting of SEQ IDs: SEQ IDs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28.

[0015] In one embodiment, the ACE2-vECD-Fc or its fusion protein binds to viruses whose native receptor is not ACE2. The viruses include, but are not limited to, SARS-CoV-1, SARS-CoV-2, MERS-CoV-1, and NL63.

[0016] The present invention provides a fusion protein comprising an isolated mutant ACE2 polypeptide, which can be further fused with a peptide, polynucleotide, or small molecule at the N or C terminus of the mutant polypeptide to form a fusion protein, where the peptide, polynucleotide, or small molecule can bind to receptors in immune system-related cells, such as lymphocytes and macrophages.

[0017] In another embodiment, such mutation sites are used for screening agonists or antagonists.

[0018] In one embodiment, the polypeptide is DNA or RNA.

[0019] In another embodiment, small molecules are screened against the catalytic domain or a mutant protein as a drug screening system.

[0020] In another embodiment, the peptide is a ligand that binds to the Fc binding receptor (FcγR) on immune cells such as lymphocytes. Lymphocytes are selected from the group consisting of T cells, B cells, and natural killer cells.

[0021] In one embodiment, the peptide is the Fc domain (Fcγ) of a human IgG antibody.

[0022] In another embodiment, ACE2 is a polypeptide having one or more mutations that cause loss of ACE2 enzyme activity, and at the same time retains the same or higher binding affinity for viral proteins compared to wild-type ACE2 or ACE2 present in the subject, where such a subject may be human. The mutations may be in the catalytic region of the ACE2 polypeptide or outside the catalytic region. The mutations can be two, three, four, or five mutations on the polypeptide.

[0023] The present invention provides an isolated polynucleotide encoding wild-type ACE2, ACE2-ECD, mutated ACE2, or ACE2-vECD.

[0024] In one embodiment, wild-type ACE2-ECD includes SEQ ID NO: 1, 2, 29.

[0025] In another embodiment, the ACE2-vECD mutant or mutant comprises a polypeptide selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and combinations thereof, or a combination with other selected amino acid mutants.

[0026] This invention provides a polynucleotide encoding a wild-type, mutant, or mutated fusion protein ACE, wherein ACE2-vECD is fused to Fc.

[0027] The present invention provides isolated polynucleotides encoding wild-type ACE2-ECD, including SEQ ID NOs: 1, 2, or 29.

[0028] The present invention provides isolated polynucleotides encoding mutated ACE2-vECD selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28.

[0029] The present invention further provides isolated wild-type ACE2-ECD polynucleotides, including SEQ ID NOs: 65 and 71.

[0030] The present invention further provides isolated mutant ACE2-vECD polynucleotides comprising SEQ ID NOs: 64, 66, 67, 68, 69, 70, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.

[0031] In one embodiment, the wild-type ACE2-ECD polynucleotide encodes a polypeptide containing SEQ ID NO: 1, 2, or 29.

[0032] In another embodiment, the mutant ACE2-vECD polynucleotide encodes a polypeptide containing SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

[0033] The present invention provides isolated angiotensin-converting enzyme 2 (ACE2) polypeptides having one or more mutations that cause loss of enzymatic activity of angiotensin-converting enzyme 2 (ACE2), wherein such ACE2 polypeptides retain the same or higher binding affinity to binding partners compared to wild-type ACE2.

[0034] In one embodiment, the enhanced binding affinity is caused by a mutation in the catalytic region of ACE2. In another embodiment, the mutation is located in a region outside the catalytic region.

[0035] In yet another embodiment, the mutation includes a combination of sites at K26, T27, L79, N330, H374, E375, H378, A386, A387, E402, G466, and L795 and two, three, four, five, six, seven, or more mutations.

[0036] In yet another embodiment, the mutations are selected from the group consisting of sites K26R, T27Y, L79S, N330F, H374A, E375Q, H378R, A386V, A387L, E402Q, G466D, L795H and combinations of 2, 3, 4, 5, 6, 7 or more mutations thereof.

[0037] The polypeptide maintains the same or higher binding affinity to its binding partner compared to wild-type ACE2. The polypeptide retains the same or higher binding affinity to its binding partner compared to wild-type ACE2, and the sequence above can further fuse with small molecules at the N or C terminus of the peptide, polynucleotide, or mutated polypeptide to form a fusion protein, which can then bind to receptors on immune system-related cells.

[0038] In yet another embodiment, the binding affinity of ACE-vECD mutants or mutants to MERS is higher than that of wild-type ACE2 or wild-type ACE2-ECD. This affinity enhancement can be 150%, 200%, 300%, 400%, 500%, 600%, or 700% higher than that of the wild-type.

[0039] In one embodiment, the introduction of the expression vector includes wild-type ACE2, ACE2-ECD, ACE2 mutant, ACE-vECD, or a fusion protein thereof.

[0040] In one embodiment, the vector is selected from either a viral vector or a non-viral vector. Viral vectors may include AAV, adenoviral, lentiviral, and HSV (viral vectors produced using insect and mammalian systems), and AAV vectors may be one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or a combination thereof. Non-viral vectors may include plasmids, nanoparticles, liposomes, PEI-derived particles, or colloidal gold particles.

[0041] The present invention also provides host cells containing an expression vector for mutant ACE2 or ACE2-vECD or a fusion protein thereof, as described herein.

[0042] In one embodiment, the host cell may be selected from a group consisting of prokaryotic cells or eukaryotes. Prokaryotic cells may be bacterial cells, and eukaryotic cells may be selected from a group consisting of mammalian and non-mammalian cell lines. Examples of cells of mammalian origin include CHO, NS0, and BHK-21.

[0043] In another embodiment, the present invention provides a composition comprising a polypeptide, a fusion protein, a vector / expression vector, or a host cell as described herein.

[0044] In one embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide, a fusion protein, a vector / expression vector or host cell as described herein, and a receptive pharmaceutical carrier.

[0045] In one embodiment, the pharmaceutical composition is administered nasally, orally, through the airway, through the ear, subcutaneously, intramuscularly, intravenously, or intrathecally.

[0046] The present invention also provides vaccine compositions comprising an ACE2 expression vector, wild-type or ACE2 mutant or ACE2-vECD or ACE2-vECD-Fc as a protein therapeutic agent or vector-mediated particle, virus or non-viral vector.

[0047] This invention provides a method for producing mutant polypeptides by synthesis or expression in host cells.

[0048] In one embodiment, the composition or pharmaceutical composition is used to treat a viral infection, including but not limited to alpha or beta coronavirus infections, SARS-CoV-1, SARS-CoV-2, MERS-CoV-1, and NL63.

[0049] In yet another embodiment, the present invention provides a method for preventing or prophylactically treating a viral infection by injecting a healthy subject with a pharmaceutical composition of wild-type ACE2, ACE2 mutant / ACE2-vECD, or a fusion protein thereof, as described herein.

[0050] The present invention provides a method for screening compositions, comprising: a) exposing a population of cells transfected with a mutated gene to various diagnostic reagents (in high-throughput screening, carried out over a period of time under conditions that allow the diagnostic reagent to affect ACE2 enzyme activity); and b) selecting a diagnostic reagent if it causes a statistically significant increase or decrease in ACE2 enzyme activity and binding affinity levels compared to pre-exposure levels. The diagnostic reagent may be either an ACE2 agonist or an antagonist.

[0051] In one embodiment, contact is performed in vitro or in vivo. [Brief explanation of the drawing]

[0052] Figure 1 is a schematic diagram showing the structure of human ACE2, which is a type I membrane-bound carboxypeptidase with 805 amino acids and contains one HEXXHE zinc-binding consensus sequence. Figure 2 is a schematic diagram of the biological activity of ACE2. Figure 3 shows the amino acid sequence of human angiotensin-converting enzyme 2. Figure 4 shows the ACE2-ECD-Fc (wild-type) sequence. Figures 5A and 5B show a summary of the amino acid composition (wild type) of the polypeptides of this disclosure. Figure 6 is a schematic diagram illustrating the loss of ACE2 enzyme activity caused by a mutation in the zinc ion binding site. Figure 7 is a schematic diagram showing how ACE2-vECD-Fc binds to virus particles. Figure 8 shows soluble ACE2-vECD-Fc bound to the virus particle. Figure 9 shows a Blast search for the ACE2 mutant extracellular domain (ACE2-vECD), with the completely substituted ECD used as the query sequence. Figure 10 shows the sequence of ACE2-vECD-Fc (with an ECD mutation). Figure 11 shows the structure of ACE2-vECD-Fc (AAV-ACE2-vECD-Fc) in a viral vector. Figure 12 shows the results of Western blots of ACE2-Fc mutants / mutants. Figure 13 shows affinity chromatography and SDS-PAGE assays of ACE2-Fc mutant preparations described in some embodiments of this disclosure. Figure 14 shows the assay results for a peptide according to this disclosure. Figures 15A and 15B show assay results for a peptide of this disclosure, demonstrating that the ACE2 activity of the ACE2-vECD-Fc mutant is completely depleted. Figure 16A shows the ELRLA assay results, demonstrating that the ACE2-Fc mutant binds to the S1 protein of β-coronavirus. Figure 16B shows a binding curve illustrating that the ACE2-Fc mutant binds to the SARS-CoV-2 B117 (N501Y)S1 protein receptor-binding domain (RBD), as detected by ELRLA. Figures 17A and 17B show binding curves illustrating that ACE2-Fc and vACE2-Fc bind to three S1 proteins, as detected by ELISA. Figure 18 shows the affinity analysis results of a peptide of this disclosure using BiaCore 3000. Figure 19 shows fluorescence micrographs and assays, which demonstrate the neutralization of GFP pseudoviral particles packaged with SARS-CoV-2 S1. Figure 20 demonstrates the neutralization of SARS-CoV-2 S1-filled GFP pseudoviral particles. Figure 21 shows the neutralization of SARS-CoV-2 wild-type virus (USA-WA1 / 2020) by a mutant of the ACE2-Fc fusion protein according to an embodiment of the present disclosure. Figure 22 shows a staining assay for a certain AAV vector. Figure 23 shows SDS-PAGE and Western blots of AAV5-ACE2-Fc and its variants from HEK293 cell culture supernatant. Detailed explanation

[0053] definition : Adeno-associated viruses (AAVs) are small, non-enveloped viruses with replication defects that infect humans and other primates. AAVs are not known to cause disease and only elicit a very mild immune response. Gene therapy vectors utilizing AAVs can infect both dividing and quiescent cells and can persist outside the chromosome without being integrated into the host cell's genome. These characteristics make AAVs an attractive viral vector for gene therapy. Currently, 11 serotypes of AAV are recognized (AAV1-11).

[0054] Administration / Administration: Providing or administering an active substance, such as a therapeutic agent (e.g., recombinant AAV), to a target via an effective route. Exemplary routes of administration include, but are not limited to, injection (subcutaneous, intramuscular, intradermal, and intravenous), oral, intratubal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0055] Binding affinity: The strength of the binding interaction between a single biomolecule (e.g., protein or DNA) and its ligand / binding partner. Binding affinity is typically measured and reported using the equilibrium dissociation constant (KD), which is used to evaluate and rank the strength of the interaction between two molecules. A smaller KD value indicates a higher binding affinity of the ligand to the target. A larger KD value indicates a weaker mutual attraction and binding between the target molecule and the ligand.

[0056] Binding region / binding center: This active site is the region of the enzyme where the substrate molecule binds and undergoes a chemical reaction. This active site consists of an amino acid residue that forms a temporary bond with the substrate (binding site) and a residue that catalyzes the reaction with the substrate (catalytic site).

[0057] Catalytic activity: An increase in the rate of a particular chemical reaction induced by an enzyme or other catalyst under specific assay conditions.

[0058] Catalytic region or catalytic center: Generally, this is a site on an enzyme that catalyzes the enzymatic conversion of its substrate into a product. This conversion is an enzymatic reaction. In ACE2, the catalytic center is formed by several amino acid residues and divalent ions, such as Zn2+, Co2+, and Mn2+.

[0059] As used herein, an effective dose means the amount that is effective for the duration and duration required to raise ACE2 levels.

[0060] Fc-binding receptors: Fc receptors are proteins found on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, eosinophils, basophils, human platelets, and mast cells, and they contribute to the protective function of the immune system.

[0061] Isolated: An “isolated” biological component (nucleic acid molecule, protein, virus, or cell) is one that has been substantially separated or purified from the cells or tissues of a living organism or other biological components of the organism itself, from which the component (other chromosomes and extrachromosomal DNA and RNA, proteins, and cells) naturally exists. “Isolated” nucleic acid molecules and proteins include those purified by standard purification methods. This term also includes nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules and proteins.

[0062] Modified: In the context of this disclosure, a “modified” ACE2 polynucleotide or polypeptide sequence that, when compared to a wild-type sequence (for example, when comparing an ACE2 mutant to ACE2 wild-type), includes at least one nucleic acid or amino acid substitution, deletion, or insertion.

[0063] Functionally linked: A first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is functionally related to the second nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence when the promoter influences the transcription or expression of the coding sequence. Generally, functionally linked DNA sequences are located close together at the site where the regions encoding two proteins need to be joined. Codon-optimized: A “codon-optimized” nucleic acid is a nucleic acid sequence that has been modified so that the codons are optimized for expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a particular mammalian species (e.g., human cells). Codon optimization does not change the amino acid sequence of the encoded protein.

[0064] Enhancer: A nucleic acid sequence that increases the transcription rate by increasing the activity of the promoter.

[0065] Reverse end repeats (ITRs): Symmetric nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs function as the starting point for viral DNA synthesis replication and are essential cis elements for AAV integration vector generation.

[0066] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (mediums) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) describes compositions and methods suitable for the pharmaceutically active substance delivery of one or more therapeutic compounds, molecules, or active substances.

[0067] To prevent, treat, or improve a disease (viral infection) means to prevent the disease from developing completely. To treat a disease or pathological condition means to improve the signs or symptoms of the disease or pathological condition after the onset of symptoms. To improve means to reduce the number or severity of the signs or symptoms of the disease.

[0068] Purified: The term "purified" does not require absolute purity. Rather, it is meant to be used as a relative term. For example, a purified peptide, protein, virus, or other active compound is one that has been isolated whole or partially from naturally occurring proteins and other contaminants. In some embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture media, or other crude preparations and subjected to fractionation to remove various components of the original preparation, such as proteins, cell fragments, and other components.

[0069] Recombinant: Recombinant nucleic acid molecules have sequences that do not exist in nature, or sequences that are artificially created by combining two normally separate segments within a sequence. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of isolated segments of nucleic acid molecules, e.g., genetic engineering techniques. Similarly, recombinant viruses are viruses that have sequences (such as genome sequences) that do not exist in nature or that are created by artificially combining at least two sequences of different origins. The term “recombinant” includes viruses, as well as nucleic acids, proteins, and viruses that have been modified solely by the addition, substitution, or deletion of parts of natural nucleic acid molecules, proteins, or viruses. As used herein, “recombinant AAV” means AAV particles in which a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule encoding mutant ACE2) is packaged.

[0070] Sequence Identity: The identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured by a percentage of identity. A higher percentage indicates greater sequence identity. Sequence similarity can be measured by a percentage of similarity (which takes into account conserved amino acid substitutions). A higher percentage indicates greater sequence similarity. Homologs or orthologues of nucleic acid sequences or amino acid sequences, when sequenced using standard methods, exhibit a relatively high degree of sequence identity / similarity. This homology is more pronounced when orthologue proteins or cDNAs originate from more closely related species (e.g., human and mouse sequences) compared to those from more distantly related species (e.g., human and C. elegans sequences).

[0071] Methods for arranging sequences for comparison are well known in this field. Various programs and sequencing algorithms are discussed in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31. This paper, published in 1994 by Altschul et al. in J. Mol. Biol. 215:403-10, presents a detailed discussion of sequence alignment methods and homology calculations.

[0072] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from various sources, including the National Center for Biotechnology Information (NCBI) and the internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found on the NCBI website.

[0073] Serotype: A group of closely related microorganisms (such as viruses) that are distinguished by a characteristic set of antigens. Subjects: Living multicellular vertebrates, including humans and non-human mammals. Synthetic: Created artificially in a laboratory; for example, synthetic nucleic acids are chemically synthesized in a laboratory.

[0074] Therapeutic or therapeutic: As used herein, means an approach to obtain a beneficial or desirable outcome, including clinical outcomes. Beneficial or desirable clinical outcomes include, but are not limited to, relief or improvement of one or more symptoms or conditions, reduction in the severity of the disease, stable (i.e., non-worsening) state of the disease, prevention of disease transmission, slowing or delaying disease progression, improvement or relief of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Therapeutic" also means an extension of survival compared to the expected survival without treatment.

[0075] Vaccine: A vaccine is a composition that provides protection against pathogenic infections (e.g., protozoan, viral, or bacterial infections), cancer, or other diseases, or treatment for pathogenic infections, cancer, or other diseases. Protection against pathogenic infections, cancer, or other diseases means completely preventing an infection or tumor or other disease, or, if the infection, tumor, or other disease develops later, reducing the severity or duration of the infection, tumor, or other disease. Treatment means improving one or more symptoms, or reducing their severity or duration. For the purposes of this specification, a vaccine is the result of an intravenous infusion (combined, sequential, or simultaneous) of an infusion agent of antigens and synthetics prepared in the manner of this specification. As used herein, improvement of symptoms of a particular disease by administration of a particular composition means relief by or associated with the administration of the synthetics described herein, whether permanent or temporary, persistent or transient.

[0076] Immunization therapy is a treatment plan in which a vaccine comprising the antigens and / or gene therapy vectors described herein (alone or in combination) is administered to the subject as an adjuvant. The administration is carried out in combination, simultaneously, separately, in the form of combined formulations, or sequentially, at different times separated by minutes, hours, or days. However, in some way, they work together to induce an enhanced immune response in the subject to the vaccine (compared to the subject's immune response without the compositions according to the present invention).

[0077] Vectors: A vector is a nucleic acid molecule that allows for the insertion of foreign nucleic acids without disrupting the vector's ability to replicate and / or be incorporated into a host cell. A vector may contain nucleic acid sequences, origins of replication, etc., that enable replication in a host cell. A vector may also contain one or more selectable labeling genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences that enable the transcription and translation of one or more inserted genes. In some embodiments herein, the vector is an AAV vector.

[0078] ACE2 polypeptide, its mutations, and fusion proteins ACE2 is a type I membrane-bound carboxypeptidase with a read sequence containing 805 amino acid residues. Its mature protein has 788 amino acid residues, including a 725-amino acid extracellular domain, a short 21-amino acid transmembrane domain, and a 44-amino acid intracellular domain. Within the extracellular domain, the "HE-XX-HE" metal ion-binding consensus sequence, motif H374E375XXH378….E402, has been identified as a catalytically essential sequence (Figure 1). Examples of specific sequences are shown in Table 1.

[0079] [Table 1]

[0080] The objective of this invention is to induce mutations (substitutions) in at least one amino acid residue H, E, in the H374E375XXH378….E402 metal ion binding motif, which is the only metal ion binding motif in the ACE2 extracellular domain and ACE2 vECD.

[0081] Mutations in the metal ion-binding motif completely depleted ACE2 endopeptidase activity, but retained coronavirus binding specificity and affinity compared to the wild type. The complete sequence of the N-terminal human ACE2 extracellular domain consists of 725 amino acid residues (18-742 aa) (Figure 3, Table 2), and is expected to have a molecular weight of 83596 Da and a decay coefficient of 16140 M-1CM-1. The estimated pI is 5.26. The human "ACE2" as used herein is a glycoprotein, and its molecular weight varies to some extent depending on the glycosylation state. The extracellular domain (ECD) of the human ACE2 amino acid sequence is shown in Sequence ID: 1.

[0082] In another embodiment, the ACE2 polypeptide mutations include sites in K26, T27, L79, N330, H374, E375, H378, A386, A387, E402, G466, L795 and combinations of 2, 3, 4, 5, 6, 7 or more mutations. The mutations are selected from sites K26R, T27Y, L79S, N330F, H374A, E375Q, H378R, A386V, A387L, E402Q, G466D, L795H and combinations of 2, 3, 4, 5, 6, 7 or more of these.

[0083] [Table 2]

[0084] The ACE2 molecule contains 1 gram atom of zinc per mole of protein. Zinc ions are cofactors of this enzyme and are essential for the catalytic activity of ACE and ACE2. ACE2 is an important member of the renin-angiotensin system, which is crucial for regulating cardiac function and blood pressure homeostasis. Chelating agents such as EDTA completely inactivate this enzyme by removing zinc ions from the catalytic center and forming a zinc-free apoenzyme. Adding Zn2+, Co2+, or Mn2+ to a metal-free apoenzyme solution restores metalloenzyme activity. Metalloenzyme activity is Zn2+ > C2+ or > Mn2+. However, adding metal ions - Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ does not restore activity. This protein binds more strongly to Zn2+ than to Co2+ or Mn2+.

[0085] Human ACE2 is expected to have six N-linked glycosylation sites, which are asparagine (N) residues at sites N53, N90, N104, N332, N432, and N546. In mammalian and human cells, the carbohydrates of membrane proteins are sialylated. At least one of these sialic acid moieties of the glycated asparagine residues contributes to coronavirus binding. The native substrate of ACE2 is angiotensin II, a short peptide molecule. Crystal structure studies indicate that these residues are not involved in the catalytic reaction that converts angiotensin II to angiotensin 1-7 (Wang, QH et all, 2020).

[0086] Human angiotensin I (Ang I) is a short peptide consisting of 10 amino acid residues, represented as H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH (or DRVYIHPFHL in single-letter abbreviation). Ang I is cleaved into Ang II by angiotensin-converting enzyme (ACE), or converted to Ang II by non-angiotensin-converting enzyme-dependent conversion. Human chymase effectively converts 10-mer Ang I to the 8-mer hormone Ang II by cleaving the Phe8-His9 bond in Ang I, producing H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH (or DRVYIHPF). Ang II is further cleaved by carboxypeptidase (exopeptidase), removing the C-terminal Phe(F) and becoming Ang I-7. The biochemical reactions and biological functions of AngI, AngII, and Ang1-7 are summarized in Figure 2. Under normal conditions, ACE2 activity is well balanced through physiological and biochemical regulation. Changes in this balance lead to pathological conditions. In SARS-CoV-2 or SARS-CoV-1 infections, ACE2 molecules on the host cell surface are depleted by viral particles, resulting in ACE2 depletion.

[0087] While using wild-type ACE2 decoy receptors for the treatment of SARS-CoV-2 infection, administering high levels of ACE2 protein preparations may lead to a significant increase in the enzyme that catalyzes the conversion of AngII to Ang1-7, potentially causing AngII depletion. A significant decrease in AngII could lead to side effects. In certain embodiments, the mutant ACE2 sequence includes the sequence summarized in Table 3:

[0088] [Table 3]

[0089] Due to the properties of the ACE2 catalytic center, at least one mutation, substitution, deletion, or alanine substitution can result in complete or dramatic depletion of its activity. This is the fundamental theory within the present invention, which utilizes the full length of the enzyme molecule without its catalytic activity. Such use may, but is not limited to, binding to viruses, namely SARS-CoV-1, SARS-CoV-2, MERS-CoV, or emerging coronaviruses.

[0090] In addition to the metal ion binding motif, several important amino acid residues contribute to enzyme activity. The key ACE2 residue, arginine 273 (R273), contributes to substrate recognition via salt bridges and hydrogen bonds. Removal of Arg273 resulted in loss of enzyme activity. The ACE2 residue histidine 345 (H345) stabilizes the substrate-enzyme intermediate, and histidine 505 also contributes significantly. Removal of histidine 505 resulted in a 300-fold decrease in enzyme activity. Other residues, such as proline 346 (P346) and histidine 515 (H515), are also important for enzyme activity. Based on the above description of mutations in the metal ion binding motif residues, mutations in one or more of the R273, H345, P346, H505, and H515 residues of the ACE2 molecule can cause complete loss of enzyme activity. (Nicola E. Clarke et al, Handbook of Proteolytical Enzymes, chapter 100, pp499-504, 3rd eds, 2013). For this reason, it is reasonable to assume that a mutation in the arginine 273 (R273) residue of ACE2 would also cause a significant decrease or depletion of enzyme activity. This is because it contributes to the positive charge of R for salt bridges to the substrate-enzyme intermediate.

[0091] ACE2 variants or mutants may be used in the methods of the present invention. Changes that result in the generation of chemically equivalent or chemically similar amino acid sequences are within the scope of the present invention. Polypeptides having sequence identity with the catalytic region of ACE2 have been examined and confirmed to be suitable for use in the methods of the present invention. Variants of the polypeptides of the present invention may exist in nature, for example, through mutation, or can be produced by polypeptide engineering techniques such as site-directed mutagenesis. This is well known in the art with respect to amino acid substitutions. For example, hydrophobic residues such as glycine can be substituted with other hydrophobic residues such as alanine. Alanine residues can be substituted with more hydrophobic residues such as leucine, valine, or isoleucine. Negatively charged amino acids such as aspartic acid can be substituted with glutamic acid. Positively charged amino acids such as lysine can be substituted with other positively charged amino acids such as arginine.

[0092] Accordingly, the present invention includes polypeptides having conservative changes or substitutions in their amino acid sequences. In a conservative substitution, one or more amino acids are inserted, and these amino acids have chemical properties similar to those of the substituted amino acids. The present invention includes sequences in which conservative substitutions are made that do not destroy the activity of the compound.

[0093] Polypeptides containing one or more d-amino acids are of great interest in this invention. Polypeptides in which one or more amino acids are acetylated at the N-terminus are also of great interest. Those familiar with this technique will recognize that various techniques are available for constructing polypeptide mimes that have the same or similar compound activity as the corresponding polypeptide compounds of this invention, but with more favorable activity in terms of solubility, stability, and / or sensitivity to hydrolysis and proteolysis. See, for example, Morgan and Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989). Examples of polypeptide mimes are described in U.S. Patent No. 5,643,873. Other patents describing the preparation and use of mimics include, for example, 5,786,322, 5,767,075, 5,763,571, 5,753,226, 5,683,983, 5,677,280, 5,672,584, 5,668,110, 5,654,276, and 5,643,873. Mimics of the polypeptides of the present invention may also be prepared according to other techniques known in the art, for example, by treating the polypeptides of the present invention with reagents that chemically alter the side groups by converting hydrogen groups to other groups such as hydroxyl or amino groups. It is desirable that the mimics consist of either a sequence consisting entirely of amino acids or a sequence that is a synthesis containing amino acids and modified amino acids or other organic molecules.

[0094] The present invention also includes synthetics and polypeptides in which, for example, a nucleotide sequence is combined with a second sequence.

[0095] The present invention also includes a method of using polypeptide fragments of ACE2, which may be used to confer activity to a compound if they retain activity.

[0096] The present invention also comprises polypeptides and polypeptide fragments, which can be used as research tools for characterizing polypeptides or their activity. Such polypeptides are preferably composed of at least five amino acids. In preferred embodiments, they may consist of 6 to 10, 11 to 15, 16 to 25, 26 to 50, 51 to 75, 76 to 100, or 101 to 250, or 250 to 500 amino acids. Fragments may include sequences from which one or more amino acids have been removed, for example, sequences from which the C-terminal amino acid of a compound sequence has been removed.

[0097] In particular, the ACE2 polypeptide has enhanced binding affinity at the mutated catalytic site. In one embodiment, the binding affinity to the ACE2-vECD mutant or mutant MERS is higher than that of wild-type ACE2 or wild-type ACE2-ECD. This affinity is 150%, 200%, 300%, 400%, 500%, 600%, or 700% higher than that of wild-type ACE2 or wild-type ACE2-ECD.

[0098] In another embodiment, the ACE2 polypeptide maintains the same or higher binding affinity to its binding partner compared to wild-type ACE2. In another embodiment, the increase / enhancement of binding affinity occurs through mutations outside the catalytic region. Several examples of mutations are shown in Table 3.

[0099] ACE2 polypeptide and its fusion The present invention provides a fusion protein (ACE-vECD) of a 723-amino acid extracellular domain fused to a human IgG (e.g., IgG1 Fc or IgG4) Fc domain via N-terminal or C-terminal fusion in the form of ACE2-vECD-Fc after a mutation process (Figure 4). When the ACE2 portion of the ACE2-Fc molecule binds to its specific virus, the Fc portion exerts biological functions such as complement activation, and Fc receptor-positive cells attack the complex. The ACE2 ECD-Fc and ACE2 vECD-Fc mutants are designed so that the ACE extracellular domain is fused to either the N-terminus or C-terminus of the human IgG1 Fc fragment. An example of the analysis of the wild-type ACE2-derived Fc fusion protein molecule, ACE2-ECD-Fc, is shown below (Figure 4, Figure 5A, 5B, Table 4):

[0100] [Table 4]

[0101] Mutant ACE2-vECD or ACE-vECD-Fc can include modifications at additional residues, as long as the protein retains enzymatic SARS-CoV binding activity while losing divalent metal ion binding activity (Figure 6). For example, mutant ACE2 may include substitutions at other residues in the HEXXE region, including positions H374, E375, H378, and E402 in ACE2 ECD. Once ACE2 loses its catalytic function, it becomes a coronavirus conjugate, blocking viral infection and its transmission (Figures 7 and 8). (Listed as wild-type ACE2 ECD under Sequence ID No. 1).

[0102] The present invention provides nucleic acid molecules encoding various ACE2 mutants or ACE2-vECDs (Figures 9 and 10). In some embodiments, the nucleic acid molecule encodes a fusion protein having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to wild-type ACE2, mutant ACE2 / ACE2-vECD mutant, or SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0103] In certain embodiments, the polypeptide of ACE2-vECD consists of or includes SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0104] In certain embodiments, the polypeptide of wild-type ACE2-vECD consists of or includes SEQ ID NOs: 1, 2, or 29.

[0105] In certain embodiments, the polypeptide of wild-type ACE2-vECD consists of or includes SEQ ID NOs: 65 or 71.

[0106] In a non-limiting embodiment, the isolated polynucleotide consists of or includes any nucleotide sequence of SEQ ID NOs: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.

[0107] In another embodiment, the isolated polynucleotide consists of or includes any nucleic acid sequence encoding SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0108] Also provided herein are vectors comprising isolated nucleic acid molecules encoding mutant ACE2 amino acid sequences. In some embodiments, the nucleic acid molecules encoding mutant ACE2 / ACE2-vECD are operably ligated to a promoter that drives ACE2 or ACE protein expression. In some embodiments, the ACE2 polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide encoding SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.

[0109] Vector and Manufacturing : Wild-type ACE2, ACE2-vECD, or ACE2-vECD-Fc are expressed, produced, purified, and homogenized using mammalian cell culture systems such as Chinese hamster ovary (CHO) and baby muster kidney (BHK) cells, and administered as a preventive measure and / or emergency treatment for coronavirus infectious diseases such as SARS, MERS, and COVID-19 or variants. Basic cloning and molecular biological methods are known in the art and can be found in the references (Green, MR et al, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press Bookstore A Division of CSHL).

[0110] Wild-type ACE2, ACE2-vECD, or ACE2-vECD-Fc are further vectorized for introduction into the human body with the intention of long-term expression of the gene of interest. The vector design and manufacturing process is briefly described in Figure 11. Vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, human formyvirus (HFV), and lentiviruses. All viral vector genomes are modified by deleting a portion of their genome to disrupt replication and enhance safety. However, this system has several problems, such as significant immunogenicity (which induces inflammation and causes degeneration of transduced tissues), toxin production (including death and mutagenesis by insertion), and limitations in the magnitude of its recombination capacity. In recent years, several viral vectors with specific receptors have been designed (retargeted) that can deliver the transgene to several other specific cells (not the natural target cells).

[0111] Nonviral systems encompass all physical and chemical systems other than viral systems and generally include either chemical methods such as cationic liposomes and polymers, or physical methods such as gene guns, electroporation, particle bombardment, ultrasound, and magnetofection. More importantly, these methods are less likely to induce an immune system compared to viral systems, have no limitations on the size of the recombinant DNA, and are therefore more effective for gene delivery than nonviral delivery systems to date.

[0112] DNA fragments encoding wild-type ACE2, ACE-vECD, or ACE2-vECD-Fc are also cloned into a gene transfer system using the viral vectors described herein or non-viral vectors. Polynucleotides encoding ACE2 or its mutants / mutants can be cloned in vectors for polypeptide expression for manufacturing purposes. Such vectors can also be used for gene therapy purposes. When using AAV vectors, the vectors may contain reverse terminal repeats (ITRs). In some embodiments, the AAV vector contains a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to nucleotide AAV.

[0113] In some embodiments, the vector contains a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the AAV vector nucleotide.

[0114] In some embodiments, the vector is an AAV vector. The AAV serotype may be any serotype suitable for introducing the transgene into the target. In some embodiments, the AAV vector is serotype 8 AAV (AAV8). In other embodiments, the AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, or 12 vector (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, or AAV12). In yet another embodiment, the AAV vector is a compound of two or more AAV serotypes (e.g., AAV2 / 1, AAV2 / 7, AAV2 / 8, or AAV2 / 9, but not limited to these). The selection of the AAV serotype depends, in part, on the type of cells being targeted by the gene therapy.

[0115] This invention provides a vector for transfecting or infecting host cells for expression. Such host cells can produce polypeptides. Alternatively, such host cells can be used for cell therapy purposes.

[0116] The present invention provides isolated host cells containing nucleic acid molecules or vectors disclosed herein. For example, the isolated host cell may be a cell (or cell line) suitable for the production of recombinant AAV (rAAV). In some examples, the host cell is a mammalian cell such as CHO, HeLa, HEK-293, BHK, Vero, RD, HT-1080, A549, Cos-7, ARPE-19, or MRC-5 cell.

[0117] The viral vector carrying ACE2-vECD-Fc can be prepared in any eukaryotic cell culture system, including mammalian cells, insect cells, and yeast cells. This method also relates to a method for creating a stockpile of recombinant viruses by producing viruses suitable for gene therapy that contain DNA encoding ACE2. This method preferably involves transfecting cells that are tolerant to viral replication (viruses containing nucleic acid molecules) and collecting the produced viruses.

[0118] The present invention also includes transformed cells containing a vector and recombinant ACE2 or ACE2-vECD nucleic acid molecular sequence.

[0119] Treatment and Immunity This invention provides the use of ACE2 vECD as a viral receptor inhibitor, wherein this molecule does not exhibit ACE2 enzyme activity. This approach prevents undesirable biological outcomes from being caused by the administration of ACE2 therapeutic agents or by the implementation of vector-mediated therapy, such as recombinant protein, DNA, or mRNA. In one embodiment, wild-type ACE2 is also used herein.

[0120] In one embodiment, when a virus enters the body and encounters the soluble form of the ACE vECD protein, the virus receptor interaction inhibitor competes with the binding of the virus to the host cell surface ACE2 molecule (viral receptor), preventing the host ACE2 from binding to the virus, thereby terminating the viral replication process. The binding of soluble cell surface ACE2 is preserved, and the normal biological function of the cell is maintained.

[0121] Also provided are recombinant AAVs (rAAVs) comprising nucleic acid molecules, as disclosed herein. In some embodiments, the rAAV is rAAV5. However, the AAV serotype may be any other suitable AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, or AAV12, or a synthesis of two or more AAV serotypes (such as, but not limited to, AAV2 / 1, AAV2 / 7, AAV2 / 8, or AAV2 / 9).

[0122] Compositions comprising rAAV and pharmaceutically acceptable carriers as disclosed herein are also provided herein. In some embodiments, the compositions are prepared for intravenous or intramuscular administration. Suitable drug formulations for the administration of rAAV can be found, for example, in U.S. Patent Publication No. 2012 / 0219528 (incorporated herein by reference). As described herein, ACE2-vECD or ACE2-vECD-Fc are soluble receptors for coronaviruses.

[0123] The invention known as ACE2-vECD or ACE2-vECD-Fc fusion protein specifically binds to the spike proteins of (SARS) coronavirus (SARS-CoV-1), Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), and the current global pandemic COVID-19, SARS-CoV-2, and HCoV-NL63.

[0124] ACE2 or ACE2-vECD polypeptides are used for cardiovascular disease, hypertension, myocardial infarction (MI), fibrosis, inflammation, and many other conditions. The present invention can be used for therapeutic purposes and can be administered for the treatment of infections caused by any of these emerging coronaviruses and other related viruses. The bound viruses can be eliminated by Fc receptor-positive immune cells.

[0125] Also provided is a method for treating subjects diagnosed with a viral infection, comprising selecting subjects with such infection and administering a therapeutically effective amount of rAAV (or a composition comprising rAAV) as disclosed herein to the subjects.

[0126] The present invention, AAV-ACE2-vECD or AAV-ACE2-vECD-Fc, allows for the transduction of non-immune cells according to the serotype of the AAV vector used, such as AAV5, and can transduce hepatocytes, muscle cells, and epithelial cells. This is of great importance to individuals with a weakened immune response, as it makes it possible to build immunity with non-immune cells transduced by vectors such as the AAVx-ACE2-vECD-Fc vector.

[0127] In one embodiment, such vector compositions can sustainably express the ACE2-vECD-Fc fusion protein for several years, thus providing long-lasting protection against viral infections.

[0128] Methods for prophylactic or prophylactic treatment of healthy subjects by using compositions having rAAV / ACE2-vECD, ACE2, and ACE-vECD are also provided in this disclosure. In some embodiments, the method involves administering a therapeutically effective amount of rAAV (or a composition comprising rAAV) disclosed herein to a subject. In some embodiments, the subject has a viral infection. Such infections may be SARS-CoV-1, SARC-CoV-2, MERS-CoV-1, or HCoV-NL63. Thus, in some embodiments, the method involves selecting subjects having different viral infections.

[0129] Mutations in the zinc ion-binding motif completely eliminate the enzymatic activity of ACE2, and its protein molecules, ACE2-ECD-Fc, ACE2-vECD-Fc, and vectors (viral or nonviral) carrying these types of DNA fragments, along with their protein products, function only as neutralizing antibodies and lack enzymatic function. Therefore, it is safe to use these products.

[0130] Furthermore, changes in several relevant amino acid residues near the N-terminus or zinc-binding motif significantly enhanced the binding of SARS-CoV-1, SARS-CoV-2, and MERS-CoV S1 proteins to the ACE2 receptor. Methods and compositions for administering ACE2 (including in gene therapy) to isolated cells or animals are described, for example, in U.S. Patent Nos. 5,672,344, 5,645,829, 5,741,486, 5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346, 5,670,488, 5,240,84, 6,322,536, 6,306,830 and 6,071,890 and U.S. Patent Publication No. 20010029040, which are incorporated herein by reference in their entirety.

[0131] The methods and compositions can be used in vivo or in vitro. The present invention also includes compositions (preferably pharmaceutical compositions for gene therapy). The compositions include vectors containing ACE2. The carrier may be a pharmaceutical carrier or a transformed host cell containing the vector. Vectors known in the art include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAVs), herpesvirus vectors such as vaccinia virus vectors, HIV and lentivirus-based vectors or plasmids. The present invention also includes helper cell lines necessary for packaging and vector preparation. Methods for vector preparation and methods for gene therapy using vectors are also included in the present invention.

[0132] Generally, the properties of the carrier depend on the specific mode of administration used. For example, parenteral formulations typically consist of an injectable liquid containing a pharmaceutically and physiologically acceptable liquid as the carrier, such as water, saline, equilibrium salt solution, glucose solution, or glycerol. Regarding solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifiers, and preservatives, as well as pH buffers such as sodium acetate or sorbitan laurate monoester.

[0133] Immunogenic or immunological compositions may also be prepared in the form of oil-in-water emulsions. Oil-in-water emulsions are based on, for example, light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oils such as squalane, squalene, eicosane™, or tetratetracontane; oils resulting from the oligomerization of alkenes, such as isobutene or decene; esters of acids or alcohols containing linear alkyl groups, such as vegetable oils, ethyl oleate, di(caprylic / capric acid) propylene glycol, tri(caprylic / capric acid) glyceryl, or propylene glycol dioleate; and esters of branched fatty acids or alcohols, such as isostearates. Oils are used in combination with emulsifiers to advantageously form emulsions. The emulsifier may be a nonionic surfactant, such as sorbitan, mannides (e.g., mannitol anhydrous oleic acid), glycerol, polyglycerol, propylene glycol, sucrose, trehalose, and oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid (these may be ethoxylated) and Pluronic.RTM. products, such as polyoxypropylene-polyoxypropylene copolymer blocks like L121. The adjuvant may be a mixture of emulsifiers, micellars, and oils, such as oils commercially available under the name Provax.RTM. (IDEC Pharmaceuticals, San Diego, Calif.).

[0134] The immunogenic compositions of the present invention may include additional substances such as wetting agents or emulsifiers, buffers, or adjuvants that enhance the efficacy of vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).

[0135] Adjuvants may also be included. Adjuvants include, but are not limited to, mineral salts (e.g., AlK(SO4)2, AlNa(SO4)2, AlNH(SO4)2, silica, alum, Al(OH)3, Ca3(PO4)2 kaolin or carbon), polynucleotides with or without immunostimulatory complexes (ISCOMs) (e.g., CpG oligonucleotides, e.g., those described in Chuang, TH et al, (2002) J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al (2002) Eur. J. Immunol. 32(7): 1958-68); polyIC or polyAU acids, polyarginines with or without CpG (also known in this work as IC31; Schellack, C. et al (2003) Proceedings of the 34th Annual Meeting of the German Society of Immunology; see Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508), JuvaVax.TM. (US Patent No. 6,693,086), certain natural substances (e.g., wax D from Mycobacterium tuberculosis, substances present in Cornyebacterium parvum, Bordetella pertussis, or substances present in the membranes of the genus Brucella), flagellin (Tolle-like receptor 5 ligand; see McSorley, SJ et al (2002) J. Immunol. 169(7): 3914-9), saponin 7 such as QS21, QS17, and QS7 (US Patent Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A, especially 3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also known in the industry as IQM and marketed as Aldara.RTM; U.S. Patent Nos. 4,689,338; 5,238,944; Zuber, AK et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (Veazey, RSet al (2003) J. Exp. Med. 198: 1551-1562). .

[0136] Aluminum hydroxide or aluminum phosphate (alum) is commonly used as a 0.05-0.1% solution when dissolved in phosphate-buffered saline. Other adjuvants that may be used in conjunction with DNA vaccines include cholera toxin, particularly CTA1-DD / ISCOMs (see Mowat, AM et al (2001) J. Immunol. 167(6): 3398-405), polyphosphazenes (Allcock, HR (1998) App. Organometallic Chem. 12(10-11): 659-666; Payne, LG et al (1995) Pharm. Biotechnol. 6: 473-93), and cytokines (e.g., IL-2, IL-4, GM-CSF, IL-12, IL-15, IGF-1, IFN-alpha, IFN-beta, and IFN-gamma, but not limited to these) (Boyer et al., (2002) J. Liposome Res. 121:137-142; These include immunomodulatory proteins, such as CD40L (ADX40; see, e.g., WO03 / 063899), and CD1a ligands for natural killer cells (also known as CRONY or alpha-galactosyl ceramide; see Green, TD et al, (2003) J. Virol. 77(3): 2046-2055), immunostimulatory fusion proteins, such as IL-2 fused with an immunoglobulin Fc fragment (Barouch et al., Science 290:486-492, 2000), and co-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can be administered as proteins on the same expression vector as the vector encoding the antigen of the present invention, or on a different expression vector, or in the form of DNA.

[0137] Immunogenic compositions can be designed to introduce nucleic acids or expression vectors to the desired site of action and release them at an appropriate, controllable rate. Methods for preparing controlled-release formulations are well known in the art. For example, controlled-release formulations can be manufactured by using polymers that complex with or absorb immunogens and / or immunogenic compositions. Controlled-release formulations can be prepared using suitable polymers (e.g., polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled-release properties or release profile. Another possible method for controlling the duration of action with controlled-release formulations is to incorporate the active ingredient into particles of polymeric material, such as polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these active ingredients into polymer particles, these materials can be encapsulated in microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules prepared by droplet formation technology or interfacial polymerization. Another example is the encapsulation of these materials in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in PEI derivatives or macroemulsions.These technologies are disclosed in *New Trends and Developments in Vaccines*, Voller et al. (eds.), University Park Press, Baltimore, Md., 1978 and *Remington's Pharmaceutical Sciences*, 16th edition.

[0138] The appropriate dose (collectively, immunogen) of the nucleic acid and expression vector of the present invention in the immunogenic composition of the present invention can be easily determined by a person familiar with this technique. For example, the dose of immunogen may vary depending on the route of administration and the size of the target. The appropriate dose can be determined by a person familiar with this technique by measuring the immune response of a target, such as an experimental animal, using conventional immunological techniques and adjusting the dose accordingly. Techniques for measuring the immune response of such a target include, but are not limited to, chromium release assays, tetramer binding assays, IFN-γ ELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, and other immunological detection assays (for example, detailed in the textbook "Antibodies: A Laboratory Manual" by Ed Harlow and David Lane).

[0139] In some embodiments, rAAV is administered at doses of approximately 1 x 10⁶ to approximately 1 x 10¹⁵ vector genome (vgvg) / kg. In some embodiments, rAAV is administered at doses of approximately 1 x 10¹¹ to approximately 8 x 10¹³ vg / kg or approximately 1 x 10¹² to approximately 8 x 10¹³ vg / kg. In other embodiments, rAAV is administered at doses of approximately 1 x 10¹³ to approximately 6 x 10¹³ vg / kg. In specific non-limiting embodiments, rAAV is administered at doses of at least approximately 1 x 10¹⁰, at least approximately 5 x 10¹⁰, at least approximately 1 x 10¹¹, at least approximately 5 x 10¹¹, at least approximately 1 x 10¹², at least approximately 5 x 10¹², at least approximately 1 x 10¹³, at least approximately 5 x 10¹³, or at least approximately 1 x 10¹⁴ vg / kg. In other non-limiting cases, rAAV is administered at doses of approximately 1 x 10¹⁰ or less, approximately 5 x 10¹⁰ or less, approximately 1 x 10¹¹ or less, approximately 5 x 10¹¹ or less, approximately 1 x 10¹² or less, approximately 5 x 10¹² or less, 1 x 10¹³ or less, 5 x 10¹³ or less, and approximately 1 x 10¹⁴ vg / kg. In one non-limiting case, rAAV is administered at a dose of approximately 1 x 10¹² vg / kg. In another non-limiting case, rAAV is administered at a dose of approximately 1 x 10¹¹ vg / kg. rAAV can be administered as a single dose or in multiple doses (2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed for the desired therapeutic outcome.

[0140] Immunological compositions can be administered using appropriate delivery methods, including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. These techniques are well known to those familiar with the art. More specific delivery methods include intramuscular injection, inhalation, spray, oral ingestion, intradermal injection, intravenous, intraperitoneal (IP), and subcutaneous injection. However, delivery is not limited to injection. Furthermore, DNA delivery to animal tissues is carried out using cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev. 38:268-274; and WO 96 / 20013), and direct injection of naked DNA into animal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960; Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994) Virology 199: 132-140; Webster et al., (1994) Vaccine 12: 1495-1498; Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et al., (1993) Hum. Mol. Gen. 2: This has been achieved by injecting DNA intradermally using the 1847–1851 (1847–1851) or “gene gun” technology (Johnston et al., (1994) Meth. Cell Biol. 43:353–365). Alternatively, the delivery route may be oral, nasal, or other suitable route. Delivery may also be achieved via mucosal surfaces such as the anus, vagina, or oral mucosa. In some embodiments of the methods disclosed herein, AAV is administered via the oral cavity, nasal cavity, ear, subcutaneous, intramuscular, intravenous, or intrathecal cavity.

[0141] Immunotherapy schemes (or regimens) are well known in animals (including humans) and can be readily determined with respect to specific subjects and immunogenic compositions. Therefore, immunogens may be administered to a subject one or more times. When immunogenic compositions are administered at intervals, it is desirable to set fixed time intervals. These intervals vary for all subjects and are typically in the range of 10 days to several weeks, often 2, 4, 6, or 8 weeks. In humans, the interval is typically 2 to 6 weeks. In particularly advantageous embodiments of the present invention, the interval is longer, preferably about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 weeks. In the most advantageous embodiment, the interval is approximately 16 weeks or approximately 53 weeks.

[0142] Immunotherapy regimens typically involve 1–6 administrations of the immunogenic composition, but may also involve only 1, 2, or 4 administrations. Methods for inducing an immune response may also include the administration of an adjuvant along with the immunogen. In some cases, additional immunizations are administered annually, every two years, or at other longer intervals (5–10 years) to supplement the initial immunization protocol.

[0143] Current methods also include various prime-boost regimens, such as the DNA prime-adenovirus boost regimen. These methods involve one or more priming immunizations followed by one or more booster immunizations. The actual immunogenic composition may be the same or different in each immunization, and the type of immunogenic composition (e.g., the protein or expression vector it contains), the route, and the method of preparing the immunogen may also vary. For example, if the expression vector is used in the priming and boosting stages, it may be of the same type or different types (e.g., DNA or bacterial or viral expression vector). One effective prime-boost regimen involves two priming immunizations 4 weeks apart, followed by two booster immunizations 4 and 8 weeks after the last priming immunization. It will be readily apparent to those familiar with the art that several permutations and combinations are included when using the DNA, bacterial, and viral expression vectors of the present invention to provide priming and booster regimens. Viral vectors expressing different antigens from different pathogens may be used repeatedly if they express US2-11.

[0144] Screening assay : The present invention provides a method for screening agonists or antagonists for wild-type ACE2, ACE2 vECD, or fusion proteins. The agonist or antagonist compound is selected if a) a population of cells transfected with a mutated gene is exposed to various diagnostic reagents (in high-throughput screening, this is done over a period of time under conditions that allow the diagnostic reagent to affect ACE2 enzyme activity); and b) the diagnostic reagent causes a statistically significant increase or decrease in ACE2 enzyme activity and binding affinity levels compared to pre-exposure levels.

[0145] The present invention also includes screening assays for detecting ACE2 activators, which may be used to treat diseases. These diseases include, but are not limited to, viral diseases. These assays may be in vitro or in vivo. In preferred embodiments, the present invention includes endothelial, kidney, lung, or cardiac cell assays for evaluating whether a candidate compound can increase ACE2 expression or activity. Cells are cultured in the presence of at least one compound, and efforts are made to determine the compound's ability to express or activate, and the cells are measured for increased ACE2 expression or activity levels. Another aspect of the present invention uses ACE2 knockout mice to identify compounds that may overcome the effects of ACE2 loss. In another embodiment, ACE2 gene expression may be enhanced by administering a substance that increases ACE2 gene expression, including a substance identified using the screening assays in this application. Polypeptides and small organic molecules are tested in these assays. The present invention includes all compounds that are identified by the screening method of the present invention and are suitable for administration to animals as pharmaceutical compositions. [Examples]

[0146] 1. Design examples of wild-type ACE2 and ACE2-vECD, molecular cloning, and mutation generation and cloning of these mutant genes. A series of ACE2 mutants were created by fusing a human antibody heavy chain secretion signal peptide at the 5' end of the protein and an IgG1 Fc fragment at the 3' end. The wild-type ACE2-Fc protein sequence was back-translated into a DNA sequence using the SnapGene program (GSL Biotech, San Diego, CA) with Homo sapiens codon output. The ACE2-Fc DNA sequence was further manually modified to adjust the GC content and sent to Twist Bioscience (South San Francisco, CA) for synthesis as three duplicated DNA fragments. Regarding the construction of the vectors, all primers were designed by experts in the field, and their names are listed in Table 5.

[0147] The constructs were prepared as shown in Table 3. To construct AMI074-pFB-CMV-SV40intron-Vh-ACE2-G449D-Fc, plasmid AMI063-pFB-CMV-hGH_intron-hCOMP-Ang1 was cleaved with EcoRI and the backbone fragment was isolated. The CMV promoter-SV40 intron fragment (748 bps) was amplified by PCR using primers A120 and A121 and AMI060 as the template. The 5'-ACE2 (1517 bps) was amplified using primers A056 and A145, the intermediate ACE2 fragment (840 bps) was amplified using primers A146 and A147, and the 3'-ACE2 fragment (660 bps) was amplified using primers A148 and A122, with synthetic DNA fragments used as templates. A second round of PCR was performed, in which the CMV-SV40 intron fragment was conjugated with the 5'-ACE2 fragment using primers A120 and A145, and the intermediate and 3'-ACE2 fragments were conjugated in another PCR reaction using primers A146 and A122. These two conjugated PCR fragments were purified and cloned into the EcoRI site of plasmid AMI063 using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA). AMI080-pSV40prom-DHFR-NeoR-CMV-ACE2-Fc was constructed by PCR amplification of the CMV-SV40-intron-ACE2-pA fragment from plasmid AMI074 using primers A098 and A161, and was ligated into the SalI and MluI sites of AMI069. To construct AMI081-pFB-CMV-SV40intron-Vh-ACE2_E402Q-G449D-Fc, plasmid AMI074 was cleaved at SfoI, and the backbone fragment was isolated. A 540bp ACE2 fragment containing the desired mutation was PCR amplified using primers A162 and A163, with AMI074 as the template. The PCR fragment was purified and cloned into the SfoI site using the NEBuilder HiFi DNA Assembly Kit.Plasmid AMI081-pFB-CMV-SV40 intron-Vh-ACE2_E402Q-G466D-Fc was cleaved with BamHI and FseI to remove the ACE2 mutation site and replaced with a wt-ACE2 fragment from AMI080-pSV40prom-DHFR-NeoR-CMV-ACE2-Fc to construct AMI089-pFB-CMV-SV40intron-Vh-ACE2-Fc. For cloning AMI090-pFB-CMV-SV40in-Vh-ACE2_E385Q-Fc, plasmid AMI081 was cleaved with AleI and FseI to isolate the backbone fragment. The 5'-ACE2 fragment was amplified with primers A156 and A170, and the 3'-ACE2 fragment was amplified with primers A169 and A158, using plasmid AMI081 as a template. Using a second round of PCR, both PCR fragments were joined, which were then cloned into the AleI and FseI sites using the NEBuilder HiFi DNA Assembly Kit. To clone AMI082-pFB-CMV-SV40intron-Vh-ACE2_H357A-E385Q-Fc, plasmid AMI090 was cut at AleI and FseI to isolate the backbone fragment. The ACE2 fragment was amplified using primers A156, A157, and A158 with plasmid AMI090 as a template. The ACE2 PCR fragment was cloned into the AleI and FseI sites of AMI090 to construct clone AMI082-pFB-CMV-SV40intron-Vh-ACE2_H357A-E385Q-Fc. ACE2 PCR fragments between AleI and FseI were PCR amplified using primers A156, A159, and A158 and plasmid AMI090 as a template. The desired E358-385Q mutation was incorporated and cloned into AMI090 to produce AMI083-pFB-CMV-SV40intron-Vh-ACE2_E358-385Q-Fc.The ACE2 fragment between AleI and FseI was amplified by PCR using primers A156, A160, and A158 and plasmid AMI090 as a template. The desired E358-385Q+H357A mutation was incorporated, and the resulting molecule was cloned into AMI090 to produce AMI084-pFB-CMV-SV40intron-Vh-ACE2_E358-385Q+H357A-Fc. The ACE2 fragment between AleI and FseI was amplified using primers A156, A160, and A158 and plasmid AMI089 as a template. The desired H357A+385Q mutation was incorporated, and the resulting molecule was cloned into AMI089 to produce AMI085-pFB-CMV-SV40intron-Vh-ACE2_H357A+385Q-Fc.

[0148] The ACE2 mutant plasmids in the second panel of Table 3, from AMI121 to AMI129, were constructed using the AMI082 plasmid as the backbone. Briefly, all mutant sequences were synthesized by Twist Biosciences as two duplicated DNA fragments. The 5'-fragment was PCR-amplified with primers A056 and A385, and the 3'-fragment was amplified with primers A386 and A158. The two PCR fragments of each mutant were purified and conjugated with primers A056 and A158. The conjugated PCR fragments were purified again and cloned into the AflII and FseI sites of AMI082 using the NEBuilder HiFi DNA Assembly Kit to construct each mutant plasmid. The mutant sequences were confirmed by DNA sequencing analysis using primers A024, A145, A169, and A148.

[0149] The plasmid constructs containing the desired ACE2-vECD-Fc mutant mutation used in this project are listed in Tables 1 and 3, and all primers used for PCR and DNA sequencing are listed in Table 5. The complete DNA sequences encoding ACE2 for all constructs are listed below.

[0150] Following the above procedure, DNA constructs encoding the remainder of the mutant protein of either ACE2-vECD or ACE2-vECD-Fc fusion protein were cloned and characterized by DNA sequencing. The DNA sequences listed are only those encoding the ACE2-vECD-Fc fusion protein.

[0151] [Table 5]

[0152] The plasmid constructs containing the desired ACE2 mutation used in this project are listed in Table 3, and the complete DNA sequences encoding ACE2 for all constructs are listed above. All primers used for PCR and DNA sequencing are listed in Table 5. All constructs were first cloned into our mammalian-specific expression vectors. The final vectors are shown in Table 4 above.

[0153] 2. Example: Transient expression of constructs in mammalian cell culture systems Human HEK293 cells were cultured in DMEM medium supplemented with 10% FBS (ATCC Manassas, VA) in a 37°C CO2 incubator. For maintenance and subculturing, cells were divided 1:10 twice weekly. For transfection, cells were seeded overnight in 10 mL of medium at a rate of 2 x 10⁶ cells / dish in 10-cm cell culture dishes (Corning, NY). 14 μg plasmid DNA and 22 μL Lipofectamine 3000 were each diluted and mixed in 0.5 mL of Opti-medium. After incubation at room temperature for 5 minutes, the mixture was added dropwise to the cells and incubated in a 37°C CO2 incubator for 48 hours. The culture medium was then collected for further experiments.

[0154] One day prior to transient transfection, HEK293 cells were seeded at a density of 2 x 10⁶ cells in a 10 cm tissue culture dish. Each transfection of either ACE2-ECD-Fc (wt) or ACE2-vECD-Fc mutant plasmid was performed using 14 μg / dish DNA containing Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. Cell culture supernatants were collected and analyzed for protein expression by Western blotting 48 hours after transfection. All transfections were performed in 3-well sets in at least three independent experiments.

[0155] ACE2-ECD-Fc(wt) or ACE2-vECD-Fc mutant proteins were determined by SDS-PAGE and Western blot analysis. HEK293 cell medium (supernatant) collected 48 hours after plasmid transfection or 72 hours after AAV5-ACE2 transduction was used for Western blot analysis. A total of 30 μL of cell supernatant was mixed with 10 μL of 4x loading buffer and loaded onto a NuPAGE 10% Tris-Glycine gel (Invitrogen) for electrophoresis. The proteins were then transferred to a PVDF membrane using the X Cell II® Blot Module (Invitrogen, Carlsbad, CA, USA). The membranes were treated with a casein blocker in PBS (Thermo Scientific, Waltham, MA, USA) at room temperature for at least 1 hour, tested with a goat anti-human IgG1 Fc antibody biotin conjugate (Abcam, Cambridge, UK), and then incubated with horseradish peroxidase (Abcam)-labeled streptavidin. The proteins were detected using the ECL® Western Blotting Kit (Amersham), and images were recorded using the iBright™ CL1500 Imaging System (Invitrogen, Carlsbad, CA) (Figure 12).

[0156] 3. Examples: Purification of ACE2-ECD-Fc (WT) and ACE2-vECD-Fc mutants The expressed ACE2-ECD-Fc(wt) or ACE2-vECD-Fc mutant proteins were purified from HEK293 cell medium samples by protein A affinity column chromatography (Mabselect™). The supernatant of the culture medium was filtered through a 0.2 μm syringe filter (Millipore). To purify the ACE2 secreted from each filtered medium, a HiTrap™ 1 mL MabSelect™ Protein A column (GE Health Care Lifesciences, Marlborough, MA 01752) was used. Column chromatography showed a sharp peak of elution from the column when the pH reached 3–4.0 (Figure 13(a)). The chromatographic protein concentration of each preparation was determined by BCA protein assay, and the results are shown in Table 6 (Thermo Scientific, Hayward, CA). The protein size of each construct was as expected ((b) Figure 13).

[0157] [Table 6]

[0158] 4. Example: Determination of the enzymatic activity of the ACE-vECD-Fc fusion protein The enzyme activity of the ACE2 ECD-Fc fusion protein mutant, purified by affinity column chromatography, was measured according to Fenxia Xiao and Kevin B. Burns (Ref: Measurement of angeiotension converting enzye 2 activity in biological fluid (ACE2), chapter 8, Hypertension: Methods and Protocols, Methods in Molecular Biology, vol. 1527, Rhian M. Touyz and Ernesto L. Schiffrin (eds.), DOI 10.1007 / 978-1-4939-6625-7_8, (copyright Springer Science+Business Media LLC 2017). The measurement mechanism was the ACE2-specific inhibitor MLN-4760 (Merck Millipore Calbiochem™ ACE2 inhibitor). The study is based on the hydrolysis of intramolecularly quenched fluorescent ACE2 substrates in the presence or absence of MLN-4760, an extremely potent ACE2 inhibitor with an IC50 of 440 pM. The specificity of the ACE2 ECD-Fc fusion protein was determined by suppression of the fluorescent signal measured using the ACE2 inhibitor MLN-4760 with an excitation wavelength of 330 nm and an emission wavelength of 450 nm filter pair. Meanwhile, the enzyme activity of ACE2 ECD-Fc mutant protein was examined in the presence and absence of the ACE2 inhibitor MLN-4760, with both wild-type and mutant proteins being examined simultaneously.

[0159] The ACE2 enzyme assay was performed using enzyme assay buffer, 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 300 mM NaCl, 10 μM ZnCl2, and pH 6.81. The ACE2 fluorescent substrate was a synthetic peptide molecule, McA-Ala-Pro-Lys(Dnp)-OH (AnaSpec, cat. # 60757, San Jose, CA, USA). The substrate was dissolved in 1% NH4OH to a concentration of 15 mM. The substrate solution was divided into 10 μL portions per vial and stored at -80°C. The protease inhibitor N-ethylmaleimide (NEM, MilliporeSigma Cat. 34115-5GM, St. Louis, MO, USA) was diluted to 100 mM in Milli Q water, and phenylmethylsulfonyl fluoride (PMSF) was diluted to 100 mM in 100% ethanol. The ACE2 inhibitor MLN-4760 (Merck MilliporeCalbiochem, San Diego, CA, USA, Catalog Number: 530616) was diluted to 10 μM in Milli Q water. The assay buffer / substrate mixture was prepared fresh according to Table 7 below.

[0160] [Table 7]

[0161] 70 μL of assay buffer / substrate mixture was added to 100 μL of reaction mixture, so the final concentration buffer components were 35 mM MES, 210 mM NaCl, and 7 μM ZnCl2. The final concentrations of the ACE2 substrate and protease inhibitor were 10.5 μM and 0.7 mM, respectively.

[0162] The wild-type ACE2 ECD-Fc (AMI080) and five mutant ACE2 ECD-Fc proteins (AMI081, AMI082, AMI083, AMI084, and AMI085) were purified as previously described. These assays were performed in 96-well microtitration plates. Each protein was diluted separately in sterile phosphate buffer (PBS, HyPure™, GE Healthcare, Hyclone Laboratories, Logan, Utah) at concentrations ranging from 500, 100, 20, 10, 5, and 2.5 ng / mL, with concentrations of 3.13 and 1.56 nM. The sample extraction method is shown in Table 8. Two blank control wells were included in the assay.

[0163] [Table 8]

[0164] The reaction was carried out in a darkened 96-well plate, with each protein tested in pairs of two wells. After all reagents and buffer mixtures were added, they were thoroughly mixed, immediately sealed, and wrapped in aluminum foil. The plates were placed on a shake platform and gently agitated at 140 rpm at room temperature for 16–20 hours. The relative fluorescence units (RFU) of the plate were read using a fluorometer, fmax (Molecular Device, Sunnyvale, CA, USA), at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The data was obtained by averaging two sets of readings. The following is a plot of RFU against the protein concentration of each ACE2 ECD-Fc protein.

[0165] As shown in Figure 14, wild-type wtACE2 ECD-Fc exhibited enzymatic activity, and relative fluorescence units (RFUs) increased with the protein concentration added to the reaction. RFUs were significantly reduced in the presence of the ACE2 inhibitor MLN4760. The ACE2 enzyme assay is specific, as the reaction can be inhibited with 0.73 μM of the ACE2-specific inhibitor. It is highly regenerative, with an inter-assay CV of 3.6% and an intra-assay CV of 1-6%. Mutations in one or more amino acid residues in the zinc-binding motif resulted in the loss of ACE2 enzymatic activity. None of the mutant ACE2-Fc proteins possessed enzymatic activity (apoenzyme). All mutant ACE2 ECD-Fc proteins did not produce significant RFUs, and the enzymatic activity of the ACE2-vECD-Fc mutant protein was depleted by a single amino acid residue mutation, specifically AMI090 with only a single mutation at E402Q, which resulted in a loss of more than 99.9% catalytic activity. To better understand the catalytic activity of each ACE2-vECD-Fc protein, the enzymatic reaction results are shown individually in Figures 15A and 15B. The correlation between sequence mutations and enzymatic activity is shown in Table 9.

[0166] [Table 9] The data presented demonstrates that a single mutation in the catalytic center, the zinc-binding motif, leads to a loss of enzyme activity. The study showed that ACE2-vECD, which had lost its activity, showed no enzymatic activity after fusion to Fc.

[0167] 5. Example: Binding of ACE2-ECD-Fc protein to coronavirus spike protein by enzyme-linked receptor-ligand assay (ELRLA) The binding of the three coronavirus spike proteins to either ACE2-ECD-Fc or ACE2-vECD-Fc was determined by enzyme-linked receptor-ligand assay (ELRLA). All buffers were prepared using sterile MQ water or sterile PBS (Cat: SH30529.03, GE Healthcare Life Science, Logan, Utah). SARS-CoV-1, SARS-CoV-2, or MERS-CoV spike protein 1 (S1) was individually diluted in sodium carbonate buffer (50 mM NaCO3, NaHCO3, pH 9.6) to 10 nM and 20 nM concentrations, and coated each 96-well microplate with 50 μL / well. The S1 proteins were purchased from Sino Biological (SARS-CoV-1 S1 cat# 40150-V08B1, SARS-CoV-2 S1 cat# 40591-V08H, MERS-CoV S1 protein cat#: 40069-V08H, Beijing, China). Microplates were tightly sealed and incubated at 2-8°C for 12 hours. They were washed three times with phosphate-buffered saline (10 phosphate buffer, 150 mM NaCl, pH 7.2, 0.01% Tween 20, PBS-T) and blocked in blocking buffer (1% BSA in PBST) at 37°C for 2 hours. After washing, serially diluted ACE2-ECD-Fc or ACE2-vECD-Fc mutant proteins at concentrations of 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.63 nM, and 0.313 nM were added to each well in pairs of two wells (50 μL / well). The plates were sealed and incubated at 37°C for 60 minutes. The plates were washed three times with PBS-T. 50 μL of goat anti-human IgG Fc-biotin complex (Abcam cat. ab98618, Cambridge, MA) was diluted 1:10000 with PBS-T-0.5% (w / v) BSA in each well and incubated at 3°C ​​for 60 minutes. The microplate was washed three times with PBS-T, then 50 μL / well of streptavidin horseradish peroxidase (HRP) diluted 1:15000 was added and incubated at 37°C for 60 minutes. The microplate was washed three times with PBS-T and once with PBS to remove any remaining Tween20. The reaction was performed in a 1-Step manner using 100 μL / well. TMThe assay was performed with ABTS substrate (Thermo Scientific REF 37615, Rockford, CA) for 30 minutes at 37°C and stopped with 50 μL / well of 2% (w / v) SDS. The plates were read using a VERSAmax Microplate Reader (Molecular Device, Sunnyvale, CA) at 405 nm. The results are shown in Figure 16A. From the binding assay, the SARS-CoV-2 spike protein bound to wild-type AMI080 (ACE2-ECD-Fc), the AMI082 (ACE2-vCECD-Fc, H274A, E402Q) mutant, and AMI090 (ACE2-vECD-Fc, E402Q) with very similar binding profiles (Figure 16A), but the affinity and Ymax values ​​did not change.

[0168] To our surprise, the MERS-CoV S1 protein barely bound to wild-type ACE2-ECD-Fc, while mutant AMI090 and AMI082 showed increased binding affinity of >200% and >150% compared to the wild type (Figure 16A). To more clearly demonstrate the reaction of individual ACE2-ECD-Fc or ACE2-vECD-Fc with SARS-CoV-1, SARS-CoV-2, and MERS-CoV S proteins, each S1 protein was coated with 20 nM and assayed precisely as described in the procedure. The reactivity of each construct protein with the three ligands is plotted in Figures 17A and 17B.

[0169] Based on the combined results of ELALA and enzyme analysis data, we concluded that mutants AMI082 and AMI090 retain their ability to bind to the viral spike protein, and that amino acids H374 and E402 do not affect the binding of SARS-CoV-1 and SARS-CoV-2 S1 proteins to their alloceptors on host cells. Both other residues, E375 and H378, are important for this binding (Figure 16A).

[0170] In the case of MERS-CoV, mutations in H374A and E402Q significantly enhanced the binding of the viral S1 protein to ACE2-vECD-Fc (AMI090 and AMI082) by approximately 500% and 800%, respectively, compared to their maximum response values ​​(Ymax values). AMI083 also showed an increase in binding of approximately 200%, while other mutations showed little to no binding to ACE2. Therefore, we hypothesized that mutant ACE2-vECD-Fc (AMI090 and AMI082) could also be used to block MERS-CoV infection.

[0171] From these data, we discovered relationships between amino acid residues, ACE2 catalytic activity, and coronavirus binding properties. These findings are summarized in Table 10. [Table 10]

[0172] The binding affinity was estimated according to Hill's formula (Mohameedyaseen Syedbasha et al, J. Visual. Exp. 2016, 1109: 4-10), and the results for binding to SARS-COV-2 S1 and SARS-COV-1 S1 proteins are shown in Tables 11 and 12, respectively. [Table 11]

[0173] The mutant ACE2-Fc AMI082 and AMI090 showed approximately 30–50 times higher affinity for the SARS-CoV-2 S1 protein than the wild-type ACE2-Fc AMI080 protein (Table 11). This increased binding affinity suggests a tighter interaction between ACE2 and SARS-CoV-2 virus particles. Meanwhile, the binding of ACE2-Fc mutants to the SARS-CoV-1 S1 protein was also evaluated using the same assay procedure. The results showed that the binding affinity of AMI082 and AMI090 was 13-fold higher than that of wild-type ACE2-Fc (Table 12).

[0174] [Table 12]

[0175] To further determine the binding affinity of these ACE-Fc mutant proteins to SARS-CoV-2 mutants, purified viral receptor-binding domain (RBD) protein of SARS-CoV-2 B117(N501Y) (Sino biologics cat #: 40592-V08H82) was used. Plates were coated with 10 nM vAC2-Fc protein, and SARS-CoV-2 B117(N501Y) RBD protein at concentrations of 0.01, 0.04, 0.13, 0.40, 1.27, 4.07, 13.02, 39.01, 125, and 400 nM were tested in pairs of two wells. Binding affinity was estimated according to Hill's formula (Mohameedyaseen Syedbasha et al, J. Visual. Exp. 2016, 1109: 4-10), and the results are shown in Figure 16B and Table 13. Of these ACE2-Fc mutant proteins, AMI090, AMI126, and AMI133 had very similar KD values ​​at <1 nM. AMI090 had the highest Ymax value (Figure 16B).

[0176] [Table 13]

[0177] In qualitative binding assays, ACE2-Fc mutants AMI080, AMI082, and AMI090 were evaluated for binding to various SARS-CoV-2 mutants. The tests clearly showed that the E484K mutant responded more strongly to the ACE2-Fc protein than the other mutants (Table 14).

[0178] [Table 14]

[0179] 6. Example: Binding of coronavirus spike protein-containing viral antigens by surface plasmon resonance (SPR) Surface plasmon resonance (SPR) was used to determine the binding ability of wt ACE2-ECD-Fc and mutant proteins to the coronavirus spike 1 protein. wt ACE2-ECD-Fc or mutant ACE2-vECD-Fc (AMI084) was bound to sensor chip protein A (GE Healthcare now Cytiva, cat 29-1275-57, Uppsala, Sweden) at a concentration of 5 μg / mL in phosphate-buffered saline containing 0.01% Tween 20 (PBS-T). The SARS-CoV-2 S1 protein and ACE2-ECD-Fc protein were able to bind to the protein ligand on the chip via the IgG1Fc region. SARS-CoV-1 and MERS-CoV were obtained from Sino biologicals (Beijing, China). The S1 protein was diluted with PBS-T to final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 nM. The program was run using a BiaCore 3000 instrument as a binding kinetics instrument. The observed clear binding affinity indicated that recombinant wt ACE2-ECD-Fc and mutant ACE2-vECD-Fc could bind separately to the S1 proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. AMI084, which has three amino acid mutations in the catalytic center of ACE2, would likely show a similar binding profile to wt ACE2 ECD-Fc. It is also hypothesized that formulations of proteins with fewer mutations could bind to the S1 proteins of these coronaviruses.

[0180] Results: The purified ACE2-Fc protein formulation was bound to the spike protein S1 of SARS-CoV-2 determined by BiaCore 3000 (Figure 18). The purified mutant ACE2-vECD-Fc protein is under inspection. The binding affinity (KD) of wild-type ACE2-Fc, AMI080 was 16.81 nM, and that of the mutant ACE2-Fc protein AMI090 was 0.49 nM, showing an increase in binding by the Biacore assay.

[0181] 7. Example: In vitro neutralization of SARS-Cov-2 pseudovirus particles An in vitro virus neutralization screening assay was performed using SARS-CoV-2 pseudovirus and SARS-CoV-2 S1 lentiviral vector. This vector expresses green fluorescent protein (GFP) when it binds to the SARS-CoV-2 receptor on the cell surface of human ACE2 (hACE2) protein, stably transfected HEK293 cells (293T-hACE2). This is a safe and specific screening method for evaluating compounds, antibodies or soluble receptors of the virus.

[0182] Briefly, 1.5x10 4 293T-hACE2 cells (CMV-hACE2) were seeded per well in a gelatin-coated 96-well plate and cultured overnight at 37 °C with 5% CO2 and 95% humidity. ACE2-ECD-Fc or ACE2-vECD-Fc mutant proteins were individually serially diluted 1:2 with PBS in another 96-well "setup" plate to 20, 10, 5, 2.5, 1.25, 0.625 and 0.313 μg / mL, and each sample was assayed in duplicate wells. The stock solution of the pseudovirus was approximately 1 million infectious units (IFU) per mL (10 [[ID=The solution was diluted to IFU / mL. 60 μL of the diluted pseudovirus solution was added to all wells containing the ACE2 mutant protein and to the wells containing the pseudovirus and cell control. The plates were thoroughly mixed and incubated at 37°C for 1 hour. 100 μL of the mixture from each well of the setup plate containing the antibody and virus diluent was carefully added to each well, replacing the medium in the corresponding wells of the HEK293T-hACE2 cell plate. Finally, 1Trans plus® (Alstem, Cat# V050, Richmond, CA) was added to each well according to the manufacturer's instructions to achieve a final 1X concentration. The plates were incubated at 37°C for 48–60 hours, after which fluorescence was measured. Fluorescence for each well was counted.

[0183] Assay results showed that 293T-hACE2 cells exhibited green fluorescent forsythia (GFF) in the absence of blocking or neutralizing agent, but no GFF was observed in the presence of a specific neutralizing reagent (19). The results of SARS-CoV-2 pseudovirus neutralization are shown in Figures 20 and 21. The 50% neutralization concentration was estimated to be approximately 5 μg / mL for ACE2-ECD-Fc, ACE2-vECD-Fc (AMI082), and ACE2-vECD-Fc (AMI090), respectively. For the other three constructs, ACE2-vECD-Fc (AMI081), ACE2-vECD-Fc (AMI083), and ACE2-vECD-Fc (AMI084), it was estimated to be 10 μg / mL (Figure 20).

[0184] 8. Example: TCID 50 Efficacy of in vitro SARS-CoV-2 neutralization by assay Toxicity neutralization assays were performed at Southern Research Institute (2000 Ninth Avenue South, Birmingham, Alabama 35205). Neutralization of SARS-CoV-2 with selected ACE2-ECD-Fc (wild-type AMI080) or vACE2-ECD-Fc mutant proteins (AMI082 and AMI090) was performed using Vero6 cell cultures infected with the highly virulent SARS-CoV-2 strain (strain name: USA-WA1 / 2020, SARS-CoV-2).

[0185] The protein preparations, wild-type ACE2-ECD-Fc (AMI080), ACE2-vECD-Fc (AMI082), and vACE2-ECD-Fc (AMI090) gene plasmid DNAs were prepared by separately transfecting monolayer cultured HEK293 cells with AMI080, AMI082, and AMI090 plasmid DNA preparations. The protein preparations are summarized in Table 15. [Table 15]

[0186] The procedure for the SARS-CoV-2 coronavirus cytopathic effect (CPE) reduction assay (neutralization) is described below. The first step was to dilute the ACE2-Fc protein. AMI080, AMI02, and AMI090 were serially diluted with PBS and transferred separately to wells on an empty ECHO plate (storage plate). The ACE2-Fc protein was 2-fold diluted by transferring 40 μL of each storage sample to an adjacent well containing 40 μL of PBS and mixing. This process was repeated to produce eight more wells of serially diluted samples. Each well contained a sample that was 3-fold diluted compared to the previous well. A 90 nL aliquot of each sample was dispensed into the corresponding assay-ready well. An ECHO 555 acoustic liquid processing system was used for this. The final assay concentration range was from 200 to 0.01 μg / mL with 3-fold serial dilutions. PBS was added to the control wells to maintain a consistent assay concentration of 0.3% in all wells.

[0187] The second step was to measure the antiviral effect of the compound: Vero E6 cells were cultured in MEM supplemented with 10% HI FBS and harvested on the day of assay in MEM supplemented with 2% HI FBS and 1% Pen / Strep. Assay-ready plates, pre-added with the test compounds AMI080, AMI082, and AMI00, were prepared in a BSL-2 laboratory by adding 5 μL of assay medium to each well. The plates and cells were then transferred to a BSL-3 facility. Cells were batch inoculated with SARS-CoV-2 (USA_WA1 / 2020; MOI~0.002), resulting in a cell viability of ~5% 72 hours post-infection. 25 μL aliquots of virus-inoculated cells (4000 Vero E6 cells / well) were added to each well in rows 3-24 of the assay plate. Wells in rows 23-24 contained only virus-infected cells as a 0% CPE reduction control. Prior to virus inoculation, 25 μL aliquots of cells were added to 1-2 rows of each plate. These rows contained only cells and served as a 100% CPE reduction control. After incubating the plates for 72 hours at 37°C with 5% CO2 and 90% humidity, 30 μL of Cell Titer-Glo (Promega) was added to each well. To measure cell viability, the plates were incubated at room temperature for 10 minutes, and then luminescence was measured using a BMG CLARIOstar plate reader. Prior to luminescence measurement, the plates were sealed with a clear cover to remove surface contamination. To ensure the reliability of the neutralization assay, several small virus inhibitors were used. [text interrupted]

[0188] Method for measuring the cytotoxic effects of ACE2-Fc protein preparations: The cytotoxicity of ACE2-Fc proteins AMI080, AMI082, and AMI090 was evaluated using a BSL-2 counterscreen as follows: Host cells from culture medium were added in 25 μl aliquots (4000 cells / well) to each well of an assay-ready plate prepared with the test proteins as described above. Cells alone (100% viability) and cells treated with a final concentration of 100 μM hyamine (0% viability) were used as high and low signal controls for the cytotoxic effect of this assay, respectively. PBS was maintained at a constant concentration for all wells according to the specified dilution factors of the storage solutions for these protein concentrations. After incubating the plates for 72 hours at 37°C with 5% CO2 and 90% humidity, 30 μL of Cell Titer-Glo (Promega) was added to each well. After incubation at room temperature for 10 minutes to measure cell viability, luminescence was measured using a BMG CLARIOstar plate reader.

[0189] Data analysis: For all assays, raw data from the plate reader is imported into ActivityBase, where the values ​​are linked to compound IDs and test concentrations. In the antiviral CPE reduction assay, the raw signal value is converted to a percentage CPE reduction using the following formula. % CPE reduction = 100 x (test protein value - average value of infected cells) / (average value of uninfected cells - average value of infected cells).

[0190] In cell survival assays that measure the cytotoxicity of compounds, the % cell survival rate is calculated as follows: % Survival rate = 100 * (Test protein value - mean low-signal control) / (mean high-signal control - mean low-signal control).

[0191] 50% inhibitory concentration of viral infection (IC) 50 ) and the concentration that causes 50% cytotoxicity (CC) 50The parameters were calculated using the ActivityBase Xlfit module with upper and lower limits restricted to 100% and 0%, respectively, from a 4-parameter logistic fit of the data. The analyzed data for SARS-CoV-2 (USA-WA1 / 2020) are shown in Figure 21, which demonstrates the effective neutralization of SARS-CoV-2 (USA-WA1 / 2020) achieved by wild-type and enzyme-isolated ACE2-Fc mutants, AMI082, and AMI090.

[0192] IC of three proteins 50 The concentrations for ACE2-Fc proteins AMI080, AMI082, and AMI090 were 5.55, 5.43, and 5.33 μg / mL, respectively. No significant differences were observed among these three constructs, however, mutant ACE2-Fc AMI082 and AMI090 showed a 30-50-fold increase in affinity (KD) in the S1 protein receptor binding assay.

[0193] 9. Example: Gene transfer vectors for ACE2-ECD-Fc and ACE2-vECD-Fc using adeno-associated virus vectors (AAV) AAV5 was selected in this invention because it can be introduced into the airway epithelium, AAV3 can be introduced into the sinuses, nose, and / or lungs, and other serotypes of AAV can also be used depending on the target tissue or cells to which the gene is introduced. Several types of animals are used, including rats, cats, guinea pigs, hamsters, mice, minks, sheep, and rabbits. AAV6 tends to preferentially introduce genes into lung cells. In this test, we start with AAV5 for vector preparation for the purpose of gene introduction.

[0194] SF9-derived insect cell line, V432A cells, were cultured in ESF AF medium (Expression Systems) supplemented with 100 units / ml penicillin and 100 μg / ml streptomycin (Corning) at 28°C in Corning storage bottles. The cell density was 7 x 10⁶. 6When the cell / ml level reached a certain point, it was divided into a 1:4 ratio for maintenance. Recombinant baculovirus (rBV) was prepared according to the Invitrogen protocol (Carlsbad, CA). In short, the constructed plasmid was used to induce DH10Bac transformation, and recombinant bacmid DNA was isolated. The bacmid DNA was transfected into V432A cells to produce rBV. rBV was quantified by QPCR.

[0195] AAV vector preparation, purification, and quantification - V432A cells were cultured in 7x10 6The cells were diluted 1:1 with fresh ESF AF medium at a concentration of cells / ml. Approximately 200 viruses per cell of rBV containing the specified rep-cap gene and 100 viruses per cell of rBV containing DNA sequences encoding ACE2-ECD-Fc or ACE2-vECD-Fc mutant proteins were added separately to infect V432A cells in a shaker incubator at 28°C for 3 days. Infected V432A cells were centrifuged at 3,000 rpm for 10 minutes and collected. The cell pellet was lysed in SF9 lysis buffer (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, 2 mM MgCl2, 1% Sarkosyl, 1% Triton X-100, and 140 units / ml Benzonase®, Millipore, Burlington, MA). Genomic DNA was incubated and digested at 37°C for 1 hour. At the end of incubation, sodium chloride was added to bring the salt concentration of the lysate to approximately 1 M, and the AAV vector was further dissociated from the cell substrate. Cellular residue was removed by centrifugation at 8,000 rpm for 30 minutes. The lysate, with impurities removed, was loaded onto a CsCl step gradient and ultracentrifuged at 28,000 rpm for 20 hours using a swing rotor. The viral band was aspirated using a syringe fitted with an 18-gauge needle and loaded onto a second CsCl step gradient, and linearly ultracentrifuged at 65,000 rpm for 20 hours. The viral band was then extracted and passed through two PD-10 desalting columns (GE HealthCare) to remove CsCl and surfactant, while simultaneously being replaced with Buffer B (1x PBS, 0.1 M Sodium Citrate, and 0.001% pluronic F-68). Quantitative real-time PCR (qPCR) was performed to determine the copy number of the AAV vector genome using the following ITR primers and probes. ITR-QPCR-F: 5'-GGAACCCCTAGTGATGGAGTT-3' (Sequence ID: 61) ITR-QPCR-R: 5'-CGGCCTCAGTGAGCGA-3' (Sequence ID: 62) ITR-FAM-2ITR-MGB: 5'- CACTCCCTCTCTGCGCGCTCG-3' (Sequence ID: 63)

[0196] SDS-PAGE and SimplyBlue staining for AAV vector purity confirmation - The AAV vector was mixed with SDS-PAGE loading buffer (Invitrogen) and heated at 95°C for 5 minutes. The vector was then loaded onto a 10% SDS-PAGE gel and electrophoresis was performed at 100 volts until the dye reached the end of the gel. The gel was stained according to the manufacturer's protocol (Invitrogen).

[0197] In this experiment, AAV5-ACE2-ECD-Fc or AAV5-ACE2-vECD-Fc mutant vectors were prepared and purified as described separately. The titer of each AAV5-ACE2-ECD-Fc or AAV5-ACE2-vECD-Fc mutant vector was determined using a primer pair and probe selected from the above ITR sequences. The titers, productivity, and protein levels of these AAV vectors are shown in Table 16.

[0198] [Table 16]

[0199] SDS-PAGE and SimplyBlue staining of AAV5 vectors The purity of the AAV vector was determined by the SimplyBlue Staining assay. Briefly, 26 μl of AAV sample was mixed with 10 μL of 4x loading buffer and 4 μL of 10x reducing agent (Invitrogen) and incubated at 95°C for 2 minutes. Approximately 1E+11vg of each AAV sample was loaded into each lane as shown in the description in Figure 22. Typical gel patterns with expected VP1, VP2, and VP3 component levels were obtained (Figure 22).

[0200] Recombinant AAV5 vector expression of ACE2-ECD-Fc and ACE2-vECD-Fc Further evaluation of the production of each construct protein using the AAV5 vectors listed in Table 11 was performed using HEK293 cells. HEK293 cells were seeded at 1.5e+5 cells / well in 24-well plates and cultured overnight in 0.5 mL of DMEM with 10% FBS. The following morning, the cells were washed with serum-free DMEM, and gene transduction was performed with the AAV5-ACE2 vector using various titers in 0.5 mL of serum-free DMEM with 20 μM etoposide. After overnight gene transduction, the inoculum was removed and replaced with 0.5 mL / well DMEM containing 10% FBS. After a total of 72 hours of gene transduction, the cell medium was collected, a proteinase inhibitor was added, and it was stored at ≤-65°C before use.

[0201] Expressed ACE2-vECD-Fc or ACE2-vECD-Fc mutant HEK293 cell medium (supernatant) collected 48 hours after plasmid transfection or 72 hours after AAV5-ACE2 transduction was used for Western blot analysis. A total of 30 μL of cell supernatant was mixed with 10 μL of 4x loading buffer and loaded onto a NuPAGE 10% Tris-Glycine gel (Invitrogen) for electrophoresis. Proteins were then X Cell II TM The proteins were transferred to a PVDF membrane using the Blot Module (Invitrogen, Carlsbad, CA, USA). The membrane was treated with a casein blocker in PBS (Thermo Scientific, Waltham, MA, USA) at room temperature for at least 1 hour, reacted with a goat anti-human IgG1 Fc antibody biotin conjugate (Abcam, Cambridge, UK), and then incubated with horseradish peroxidase (Abcam)-labeled streptavidin. The proteins were then transferred to ECL. TM Detection was performed using a Western blotting kit (Amersham), and images were recorded using the iBright™ CL1500 Imaging System (Invitrogen, Carlsbad, CA).

[0202] Western blotting images showed a single, sharp band of approximately 250 kDa for each construct, which was detected using a non-reducing gel. The vACE2-Fc construct was shown to be expressed by HEK293 cells transduced with the AAV5-ACE2-Fc viral vector. Furthermore, a small fraction of the smaller protein (approximately 10-30%) was present in each lane, suggesting that the smaller protein was not glycosylated (Figure 23).

[0203] Anti-coronavirus emergency treatment Emergency treatment of coronavirus infection using recombinant ACE2-ECD-Fc or ACE2-vECD-Fc proteins. The ACE2-ECD-Fc or ACE2-vECD-Fc compositions were manufactured using recombinant technology as part of the production process. The viruses include, but are not limited to, β-group coronaviruses, and include severe respiratory syndrome (SARS) coronavirus (SARS-CoV-1), Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), the pathogen that caused the recent global pandemic CoVID-19, SARS-CoV-2, and the low-pathogenic HCoV-NL63.

[0204] Generic vaccines for SARS-CoV-1, SARS-CoV-2, MERS-CoV, and HCoV-NL63, etc. Preventing coronavirus infection by transducing many types of non-immune cells and administering a single injection of an AAV5-ACE2-vECD-Fc vector product that produces sufficient levels of ACE2-vECD-Fc in vivo.

[0205] When viral particles enter the body, ACE2-vECD-Fc acts as a neutralizing antibody, binding to the virus and forming an ACE2-vECD-Fc-SARS-CoV-2 complex. This complex can be eliminated by inactive and active immune cells. This is particularly important for the elderly, whose immune function is impaired and who may not achieve antibody levels when using inactivated viral vaccines, RNA vaccines, cDNA vaccines, and recombinant vaccines currently under development in the industry.

[0206] AAV can produce protective levels of ACE2-vECD-Fc over many years. This approach is superior for any type of vaccine under development. ACE2-vECD-Fc DNA can be cloned into protein expression plasmids for transfection into mammalian cell lines, yeast, or other fungal expression systems. The resulting cell lines can be used for the production of the ACE2-vECD-Fc protein product via large-scale fermentation and a series of purification steps. This product has been used for the emergency treatment of viral infections caused by SARS-CoV-1, SARS-CoV-2, MERS-CoV-1, or HCoV-NL63 infection, and has potential against future emerging coronaviruses that use the same receptor for entry. Furthermore, the virus:ACE2-vECD-Fc can be eliminated through an immune response pathway controlled by cells possessing the IgG1 receptor, ultimately terminating the viral replication cycle.

[0207] The selected substitution mutant is cloned into an adeno-associated virus (AAV) packaging plasmid. The AAV carrying the gene of interest (GOI) ACE2-vECD-Fc is called AAV5-ACE2-vECD-Fc and is manufactured and used for the treatment and prevention of coronavirus infection.

[0208] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by technicians of the art to which this disclosure belongs. Singular terms, “a,” “an,” and “the” include plural references unless otherwise clearly stated in the context. “Equipped with A or B” means including A, or B, or A and B. Furthermore, all base sizes or amino acid sizes, and the total molecular weight or molecular mass values ​​given for nucleic acids or polypeptides should be understood to be approximations and provided for illustrative purposes only. Methods and materials similar to or equivalent to those described herein may be used in the execution or examination of this disclosure, but suitable methods and materials are listed below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, including definitions of terms, this specification shall take precedence. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting.

[0209] Considering the many possible embodiments to which the disclosed principles of the present invention are applied, it should be recognized that the described embodiments are merely preferred embodiments of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, we claim as our invention everything that falls within the scope and spirit of these claims. Array List Sequence ID: 1. Extracellular domain (ECD) of the human ACE2 amino acid sequence qst ieeqaktfld kfnheaedlf yqsslaswny ntniteenvq 61 nmnnagdkws aflkeqstla qmyplqeiqn ltvklqlqal qqngssvlse dkskrlntil 121 ntmstiystg kvcnpdnpqe clllepglne imansldyne rlwaweswrs evgkqlrply 181 eeyvvlknem aranhyedyg dywrgdyevn gvdgydysrg qliedvehtf eeikplyehl 241 hayvraklmn aypsyispig clpahllgdm wgrfwtnlys ltvpfgqkpn idvtdamvdq 301 awdaqrifke aekffvsvgl pnmtqgfwen smltdpgnvq kavchptawd lgkgdfrilm 361 ctkvtmddfl tahhemghiq ydmayaaqpf llrnganegf heavgeimsl saatpkhlks 421 igllspdfqe dneteinfll kqaltivgtl pftymlekwr wmvfkgeipk dqwmkkwwem 481 kreivgvvep vphdetycdp aslfhvsndy sfiryytrtl yqfqfqealc qaakhegplh 541 kcdisnstea gqklfnmlrl gksepwtlal envvgaknmn vrpllnyfep lftwlkdqnk 601 nsfvgwstdw spyadqsikv rislksalgd kayewndnem ylfrssvaya mrqyflkvkn 661 qmilfgeedv rvanlkpris fnffvtapkn vsdiiprtev ekairmsrsr indafrlndn 721 sleflgiqpt lgppnqppvs Sequence ID: 2 ctkvtmddfl tahhemg h iq ydmayaaqpf llrnganegf h e avgeimsl Sequence ID: 3 ctkvtmddfl tahhemghiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 4 ctkvtmddfl tahAemghiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 5 ctkvtmddfl tahhemgAiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 6 ctkvtmddfl tahAemghiq ydmayaaqpf llrnganegf heavgeimsl Sequence ID: 7 ctkvtmddfl tahhemgAiq ydmayaaqpf llrnganegf heavgeimsl Sequence ID: 8 ctkvtmddfl tahAemgAiq ydmayaaqpf llrnganegf heavgeimsl Sequence ID: 9 ctkvtmddfl tahhQmghiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 10 ctkvtmddfl tahAQmghiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 11 ctkvtmddfl tahhQmgAiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 12 ctkvtmddfl tahAQmgAiq ydmayaaqpf llrnganegf hQavgeimsl Sequence ID: 13 ctkvtmddfl tahAQmghiq ydmayaaqpf llrnganegf heavgeimsl Sequence ID: 14 ​​ctkvtmddfl tahhQmgAiq ydmayaaqpf llrnganegf heavgeimsl Sequence ID: 15 ctkvtmddfl tahAQmgAiq ydmayaaqpf llrnganegf heavgeimsl Sequence ID: 16 AMI074 / G466D CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSL Sequence ID: 17 AMI081 CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHQAVGEIMSL SAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKD Sequence ID: 18 AMI083 CTKVTMDDFLTAHHQMGHIQYDMAYAAQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 19 AMI085 CTKVTMDDFLTAHAQMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSL Sequence ID: 20 AMI121 CTKVTMDDFLTAHAEMGRIQYDMAYVAQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 21 AMI122 CTKVTMDDFLTAHAEMGRIQYDMAYVAQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 22 AMI123 CTKVTMDDFLTAHAEMGHIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 23 AMI124 CTKVTMDDFLTAHAEMGRIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 24 AMI125 CTKVTMDDFLTAHAEMGAIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 25 AMI126 CTKVTMDDFLTAHAEMGRIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 26 AMI127 CTKVTMDDFLTAHLEMGHIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 27 AMI128 CTKVTMDDFLTAHAEMGHIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 28 AMI129 CTKVTMDDFLTAHLEMGRIQYDMAYALQPFLLRNGANEGFHQAVGEIMSL Sequence ID: 29 ACE2-ECD-Fc (Wildtype wild type) 18 qst ieeqaktfld kfnheaedlf yqsslaswny ntniteenvq 61 nmnnagdkwsaflkeqstlaqmyplqeiqnltvklqlqalqqngssvlse dkskrlntil 121 ntmstiystg kvcnpdnpqe clllepglne imansldyne rlwaweswrs evgkqlrply 181 eeyvvlknem aranhyedyg dywrgdyevn gvdgydysrg qliedvehtf eeikplyehl 241 hayvraklmn aypsyispig clpahllgdm wgrfwtnlys ltvpfgqkpn idvtdamvdq 301 awdaqrifke aekffvsvgl pnmtqgfwen smltdpgnvq kavchptawd lgkgdfrilm 361 ctkvtmddfl tahhemghiq ydmayaaqpf llrnganegf heavgeimsl saatpkhlks 421 igllspdfqe dneteinfll kqaltivgtl pftymlekwr wmvfkgeipk dqwmkkwwem 481 kreivgvvep vphdetycdp aslfhvsndy sfiryytrtl yqfqfqealc qaakhegplh 541 kcdisnstea gqklfnmlrl gksepwtlal envvgaknmn vrpllnyfep lftwlkdqnk 601 nsfvgwstdw spyadqsikv rislksalgd kayewndnem ylfrssvaya mrqyflkvkn 661 qmilfgeedv rvanlkpris fnffvtapkn vsdiiprtev ekairmsrsr indafrlndn 721 sleflgiqpt lgppnqppvs dkthtcppcpapellggpsvflfppkpkdtlmisr tpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmh Accession number: 33 A024 ATCCAGCCTCCGGACTCTAGAGTTAACTGGTAAGTTTAGT Accession number: 34 A056 GTTGCCTTTACTTCTAGGCCTGCCGCCACCatgGAGTTCGGCCTGAGCTGGCTGTTCCT Accession number: 35 A074 AACAGCTATGACCATG Sequence ID: 36 A098 ATGTACGGGCCAGATATACGCGTTCGTTACATAACTTACGGTAAA Sequence ID: 37 A120 TGATTATTGACTAGTATCTGCGTTACATAACTTACGGTAA Sequence ID: 38 A121 ACTCcatGGTGGCGGCAGGCCTAGAAGTAAAGGCAACATC Sequence ID: 39 A122 ATAAAGATATTTTATTTTCGAATTCTCAGC Sequence ID: 40 A123 CTGTTCTACCAGAGCAGCCTGGCCA Sequence ID: 41 A124 CTGGGAGAACAGCATGCTGACCGAC Sequence ID: 42 A125 AGAGCATCAAGGTGAGAATCAGCCT Sequence ID: 43 A126 CGGCCAGCCCGAGAACAACTACAAG Sequence ID: 44 A145 TCGTGGGGCACGGGCTCCACCACGC Sequence ID: 45 A146 GCGTGGTGGAGCCCGTGCCCCACGA Sequence ID: 46 A147 TGGGGGGGAACAGGAACACGCTGGG Sequence ID: 47 A148 GCGGCCCCAGCGTGTTCCTGTTCCC Sequence ID: 48 A156 GAATCCTGATGTGCACCAAGGTGACCATGGACGACTTCC Sequence ID: 49 A157 GGTGACCATGGACGACTTCCTGACCGCCCACGCCGAGATGGGCCACATC Sequence ID: 50 A158 GCATGTTGAACAGCTTCT Sequence ID: 51 A159 GACCATGGACGACTTCCTGACCGCCCACCACCAGATGGGCCACATCCAG Sequence ID: 52 A160 GACCATGGACGACTTCCTGACCGCCCACGCCCAGATGGGCCACATCCAG Sequence ID: 53 A161 CGCCAAGCTCTAGCTAGAGGTCGACGCGGCCGCTCGGTCCGCAC Sequence ID: 54 A162 TTCCTGCTGAGAAACGGCGCCAACGAGGGCTTCCACcAGGCCGTGGGCG Sequence ID: 55 A163 GGGGTCTCACGTTCATGTTC Sequence ID: 56 A169 GAGATGGATGGTGTTCAAGGGCGAGATCCCCAAGGACCAG Sequence ID: 57 A170 CTGGTCCTTGGGGATCTCGCCCTTGAACACCATCCATC Sequence ID: 58 A385 CCGAAGGGCACGGTCAGGCTGTACA Sequence ID: 59 A386 TGTACAGCCTGACCGTGCCCTTCGG ITR-QPCR-F: 5'-GGAACCCCTAGTGATGGAGTT-3' (Sequence ID: 61) ITR-QPCR-R: 5'-CGGCCTCAGTGAGCGA-3' (Sequence ID: 62) ITR-FAM-2ITR-MGB: 5'- CACTCCCTCTCTGCGCGCTCG-3' (Sequence ID: 63) Attached: Complete DNA sequences for each ACE2-ECD-Fc or ACE2-vECD-Fc variant. Sequence ID: 64 AMI074 Sequence number: 65 AMI080 Sequence ID: 66 AMI081 Sequence ID: 67 AMI082 Sequence ID: 68 AMI083 Sequence ID: 69 AMI084 Sequence ID: 70 AMI085 Sequence ID: 71 AMI089 Sequence ID: 72 AMI090 Sequence ID: 73 AMI121 Sequence ID: 74 AMI122 Sequence ID: 75 AMI123 Sequence ID: 76 AMI124 Sequence ID: 77 AMI125 Sequence ID: 78 AMI126 Sequence ID: 79 AMI127 Sequence ID: 80 AMI128 Sequence ID: 81 AMI129

Claims

1. An isolated angiotensin-converting enzyme 2 (ACE2) polypeptide having one or more mutations relative to wild-type ACE2 that cause loss of ACE2 enzyme activity compared to wild-type ACE2, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 10, 17, and 18.

2. A fusion protein comprising an isolated mutant ACE2 polypeptide according to claim 1, wherein the mutant ACE2 polypeptide is fused at its N-terminus or C-terminus to a peptide capable of binding to a receptor on an immune system-associated cell.

3. The fusion protein according to claim 2, characterized in that the peptide is a ligand that binds to the Fc-binding receptor (FcγR) on immune cells of lymphocytes.

4. The fusion protein according to claim 3, characterized in that the immune cells of the lymphocytes are selected from the group consisting of T cells, B cells, and natural killer cells.

5. The fusion protein according to claim 2, characterized in that the peptide is the Fc domain (Fcγ) of a human IgG antibody.

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