Monomer polypeptide comprising engineered form of angiotensin-converting enzyme 2

Abstract

A monomer polypeptide comprising an engineered form of Angiotensin-Converting Enzyme 2 (mp- eACE2) comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 2. The mp-eACE2 may be encoded by a nucleic acid sequence set forth in SEQ ID NO: 3..

Description

Ref- 1403008MONOMER POLYPEPTIDE COMPRISING ENGINEERED FORM OF ANGIOTENSINCONVERTING ENZYME 2TECHNICAL FIELD

[0001] The present disclosure is generally related to an engineered form of Angiotensin- Converting Enzyme 2 (ACE2), polynucleotides encoding the same, methods for producing the same, compositions comprising the same, and more particularly to an engineered form of ACE2 that may not have an enzymatic function.BACKGROUND

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a non-segmented positive-sense Ribonucleic acid (RNA) virus classified under the genus Beta Coronavirus in the Coronaviridae family. Its genome, consisting of approximately 30 kilobase pairs, encodes various structural and non-structural proteins. The trimeric spike (S) protein, which is anchored to the envelope of this virus, is regarded as the crucial protein facilitating the entry of the virus into host cells. The Angiotensin-Converting Enzyme 2 (ACE2) protein, has been identified as the primary human receptor for SARS-CoV-2 by binding ACE2 to S protein. This receptor is chiefly involved in regulating physiological functions related to vasoconstriction and blood pressure. As a type I membrane protein, ACE2 is expressed in various organs, including the lungs, heart, kidneys, and intestines. Research has also indicated that cardiovascular diseases are linked to decreased expression of ACE2. It has been reported that SARS-CoV-2 is capable of infecting only those cells that express ACE2. The significant importance of the interaction between ACE2 and receptorbinding domain (RBD) in the pathogenesis of SARS-CoV-2 suggests that the introduction of inhibitory agents to disrupt this interaction could effectively prevent the virus from entering host cells. Targeting the interfaces between ACE2 and (RBD) through the use of antibodies or other small therapeutic molecules has been extensively explored in various studies.Ref- 1403008

[0003] The development of such conventional therapeutics is inherently time-consuming and requires significant resources. Moreover, none of them has been a functional product that can bind to the RBD effectively. With this context, there is a need to develop new structures of molecules that may compete with the ACE2 receptor on the cell membrane for binding to the RBD with higher binding affinity to the RBD, without enzymatic activity and higher thermostability.SUMMARY

[0004] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more exemplary aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0005] In all contexts of the present disclosure, the abbreviation “mp-eACE2” is used instead of “monomer polypeptide comprising an exemplary engineered form of Angiotensin-Converting Enzyme 2”, and the abbreviation “n-ACE2” is used instead of “native form of Angiotensin-Converting Enzyme 2”. One general aspect describes an exemplary monomer polypeptide comprising an exemplary engineered form of Angiotensin-Converting Enzyme 2 (mp-eACE2). In one or more exemplary embodiments, an exemplary mp-eACE2 may comprise a plurality of mutations, compared to an exemplary native form of Angiotensin-Converting Enzyme 2 (n-ACE2), that may have an exemplary amino acid sequence set forth in SEQ ID NO: 1. In an exemplary embodiment, an exemplary mutation may be selected from the group consisting of one or more exemplary mutations for decreasing binding affinity to Angiotensin-2 (ANG2), one or more exemplary mutations for reducing enzymatic activity, one or more exemplary mutations for increasing thermostability, one or more exemplary mutations for increasing binding affinity to receptor-binding domain (RBD), and a combination thereof.Ref- 1403008

[0006] In one or more exemplary embodiments, an exemplary mp-eACE2 may comprise an exemplary truncation, compared to an exemplary n-ACE2 that may have an exemplary amino acid sequence set forth in SEQ ID NO: 1. In one or more exemplary embodiments, an exemplary truncation may include exemplary truncation of amino acid residues 694 to 805 of SEQ ID NO: 1. In an exemplary embodiment, an exemplary mp-eACE2 may comprise an exemplary amino acid sequence set forth in SEQ ID NO: 2. In an exemplary embodiment, an exemplary mp-eACE2 may be encoded by an exemplary nucleic acid sequence set forth in SEQ ID NO: 3.

[0007] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential / key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which an exemplary embodiment will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of one or more exemplary embodiments. One or more exemplary embodiments will now be described by way of example in association with the accompanying drawings in which:

[0009] FIG. 1 shows an exemplary flowchart of exemplary method for producing an exemplary monomer polypeptide comprising an exemplary engineered form of Angiotensin- Converting Enzyme 2 (an exemplary mp-eACE2) set forth in SEQ ID NO: 2, consistent with one or more exemplary embodiments of the present disclosure;Ref- 1403008

[0010] FIG. 2 illustrates the amino acids that participate in the interaction between the ps-ACE2 and the receptor-binding domain (RBD), consistent with one or more exemplary embodiments of the present disclosure;

[0011] FIG. 3 illustrates the plots of root mean square deviation (RMSD) for an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure;

[0012] FIG. 4 illustrates the plots of Root mean square fluctuation (RMSF) for an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure;

[0013] FIG. 5 illustrates the plots of radius of gyration (Rg) for an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure;

[0014] FIG. 6 illustrates the plot of flow cytometry carried out for evaluating the transfection rate in human embryonic kidney (HEK293T) cells using lentiviral vector, consistent with one or more exemplary embodiments of the present disclosure;

[0015] FIG. 7 illustrates the image of Bright-field and fluorescence microscopy capturing from the transfected cells, consistent with one or more exemplary embodiments of the present disclosure;

[0016] FIG. 8 illustrates the plot of flow cytometry to identify the best viral titer after the transduction, consistent with one or more exemplary embodiments of the present disclosure;

[0017] FIG. 9 illustrates the result of fluorescence microscopy and the transduction rate quantified by flow cytometry after the transduction, consistent with one or more exemplary embodiments of the present disclosure;

[0018] FIG. 10 illustrates the plot of flow cytometry in order to evaluate the Cluster of differentiation (CD) marker expression in Mesenchymal stem cells (MSCs) before transduction, consistent with one or more exemplary embodiments of the present disclosure;Ref- 1403008

[0019] FIG. 11 illustrates the image of flowcytometry in order to evaluate differentiation of MSCs into osteocytes and adipocytes before transduction, consistent with one or more exemplary embodiments of the present disclosure;

[0020] FIG. 12 illustrates the information of karyotyping in MSCs before transduction, consistent with one or more exemplary embodiments of the present disclosure;

[0021] FIG. 13 illustrates the image of fluorescence microscopy of the transduced cells in order to prove the presence of an exemplary mp-eACE2 gene, consistent with one or more exemplary embodiments of the present disclosure;

[0022] FIG. 14 illustrates the curve of cell death in MSCs after the process of puromycin selection, consistent with one or more exemplary embodiments of the present disclosure;

[0023] FIG. 15 illustrates the plot of flow cytometry carried out after puromycin selection of the transduced MSCs cells, consistent with one or more exemplary embodiments of the present disclosure;

[0024] FIG. 16 illustrates the image of osteogenic differentiation and adipocyte differentiation in MSCs after the transduction, consistent with one or more exemplary embodiments of the present disclosure;

[0025] FIG. 17 illustrates the information of karyotyping in the transduced MSCs, consistent with one or more exemplary embodiments of the present disclosure;

[0026] FIG. 18 illustrates the plot of flow cytometry in order to evaluate the CD marker expression in the transduced MSCs, consistent with one or more exemplary embodiments of the present disclosure;

[0027] FIG. 19 illustrates the result of polymerase chain reaction (PCR) by primer of ACE2 on genomic DNA of un-transduced MSCs, MSCs expressing the ps-ACE2 (ps-MSCs) (lane 1&2, under lOObp band), and MSCs expressing an exemplary mp-eACE2 (mp-MSCs) (lane 3,4,&5, under lOObp band), consistent with one or more exemplary embodiments of the present disclosure;Ref- 1403008

[0028] FIG. 20 illustrates the result of qRT-PCR by primer of ACE2 on cDNA of transduced and un-transduced MSCs, consistent with one or more exemplary embodiments of the present disclosure;

[0029] FIG. 21 illustrates the chart of Real Time qRT-PCR for evaluating the ACE2 expression, consistent with one or more exemplary embodiments of the present disclosure;

[0030] FIG. 22 illustrates the level of the ACE2 in un-transduced, mp-MSC, and ps-MSC evaluating by western blot, consistent with one or more exemplary embodiments of the present disclosure;

[0031] FIG. 23 illustrates the sandwich enzyme-linked immunosorbent assay (ELISA) result for evaluating the affinity to the spike (S) protein of Coronavirus disease 2019 (COVID- 19) in both groups of an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure;

[0032] FIG. 24 illustrates the thermo- stability of an exemplary mp-eACE2 and an the ps- ACE2 evaluated in different temperatures by sandwich ELISA, consistent with one or more exemplary embodiments of the present disclosure;

[0033] FIG. 25 illustrates the enzyme activity of an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure; and

[0034] FIG. 26 illustrates the cytopathic effects (CPE) as a result of COVID-19 infection and proliferation in Vero 76-clone E6 (Vero E6 cells) (cell death and cell fusion), consistent with one or more exemplary embodiments of the present disclosure.DETAILED DESCRIPTION

[0035] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitryRef- 1403008have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0036] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in one or more exemplary embodiments. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of one or more exemplary embodiments. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of one or more exemplary embodiments. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0037] Provided herein is an exemplary monomer polypeptide comprising an exemplary engineered form of Angiotensin-Converting Enzyme 2, an exemplary polynucleotide or exemplary polynucleotides encoding an exemplary engineered form of Angiotensin-Converting Enzyme 2, an exemplary method for producing an exemplary engineered form of Angiotensin-Converting Enzyme 2, and an exemplary method for producing an exemplary polynucleotide encoding an exemplary engineered form of Angiotensin-Converting Enzyme 2. Furthermore, one or more exemplary embodiments may be directed to an exemplary vector that may harbor an exemplary polynucleotide encoding an exemplary engineered form of Angiotensin-Converting Enzyme 2. In one or more exemplary embodiments, exemplary host cells may carry an exemplary vector that may harbor an exemplary polynucleotide encoding an exemplary engineered form of Angiotensin-Converting Enzyme 2. “Monomer polypeptide” may refer to a peptide chain that has only one peptide chain. In all context of the present disclosure, the abbreviation of “mp-eACE2” is used instead of “monomer polypeptide comprising an exemplary engineered form of Angiotensin-Ref- 1403008Converting Enzyme 2”, and the abbreviation of “n-ACE2” is used instead of “native form of Angiotensin-Converting Enzyme 2”.

[0038] An exemplary mp-eACE2, disclosed in one or more exemplary embodiments, may include one or more exemplary truncations compared to an exemplary n-ACE2. “Native” may refer to an exemplary gene, an exemplary allele, or an exemplary polypeptide / protein that may be the most commonly noted genotype and / or phenotype in nature. Therefore, a native gene may be defined as an exemplary sequence, whether it be a nucleic acid or an amino acid / protein sequence, that is typically present in an exemplary organism in its natural or wild form. “Truncation” may refer to the elimination of one or more fragments / segments of an exemplary polynucleotide (e.g., an exemplary gene) and / or an exemplary polypeptide (e.g., an exemplary protein). Hence, an exemplary truncated protein enzyme and / or gene may lack one or more exemplary truncations.

[0039] In one or more exemplary embodiments, an exemplary mp-eACE2 may lack one or more exemplary domains compared to an exemplary n-ACE2. In an exemplary embodiment, an exemplary mp-eACE2 may lack one exemplary domain compared to an exemplary n-ACE2, and that exemplary domain may be related to an exemplary enzyme function of ACE2. In an exemplary embodiment, an exemplary n-ACE2 may refer to an exemplary native form of ACE2 that may have an exemplary amino acid sequence set forth in SEQ ID NO: 1 (also set forth in GenBank: NM_021804.3). Furthermore, an exemplary n-ACE2 may refer to one or more exemplary amino acid sequences with at least 80%, at least 85%, at least 90%, at least 95%, and / or at least 98.5% sequence identity to SEQ ID NO: 1.

[0040] In one or more exemplary embodiments, an exemplary n-ACE2 may include three regions comprising an exemplary extracellular domain, an exemplary transmembrane domain, and an exemplary cytoplasmic domain. In one or more exemplary embodiments, i) residue 18-740 of SEQ ID NO:1 may compose an exemplary extracellular domain of an exemplary n-ACE2 (SEQ ID NO: 1); ii) residue 741-761 of SEQ ID NO:1 may compose an exemplary transmembrane domain of an exemplary n-ACE2 (SEQ ID NO: 1); and iii) residue 762-805 of SEQ ID NO:1 mayRef- 1403008compose an exemplary cytoplasmic domain of an exemplary n-ACE2 (SEQ ID NO: 1). In one or more exemplary embodiments, an exemplary extracellular domain of an exemplary n-ACE2 (SEQ ID NO: 1) may include an exemplary receptor-binding domain (RBD) binding domain and an exemplary enzymatic domain.

[0041] An exemplary end of an exemplary polypeptide or polynucleotide may not be limited only to exemplary first or final sites of a polypeptide or polynucleotide but may comprise extra amino acids or nucleotides in exemplary terminal segments of a polypeptide or polynucleotide. A polypeptide may have both a C-terminus, which is concluded by an amino acid containing a free carboxyl group (COOH), and an N-terminus, which is concluded by an amino acid featuring a free amino group (NH2). In an exemplary embodiment, exemplary C-termini and / or N-termini of exemplary polypeptides may be altered to either conclude or initiate with an exemplary nonpolypeptide moiety, such as an organic conjugate.

[0042] In one or more exemplary embodiments, an exemplary mp-eACE2 may have an exemplary amino acid sequence of about 626 amino acids. In an exemplary embodiment, an exemplary mp-eACE2 may include an exemplary amino acid sequence set forth in SEQ ID NO: 2. In one or more exemplary embodiments, an exemplary mp-eACE2 (SEQ ID NO: 2), compared to an exemplary n-ACE2 (SEQ ID NO: 1), may include an exemplary truncation of amino acid residues 616 to 805. In one or more exemplary embodiments, an exemplary mp-eACE2 (SEQ ID NO: 2), compared to an exemplary n-ACE2 (SEQ ID NO: 1), may be different in one or more residues. In one or more exemplary embodiments, an exemplary mp-eACE2 (SEQ ID NO: 2), compared to an exemplary n-ACE2 (SEQ ID NO: 1), may include one or more exemplary mutations. In an exemplary embodiment, an exemplary mutation may be selected from the group consisting of one or more exemplary mutations for decreasing binding affinity to Angiotensin-2 (ANG2), one or more exemplary mutations for reducing enzymatic activity, one or more exemplary mutations for increasing thermostability, one or more exemplary mutations for providing a higher binding affinity to RBD, and a combination thereof.Ref- 1403008

[0043] In one exemplary embodiment, exemplary mutations for decreasing binding affinity to ANG2 may include an exemplary mutation on residue 273 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, and an exemplary mutation on residue 445 of an exemplary n-ACE2 set forth in SEQ ID NO: 2. In an exemplary embodiment, an exemplary mutation on residue 445 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for decreasing binding affinity to ANG2 may include substituting residue 445 comprising threonine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glycine. In an exemplary embodiment, an exemplary mutation on residue 273 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for decreasing binding affinity to ANG2 may include substituting residue 273 comprising arginine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glutamine.

[0044] In one exemplary embodiment, exemplary mutations for reducing the enzymatic activity may include an exemplary mutation on residue 273 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, and an exemplary mutation on residue 445 of an exemplary n-ACE2 set forth in SEQ ID NO: 2. In an exemplary embodiment, an exemplary mutation on residue 445 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for reducing enzymatic activity may include substituting residue 445 comprising threonine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glycine. In an exemplary embodiment, an exemplary mutation on residue 273 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for reducing enzymatic activity may include substituting residue 273 comprising arginine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glutamine.

[0045] In one exemplary embodiment, exemplary mutations for increasing binding affinity to RBD may include an exemplary mutation on residue 27 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 28 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 31 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, and an exemplary mutation on residue 38 of an exemplary n-ACE2 set forth in SEQ ID NO: 2. In an exemplary embodiment, an exemplary mutation on residue 27 of an exemplary n-ACE2 set forthRef- 1403008in SEQ ID NO: 2 for increasing binding affinity to RBD may include substituting residue 27 comprising threonine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with arginine. In an exemplary embodiment, an exemplary mutation on residue 28 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing binding affinity to RBD may include substituting residue 28 comprising phenylalanine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with tryptophane. In an exemplary embodiment, an exemplary mutation on residue 31 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing binding affinity to RBD may include substituting residue 31 comprising lysine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glutamine. In an exemplary embodiment, an exemplary mutation on residue 38 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing binding affinity to RBD may include substituting residue 38 comprising aspartic acid of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with valine.

[0046] In one exemplary embodiment, exemplary mutations for increasing thermostability may include an exemplary mutation on residue 448 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 489 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 481 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 396 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 574 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 355 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 590 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 113 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 430 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 339 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 451 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 301 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 551 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 507 of an exemplary n-ACE2 set forth in SEQ ID NO: , anRef- 1403008exemplary mutation on residue 526 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 178 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 500 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 254 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, an exemplary mutation on residue 316 of an exemplary n-ACE2 set forth in SEQ ID NO: 2, and an exemplary mutation on residue 299 of an exemplary n-ACE2 set forth in SEQ ID NO: 2.

[0047] In an exemplary embodiment, an exemplary mutation on residue 448 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 448 comprising glycine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with tryptophane. In an exemplary embodiment, an exemplary mutation on residue 489 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 489 comprising glutamine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with proline. In an exemplary embodiment, an exemplary mutation on residue 481 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 481 comprising lysine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with leucine. In an exemplary embodiment, an exemplary mutation on residue 396 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 396 comprising alanine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with tyrosine. In an exemplary embodiment, an exemplary mutation on residue 574 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 574 comprising valine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with asparagine. In an exemplary embodiment, an exemplary mutation on residue 355 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 355 comprising aspartic acid of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with threonine. In an exemplary embodiment, an exemplary mutation on residue 590 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 590 comprising proline of an exemplary n-ACE2Ref- 1403008set forth in SEQ ID NO: 2 with glutamine. In an exemplary embodiment, an exemplary mutation on residue 113 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 113 comprising serine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with leucine. In an exemplary embodiment, an exemplary mutation on residue 430 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 430 comprising glutamine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with tryptophane. In an exemplary embodiment, an exemplary mutation on residue 339 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 339 comprising valine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glycine. In an exemplary embodiment, an exemplary mutation on residue 451 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 451 comprising proline of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with methionine. In an exemplary embodiment, an exemplary mutation on residue 301 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 301 comprising alanine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glycine. In an exemplary embodiment, an exemplary mutation on residue 551 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 551 comprising glycine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with tryptophane. In an exemplary embodiment, an exemplary mutation on residue 507 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 507 comprising serine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with phenylalanine. In an exemplary embodiment, an exemplary mutation on residue 526 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 526 comprising glutamine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with isoleucine. In an exemplary embodiment, an exemplary mutation on residue 178 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 178 comprising proline of anRef- 1403008exemplary n-ACE2 set forth in SEQ ID NO: 2 with glutamine. In an exemplary embodiment, an exemplary mutation on residue 500 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 500 comprising proline of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with tryptophane. In an exemplary embodiment, an exemplary mutation on residue 254 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 254 comprising serine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with glycine. In an exemplary embodiment, an exemplary mutation on residue 316 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 316 comprising valine of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with arginine. In an exemplary embodiment, an exemplary mutation on residue 299 of an exemplary n-ACE2 set forth in SEQ ID NO: 2 for increasing thermostability may include substituting residue 299 comprising aspartic acid of an exemplary n-ACE2 set forth in SEQ ID NO: 2 with alanine.

[0048] Angiotensin-converting enzyme 2 (ACE2) has a short half-life of only a few hours in both humans and mice, according to pharmacokinetic studies. One of the most crucial elements influencing the protein half-life is resistance to proteolytic degradation. Important factors that influence proteolytic stability include the thermal and structural stability of proteins. In their natural shape, proteins are resistant to being broken down by proteases. After unfolding, they are quite susceptible to proteases. Even in the very compact and thermostable native conformation, unspecific proteases have the ability to target the accessible and flexible loop regions of proteins. To prevent any detrimental impacts on the binding affinity, exemplary mutations may be chosen for locations that are distant from the RDB binding site. Increasing the thermodynamic stability and thermostability of ACE2 may improve the half-life of the protein. An exemplary mp-eACE2, which is more thermostable and devoid of enzymatic activity, may bind to RBD with greater affinity. Since ACE2 is thought to be the primary RBD binding receptor, using it as an inhibitory drug gives it an evolutionary advantage over other treatments that are often used. Utilizing ACE2'sRef- 1403008extracellular domain also lessens the likelihood of negative side effects and immune reactions to this medicinal molecule.

[0049] In one or more exemplary embodiments, an exemplary mp-eACE2 (SEQ ID NO: 2) may include an exemplary amino acid sequence with at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 2. In an exemplary embodiment, an exemplary amino acid sequence of an exemplary mp-eACE2 may be at least 95 to 99.5% identical to SEQ ID NO: 2. In one or more exemplary embodiments, an exemplary mp-eACE2 (SEQ ID NO: 2) set forth in SEQ ID NO: 2 may have a molecular weight between 50 and 100 kiloDalton (kDa) compared to exemplary n-ACE2 (SEQ ID NO: 1) that may have a molecular weight of about 92 kDa. In one exemplary embodiment, an exemplary mp-eACE2 (SEQ ID NO: 2) set forth in SEQ ID NO: 2 may have a molecular weight of 72 kDa. “Identity” and “identical” may refer to a relationship that exists among exemplary nucleic acid sequences of two or more polynucleotides, which may be established through the comparison of exemplary sequences. Identity may also refer to the extent of relationship among sequences, as recognized by experts in the field, by assessing the number of matches between the residues of two or more nucleic acid strings. Identity may be determined by assessing the quantity of matching elements between two or more reference sequences, ideally focusing on the smaller sequence. This process may also take into account any gap alignments, which can be analysed using a computer program, such as algorithms, or through a specific mathematical model. The concept of identity, when applied to two or more exemplary polynucleotide sequences, pertains to the proportion of residues (specifically, nucleic acid residues) in a candidate nucleic acid sequence that are identical to those in a second exemplary nucleic acid sequence. This assessment is conducted following the alignment of the sequences, including any necessary gap alignments, to determine the highest possible percentage of identity. In one or more exemplary embodiments,Ref- 1403008exemplary variants of an exemplary polynucleotide (i.e., an exemplary reference polynucleotide) may exhibit a sequence identity ranging from a minimum of 40% to a maximum of 99% while remaining below 100% in comparison to an exemplary reference polynucleotide.

[0050] In one or more exemplary embodiments, an exemplary mp-eACE2 may also include an exemplary amino acid sequence with at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence homology to SEQ ID NO: 2. In an exemplary embodiment, an exemplary amino acid sequence of an exemplary mp-eACE2 may be at least 95 to 99.5% homologous to SEQ ID NO: 2. “Homology” may mean that exemplary sequences that are aligned or compared may have originated from a common origin. Homologous sequences suggest that a primary nucleic acid sequence or amino acid sequence (such as a protein or gene sequence, whether Deoxyribonucleic acid (DNA) or Deoxyribonucleic acid (RNA)) may be connected to a secondary nucleic acid sequence or amino acid sequence due to their derivation from a shared ancestral sequence. Homolog may also denote a connection between proteins and / or genes that have diverged due to speciation, or it may refer to a relationship between proteins and / or genes that have arisen from genetic duplication. Two exemplary nucleic acid sequences may be regarded as homologous if exemplary polypeptides they encode contain one or more segments of at least 20 amino acids that exhibit a degree of identity of at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99%.

[0051] In one or more exemplary embodiments, exemplary polynucleotides may include one or more exemplary modifications or segments (for instance, a specific sequence) that may result in advantageous characteristics, such as enhanced stability, durability, expression, or translation. Additionally, exemplary polynucleotides may contain an extra moiety for subcellular targeting or tracking purposes, such as a detectable label or tag, like a fluorescent marker, as well as a binding site for a protein or protein complex. In one or more exemplary embodiments, exemplaryRef- 1403008modifications may include, but are not limited to, modifications that may provide alterations that introduce various chemical groups that may enhance or incorporate additional characteristics such as charge, hydrophobicity, polarizability, electrostatic interactions, hydrogen bonding, and fluxionality to the polynucleotide bases or to the polynucleotide in its entirety. In one or more exemplary embodiments, exemplary modifications may yield oligonucleotides that are resistant to nuclease activity; such exemplary modifications may include one or more altered sugars, altered bases, inter-nucleotide linkages, and a combination thereof. Exemplary modifications may further include, but are not limited to, 2'-position sugar modifications, '5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, phosphorothioate or alkyl phosphate modifications, 4-thiouridine substitution, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylations, and unusual base-pairing combinations such as isobases isocytidine and isoguanidine. Modifications may further include 3' and 5' modifications, such as capping. Other non-limiting examples of modification may include a stability control sequence, addition of a 5' cap (e.g., a 3' polyadenylated tail (i.e., a 3' poly-Adenosine tail), a riboswitch sequence to facilitate controlled access by exemplary proteins and / or protein complexes and / or regulated stability, a 7-methylguanylate cap (m7G)), an exemplary sequence that may form a dsRNA secondary structure (e.g., a hairpin), an exemplary modification or an exemplary sequence that may create an exemplary binding site for proteins (e.g., exemplary proteins that may execute an exemplary function when landing on a DNA — including transcriptional repressors, an exemplary sequence or modification that may target an exemplary RNA to a subcellular location (such as nucleus, mitochondria, chloroplasts, etc.), transcriptional activators, histone acetyltransferases, DNA methyltransferases, histone deacetylases, DNA demethylases, etc.), an exemplary modification or sequence that may be used for tracking (e.g., an exemplary sequence that may provide conjugation to a moiety that could enable fluorescent detection, fluorescent detection, direct conjugation to a fluorescent molecule, etc.), and combinations thereof.Ref- 1403008

[0052] In one or more exemplary embodiments, exemplary polynucleotides may further include exemplary non-natural nucleotide(s). In an exemplary embodiment, an exemplary nonnatural nucleotide may refer to a nucleotide that has been artificially synthesized and may exhibit similarities in chemical properties and / or structure to a natural nucleotide. Examples of non-natural nucleotide may include — but are not limited to arabino-nucleoside, a-deoxyribonucleoside, a basic nucleoside, -L-deoxyribonucleoside, 2'-deoxyuridine, and other glycosylated nucleosides. In an exemplary embodiment, exemplary glycosylated nucleosides may comprise glycosylated nucleosides having arabinose, substituted pentose (2Z-O-methyl ribose, 2'-deoxy-2'-fluororibose, 3'-O-methyl ribose, or l',2'-deoxyribose), substituted arabinose sugar, alpha anomer, or substituted hexose. Exemplary non-natural nucleosides may further comprise an artificially created base analogue or a chemically altered base that has been artificially constructed. In an exemplary embodiment, examples of base analogue may comprise a 5-substituted 2-oxo(lH)-pyridine-3-yl group, a 2-oxo(lH)-pyridine-3-yl group, a 2-oxo(lH)-pyridine-3-yl group, a 2-amino-6-(2-oxazolyl) purine-9-yl group, and a 2-amino-6-(2-thiazolyl) purine-9-yl group. Examples of the chemically altered base that has been artificially constructed may include modified purine (e.g., 6-methyladenine and 6-thioguanosine), modified pyrimidine (e.g., 5-hydroxycytosine, 5-fluorouracil, and 4 -thiouracil), and other heterocyclic bases.

[0053] It is acknowledged that any exemplary nucleic acid sequences disclosed in one or more exemplary embodiments may be synthesized using commercially available poly / oligonucleotide synthesizers. Methods of synthetic oligonucleotide synthesis include, but are not limited to, liquidphase oligonucleotide synthesis, solid-phase oligonucleotide synthesis, and additional methods recognized in the art.

[0054] FIG. 1 shows an exemplary flowchart of exemplary method 100 for producing an exemplary mp-eACE2 set forth in SEQ ID NO: 2, consistent with one or more exemplary embodiments of the present disclosure. Exemplary method 100 may comprise one or more steps in relation to the exemplary features and embodiments outlined above. In an exemplaryRef- 1403008embodiment, exemplary method 100 may include: providing an exemplary polynucleotide encoding an exemplary mp-eACE2 having an exemplary amino acid sequence set forth in SEQ ID NO: 2 (e.g., providing an exemplary polynucleotide set forth in SEQ ID NO: 3) (step 102); cloning of exemplary polynucleotide encoding an exemplary mp-eACE2 with an exemplary amino acid sequence set forth in SEQ ID NO: 2 into an exemplary expression vector (step 104); transferring an exemplary expression vector into an exemplary host cell to express an exemplary mp-eACE2 (SEQ ID NO: 2) (step 106); and extracting and purifying an exemplary mp-eACE2 (SEQ ID NO: 2) from an exemplary host cell using an exemplary extraction and purification method (step 108).

[0055] Referring to FIG.l, step 102 may include providing an exemplary polynucleotide encoding an exemplary mp-eACE2 having an exemplary amino acid sequence set forth in SEQ ID NO: 2. In an exemplary embodiment, an exemplary polynucleotide encoding an exemplary mp-eACE2 may have an exemplary nucleotide sequence set forth in SEQ ID NO: 3. In an exemplary embodiment, an exemplary polynucleotide encoding an exemplary mp-eACE2 may be obtained utilizing an exemplary nucleotide sequence of SEQ ID NO: 2 as a template and at least one pair of specific primers. For example, in order to obtain an exemplary nucleotide sequence of SEQ ID NO: 3, an exemplary forward primer set forth in SEQ ID NO: 4 and an exemplary reverse primer set forth in SEQ ID NO: 5 may be utilized. In one or more exemplary embodiments, an exemplary polynucleotide encoding an exemplary mp-eACE2 may be produced synthetically, e.g., by using an exemplary commercially available gene / oligo synthesizer.

[0056] Referring to FIG.l, step 104 may include cloning of an exemplary polynucleotide encoding an exemplary mp-eACE2 with an exemplary amino acid sequence set forth in SEQ ID NO: 2 in an exemplary expression vector. In an exemplary embodiment, step 104 may include cloning of an exemplary polynucleotide set forth in SEQ ID NO: 3 in an exemplary expression vector. In an exemplary embodiment, an exemplary molecular cloning method may be used to provide an exemplary expression vector harboring an exemplary nucleic fragment of interest. Exemplary molecular cloning methods may include amplification of an exemplary interest-relatedRef- 1403008DNA fragment prior to being placed into an exemplary vector. After that, an exemplary expression vector may be inserted into an exemplary host cell or organism. Then, exemplary host cells that carry out an exemplary expression vector (that may harbor an exemplary interest related DNA fragment) may be screened. “Vector” may refer to a device that may facilitate an exemplary nucleic acid entity to move from one environment to another. In one or more exemplary embodiments, an exemplary vector may be replicated when linked to one or more exemplary control elements. A vector may refer to a nucleic acid molecule that may have the potential to carry another nucleic acid molecule, to which it is linked, into a host cell. Exemplary vectors may include, but are not limited to: i) nucleic acid molecules that possess either no free ends, such as circular forms, or one or more free ends; ii) single-stranded nucleic acid molecules, double- stranded nucleic acid molecules, or not fully double-stranded nucleic acid molecules, iii) nucleic acid molecules including DNA, RNA, or both, and iv) other groups of nucleic acid molecules. A specific category of vectors may include plasmids, which are defined as exemplary circular double- stranded DNA loops that allow for the insertion or cloning of additional DNA fragments, typically utilizing exemplary standard molecular cloning methods. Another category of vectors may include an exemplary viral vector including, exemplary virally -derived DNA or RNA, which may be necessary for packing into an exemplary virus (e.g., replication defective retroviruses, adenoviruses, adeno-associated viruses, retroviruses, and replication defective adenoviruses). Exemplary viral vectors may also comprise exemplary polynucleotides that may be usually transferred by viruses for transfection into an exemplary cell or an exemplary host cell. Some vectors may be able to replicate independently in an exemplary host cell into which they have been inserted (e.g., bacterial vectors and / or episomal mammalian vectors / plasmids containing a bacterial origin of replication (Ori)). Other vectors, including non-episomal mammalian vectors, may be unified into a genome upon entering into an exemplary host cell and may be replicated beside of an exemplary host cell's genome. In one or more exemplary embodiments, selection of a suitable exemplary vector may rely on the type of exemplary host cell / host organism in which anRef- 1403008exemplary vector may be inserted. “Host organism / host cell” may refer to an exemplary cell which may have been transformed or may be able of being transformed, by an exemplary exogenous sequence of nucleic acid. An exemplary host organism / host cell may include, but is not limited to, prokaryotic cells such as Escherichia coli (E. coli) cells, eukaryotic cells such as yeast cells and insect cells, animal cells (e.g., mammalian cells, such as human cells, mouse cells, etc.), and plant cells. In an exemplary embodiment, an exemplary vector may be an independently replicating vector (e.g., an exemplary plasmid). In one or more exemplary embodiments, as mentioned above, an exemplary vector may be able to be unified into the genome of an exemplary host cell / host organism — partially or in its entirety — and may be replicated along with an exemplary chromosome into which it has been unified. In one or more exemplary embodiments, an exemplary expression vector may further include an exemplary antibiotic-resistance marker, one or more exemplary Ori, an exemplary promoter that may supply a binding site for starting transcription of a gene of interest (e.g., an exemplary gene of an exemplary mp-eACE2 gene set forth in SEQ ID NO: 3), a ribosomal binding site (RBS), one or more exemplary regulatory components, a Poly-A (polyadenylation) tail (adjusted to protect an mRNA from modification by nucleases and for finishing translation and / or transcription processes), an exemplary transcription finishing site, an exemplary reporter gene (adjusted to make a reporter protein that may be quantified and / or identified with an exemplary assay), and an exemplary multiple cloning site (MCS) / poly linker having one or more exemplary restriction sites. Multiple cloning site (MCS), cloning site, or polylinker may refer to a fragment on an exemplary vector that may be originated with the purpose of transferring one or more nucleic acid sequences into an exemplary vector of interest — for example, to insert a nucleic acid sequence having an ORF encoding a definite polypeptide (e.g., the polynucleotide (SEQ ID NO: 3) encoding an exemplary mp-eACE2 set forth in SEQ ID NO: 2). In one or more exemplary embodiments, an exemplary promoter may include — but is not limited to — lac, trp, GAP (glucose aldehyde 3-phosphate), tac, A0X1, Z.PL, GALI, GAL10, nmt81, nmtl, and nmt42 promoters. An exemplary antibiotic -resistance marker may prepare antibiotic -resistanceRef- 1403008to an exemplary bacteria harboring an exemplary antibiotic -resistance marker and may make detection of an exemplary transformed bacteria possible on an exemplary selective media (i.e., an exemplary antibiotic-supplemented growth media). In one or more exemplary embodiments, an exemplary reporter gene may be used to detect localization of intracellular of an exemplary expressed protein (e.g., an exemplary mp-eACE2 set forth in SEQ ID NO: 2), and also to evaluate efficiency of gene expression. An exemplary reporter gene may include — but is not limited to — Luciferase encoding gene, lac Z gene, CAT (chloramphenicol acetyltransferase) gene, etc. In one or more exemplary embodiments, one or more exemplary sequence tags may be utilized for detection, purification, and / or localization of an exemplary protein of interest (e.g., an exemplary mp-eACE2 set forth in SEQ ID NO: 2).

[0057] With further reference to FIG. 1, step 106 may include transferring an exemplary expression vector into an exemplary host cell to express an exemplary mp-eACE2 (SEQ ID NO: 2). Expressing an exemplary mp-eACE2 may refer to the synthesis of an exemplary mp-eACE2 inside an exemplary host cell, including a yeast, bacteria, animal, or plant cell. For example, in an exemplary embodiment, an exemplary host cell / host organism may be an exemplary E. coli strain. In one or more exemplary embodiments, transferring an exemplary expression vector into an exemplary host cell (eukaryote, prokaryote, etc) / host organism may be carried out using any procedure by which nucleic acids may be transferred into an exemplary host cell / host organism. Exemplary procedures may include, but are not limited to, electroporation fusion, protoplast, calcium phosphate (CaPCU) precipitation, calcium chloride (CaCh) precipitation, lipofectamine-mediated transformation, polyethylene glycol (PEG)-mediated transformation, agrobacterium-mediated transformation, desiccation / inhibition-mediated transformation dextran, sulphate -mediated transformation, and agitation with silicon carbide fiber, etc.

[0058] With further reference to FIG. 1, step 108 may include extracting and purifying an exemplary mp-eACE2 (SEQ ID NO: 2) from an exemplary host cell / organism using an exemplary extraction and purification method. In an exemplary embodiment, extraction of an exemplary mp-Ref- 1403008eACE2 (SEQ ID NO: 2) may be carried out by disturbing an exemplary host cell / host organism using an exemplary technique including, but not limited to, homogenizer disrupting, ultrasonic treatment, grinding, high pressure extrusion, lysozyme treatment, etc. In one or more exemplary embodiments, exemplary purification method set forth in step 108 may include, but is not limited to, precipitation and differential solubilization, ultracentrifugation, gradient centrifugation, and chromatography. In one or more exemplary embodiments, step 108 may further include deleting reductant from an exemplary mp-eACE2 (SEQ ID NO: 2) using an exemplary method including, but not limited to, chromatography, dialysis, and ultrafiltration.

[0059] In one or more exemplary embodiments, an exemplary mp-eACE2 may be applied as an exemplary treatment for curing COVID-19 disease. For this purpose, an exemplary mp-eACE2 may be expressed on an exemplary surface of an exemplary eukaryote host cell (e.g., mesenchymal stem cell). After administrating an exemplary host cell expressing an exemplary mp-eACE2, the RBD domain of the spike (S) protein may bind to an exemplary mp-eACE2. Therefore, exemplary n-ACE2 of the human body may survive from binding to the RBD.

[0060] It is to be understood that, in one or more exemplary embodiments, exemplary amino acid sequences may be provided by in vitro translation / transcription or by a recombinant way. Exemplary amino acid sequences may also be produced synthetically, e.g., using a commercially peptide synthesizer. Methods of synthetic peptide producing may include, but are not limited to, liquid-phase peptide synthesis, solid-phase peptide synthesis, etc.EXAMPLES

[0061] Hereinafter, one or more exemplary embodiments will be described further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of the present disclosure. In all contexts of the present disclosure, the abbreviation “mp-eACE2” was used instead of “monomer polypeptide comprising an exemplary engineered form of Angiotensin-Ref- 1403008Converting Enzyme 2”, and the abbreviation “n-ACE2” was used instead of “native form of Angiotensin-Converting Enzyme 2”.Example 1: Obtaining an exemplary sequence encoding an exemplary mp-eACE2

[0062] The protein sequences corresponding to the receptor-binding domain (RBD) of the Coronavirus disease 2019 (COVID-19) spike (S) protein and the n-ACE2 were retrieved from databases. The three-dimensional (3D) structures of these proteins, as well as their interacting complexes, were sourced from the Protein Data Bank, utilizing the most recent and highest resolution structures available. “PDB editor” was used to delete the unnecessary domains of the optimum structures. The sequence of the RBD and the n-ACE2 was achieved from NCBI with NO: QHD43416, and 001358344, respectively. The 3D structure for interactions between the complex of the RBD and the extracellular domain of the n-ACE2 was saved with NO: 6LZG. The unnecessary domains of 6LZG were deleted. The extracellular domain is composed of two main domains, comprising the Peptidase M2 domain, which includes the enzymatic domain for binding to RBD, and the Collectrin domain. To save the necessary structural characterization, the whole sequence domain of Peptidase M2 was selected for mutation. The domain of Collectrin like, including residues 615 to 805 of the n-ACE2, was deleted. Three complementary sequences were added to an exemplary sequence encoding Peptidase M2 domain and the final sequences was called ps-ACE2 with an exemplary SEQ ID NO:6.

[0063] The structure of Angiotensin-2 (ANG2) was achieved from the structure complex with NO: 6OS0. Due to the absence of resolved structures for the complex between the ps-ACE2 and the RBD, the molecular structures were docked using servers. The resulting complex between the ps-ACE2 and the RBD was then utilized to identify the most promising candidates for point mutations. The molecular docking studies conducted between the ps-ACE2 and the ANG2 revealed that the ANG2 is capable of establishing a strong interaction with the ps-ACE2 at the anticipated central cleavage site of the enzyme.Ref- 1403008

[0064] The residue interaction network involved in the complex between the ps-ACE2 and the ANG2 was derived from the tertiary structure of their complex. To ensure robust and dependable interactions, a stringent distance threshold was applied. The methodology for identifying interacting pairs relied on measuring the distance between the mass centres of two residues in contact. All interaction types were included in this comprehensive analysis. The resulting network was subsequently analysed and visualized. To identify critical residues essential for the proper folding and functionality of the protein, a centrality analysis was performed. This centrality analysis focused on the variation in average shortest path length resulting from the removal of each residue (node) in the network and was incorporated into RINSpector.

[0065] The shortest path length represents the minimum number of edges required to connect one node to another within the network. The average shortest path is defined as the average of these values across all potential residue pairs in the interaction network. The significance of the average shortest path length is assessed through the calculation of the Z-score, with Z-scores exceeding 2 considered significant. The complex between the ANG2 and the ps-ACE2 demonstrates that several amino acids participate in the hydrophobic interactions and the hydrogen bonding. Specifically, the arginine 273 and threonine 445 residues of the ps-ACE2 molecule are involved in the hydrogen bonding. Given the crucial role of hydrogen bonds in maintaining the stability of protein-peptide interactions, these amino acids are likely to play essential roles in the stability of the complex between the ps-ACE2 and the ANG2. According to the result of centrality analysis, arginine 273 and threonine 445 have a high Z-score. Moreover, threonine 445 is bound to arginine 2 of the ANG2 by a hydrogen bond. However, arginine 273 is bound to aspartic acid 1 of the ANG2 by a hydrogen bond. Arginine 2 of ANG2 is defined as a central amino acid.

[0066] The molecular docking studies revealed that the ANG2 is capable of establishing a strong interaction with the ps-ACE2 at the anticipated central cleavage site of the enzyme. The proper positioning of the ANG2 within the active site of the ps-ACE2 suggests the effectiveness of the docking methodology utilized. The complex between the ps-ACE2 and the ANG2 wasRef- 1403008submitted to evaluate the impact of single-point mutations on protein-protein interactions. Each mutation site underwent all potential mutations to identify the mutation that most effectively deactivates the interaction. The analyses conducted to identify the critical amino acids whose mutations would lead to the cessation of enzymatic activity in the ACE2. Candidate mutations were introduced using the protein preparation tool. Table 1 illustrates the effect of threonine (THR) 445 and arginine (ARG) 273 saturation mutations on the binding affinity of the complex between the ANG2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure.Table 1: The effect of threonine 445 and arginine 273 saturation mutations on the binding affinity of the complex between the ANG2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosureRef- 1403008Ref- 1403008

[0067] The mutated amino acids, along with their adjacent residues, underwent energy minimization to achieve optimal conformations. Additionally, Molegro VIRTUAL DOCKER was used to assess the cavity sizes within the ps-ACE2 structure both prior to and following the introduction of the mutations. The docking analysis was carried out on the structure of the ANG2 and the ps-ACE2 using HPEPDOCK and ClusPro servers. The graph of 2D interaction between the ps-ACE2 and the ANG2 before and after mutation was drawn and analysed with LigPlot.

[0068] A re-examination of the molecular docking between an exemplary mp-eACE2 and the ANG2 molecule reveals that the mutations have compromised the ANG2 molecule's capacity to maintain a stable interaction with the active site of the altered enzyme. An analysis of the cavity within the ACE2 structures, both prior to and following the mutations, demonstrates that the volume of the central cavity (active site) remains relatively unchanged by the mutations, measuring 4764.67 A3 before and 4819.46 A3 after the mutation. An exemplary mp-eACE2 engages with the ANG2 in a notably different orientation. Thus, the mutations were likely to disrupt the enzymatic activity of an exemplary mp-eACE2 by eliminating crucial stabilizing interactions that were originally provided by the amino acids at positions 273 and 445.Ref- 1403008

[0069] The structure of the complex between the ps-ACE2 and the RBD was extracted from the RDB with NO: 6M17. The outcomes of the Saturation Mutagenesis analysis have been compiled and categorized based on their influence on enhancing the binding affinity between the ps-ACE2 and the RBD. Table 2 illustrates the mutations that exhibit the greatest potential to enhance the binding affinity between the ps-ACE2 and the RBD, consistent with one or more exemplary embodiments of the present disclosure.Table 2: The mutations that exhibit the greatest potential to enhance the binding affinity between the ps-ACE2 and the RBD, consistent with one or more exemplary embodiments of the present disclosure.

[0070] The mutations with a high probability of enhancing the binding affinity of the ps-ACE2 to the ANG2 were applied to the structure of the ps-ACE2 using Molegro VIRTUAL DOCKER. In order to achieve a suitable conformation, energy minimization for amino acids and their neighbors was performed. After applying mutations, the interactions of an exemplary mp-eACE2 and the RBD were redocked using molecular docking. The molecular docking result was analysed using the HADDOCK server. PRODIGY was used to evaluate the rate of change in binding energy before and after the mutation. The root mean square deviation (RMSD) was calculated to evaluateRef- 1403008the rate of change in binding energy before and after mutation. Additionally, the impact of these mutations was assessed by generating 2D plots illustrating the interactions between the ACE2 and the RBD following each mutation.

[0071] Moreover, the critical amino acids involved in the interaction between the ps-ACE2 and the RBD were illustrated. FIG. 2 illustrates the amino acids 200 that participate in the interaction between the ps-ACE2 and the RBD, consistent with one or more exemplary embodiments of the present disclosure. With further reference to FIG. 2, the structural representation of the interactions at the interface is illustrated in Part 202. Part 204 presents a two-dimensional interaction plot of these interactions. The horizontal dashed line signifies the interface, with amino acids situated above the line corresponding to the RBD, while those below the line are associated with the ACE2. Hydrogen bonds are denoted by dashed lines connecting the involved atoms, and hydrophobic interactions are depicted by an arc with spokes extending towards the contacting atoms.

[0072] Evaluations of different single or multiple mutations were conducted using their two-dimensional interaction plots. Mutations that maintained the original interacting amino acids at the interface of the ACE2 and the RBD while also increasing the number of participating amino acids were deemed appropriate. The results indicated that the predicted mutations for applying in the interaction between the ps-ACE2 and the RBD resulted in elevating energy bonding between the ps-ACE2 and the RBD and saving their main orientation. The result of the evaluation of bonding energy between the ps-ACE2 and the RBD before and after mutations indicates that an exemplary mp-eACE2 may participate in the interaction with the RBD with a better bonding energy (-13.8 kcal mol1) than the ps-ACE2 (-13.1 kcal mol1).

[0073] The result of the structural conformity analysis indicated that an exemplary mp-eACE2 may interact with the RBD with a suitable orientation or conformation. A low RMSD (0.445 A) between the ps-ACE2 and an exemplary mp-eACE2 protein indicated that the mutations do not cause severe changes in the structure of the protein. PoPMuSiC and HotMuSiC were employed toRef- 1403008predict changes in the thermodynamic stability of proteins and temperature changes at the mutations of one site, respectively. The best mutations causing an increase in thermostability were performed and optimized with Molegro VIRTUAL DOCKER. Table 3 illustrates the predicted mutations for enhancing the thermostability of the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure.Table 3: The predicted mutations for enhancing the thermostability of the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosureRef- 1403008

[0074] The stability of an exemplary mp-eACE2 was evaluated by molecular dynamics analysis. The RMSD and Root mean square fluctuation (RMSF) graphs were generated based on the results of molecular dynamics analysis. FIG. 3 illustrates the plots of RMSD 300 for an exemplary mp-eACE2 302 and the ps-ACE2 304, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG.3, the flexibility of mutated amino acids along the simulation was not great. This indicated that there is no conformation transmission at a large scale of the protein system in the mutated sites. Moreover, an exemplary mp-eACE2 was reached to stability earlier than the ps-ACE2 and remained in the stable form longer than the ps-ACE2. The RMSD value may be fixed between 0.3 and 0.4 for an exemplary n-ACE2. FIG. 4 illustrates the plots of RMSF 400 for an exemplary mp-eACE2 402 and the ps-ACE2 404, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG.4, in the most amino acids, the RMSF value was not changed significantly, before and after mutations was similar. Large changes were seen in the sites of mutations; that was reasonable based on the substitution of amino acids. FIG.5 illustrates the plots of radius of gyration (Rg) 500 for an exemplary mp-eACE2 502 and the ps-ACE2 504, consistent with one or more exemplaryRef- 1403008embodiments of the present disclosure. As shown in FIG. 5, the Rg value in an exemplary mp-eACE2 was decreased as time progressed.

[0075] The immunological assessments revealed that an exemplary mp-eACE2 does not exhibit any newly formed epitopes resulting from the mutations. Furthermore, there were no additions or deletions to the glycosylation sites in the ACE2 protein's glycosylation profile following the mutations. Positions 55, 92, 105, 324, 424, and 548 were identified as glycosylation sites in both an exemplary mp-eACE2 and the ps-ACE2. The preservation of the glycosylation pattern will facilitate the classification of an exemplary mp-eACE2 as a self-protein during clinical applications.Example 2: Obtaining exemplary Mesenchymal stem cells (MSCs) expressing an exemplary mp-eACE2 (exemplary mp-MSCs) and exemplary MSCs cells expressing the ps-ACE2 (exemplary ps-MSCs)

[0076] Exemplary DNA sequences of both an exemplary mp-eACE2 and the ps-ACE2 were optimized for codon usage to enhance eukaryotic expression. Each exemplary gene was modified to include the tissue plasminogen activator (tPA) secretion signal along with additional sequence elements necessary for effective expression. Consequently, the lengths of exemplary genes are identical, with their variations lying in the nucleotide sequences, which ultimately affect exemplary protein sequences. These two exemplary genes were individually subcloned into an exemplary pCDH- green fluorescent protein (GFP)-Puro lentiviral vector, designated as pCDH-(mp-eACE2)-GFP-Puro and pCDH-(ps-ACE2)-GFP-Puro, respectively. An exemplary DNA sequence encoding an exemplary mp-eACE2 is set forth in SEQ ID NO: 3. An exemplary DNA sequence encoding ps-ACE2 is set forth in SEQ ID NO: 6.

[0077] The human embryonic kidney (HEK293T) cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) with a solution of penicillin 1% (100 U / ml), a solution of streptomycin (100 pg / ml), and fetal bovine serum (FBS) 10%, at a temperature level of 37 °C, in the presence of CO2 (5%). About 4xlO6HEK293T cells were seeded on diameter plates (10 cm)Ref- 1403008for a time duration of about 24 h to reach a confluency of about 70%-80%. The cell line of HEK293T was transfected with 21 pg of pCDH-(ps-ACE2)-GFP-Puro and pCDH-(mp-eACE2)-GFP-Puro, 21 pg of psPAX2, and 10.5 pg of pMD2. For the transfection, the conventional calcium phosphate method was carried out. The culture media of transfected cells were collected and replaced with about 10 ml of complete media after 16 hours. To evaluate the transfection, a GFP reporter gene is used. pCDH-(mp-eACE2)-GFP-Puro comprises a GFP reporter gene; therefore, the transfected cells appeared with a green fluorescent. About 90% of cultured HEK293T cells expressed GFP, therefore 90% is an estimation of transfection efficiency. FIG.6 illustrates the plot of flow cytometry 600 carried out for evaluating the transfection rate in HEK293T cells using lentiviral vector, consistent with one or more exemplary embodiments of the present disclosure.FIG. 7 illustrates the image 700 of Bright-field 702 and fluorescence microscopy 704 capturing from the transfected cells, consistent with one or more exemplary embodiments of the present disclosure.

[0078] The viral particle-containing supernatant was collected after 24, 48, and 72 hours of the transfection. Then, the culture media of transfected cells were centrifuged (500 g, 5 minutes), and the cell pellet was discarded. After centrifuging, the supernatant was subsequently centrifuged (2,000 g for 20 min) to remove cell debris. Finally, the supernatant was filtered through a 0.45pm (low protein attachment) membrane to remove impurities. Then these harvested supernatants, including engineered lentiviral particles, was concentrated by polyethylene glycol-8000 (PEG-8000). For this purpose, PEG-8000 (with a purity of 50 wt%) and a solution of sodium chloride (NaCl) (with a concentration of 5 Molar) were added to the harvested viral supernatant. The final solution comprising harvested viral supernatant, PEG-8000, and NaCl was incubated on a rotator at 4°C overnight. Then, the final solution was centrifuged, and the fluffy white pellet containing viral particles was obtained. To validate the activity of engineered lentiviral particles in transduced cells, flow cytometry titration was carried out. The best viral titer was for supernatant of cells after 48 h of the transfection (near 60% GFP+) and was evaluated at about 6xl08Transduction UnitsRef- 1403008per ml (TU / ml). For the purpose of titration, about 6xl04HEK293T cells were seeded in a plate (24-well) and were transduced by viral particles (1, 5, and 10 pl). After 72 h, the cells were assessed with a fluorescence microscope and flow cytometry device. The value of TU / ml was calculated by formula 1.Formula 1: TU = (1 x 102seeded cells x %GFP — positive cells) x 1000 / pl of vector

[0079] FIG. 8 illustrates the plot 800 of flow cytometry to identify the best viral titer after the transduction, consistent with one or more exemplary embodiments of the present disclosure. FIG.9 illustrates the result 900 of fluorescence microscopy 902 and the transduction rate 904 quantified by flow cytometry after the transduction, consistent with one or more exemplary embodiments of the present disclosure. Data were presented as means ± SD of three separate groups, n = 3, *p<0.05. When the cell density reached l-2xl04per well, MSCs were plated in a 24-well plate. Viral particles with a multiplicity of infection (MOI) of 50 and 100 were introduced to each well, accompanied by 8 pg / ml of polybrene, and the media were replaced after 24 h. To enhance the transduction efficiency, the process was repeated three times. One week later, the transduced mesenchymal stem cells (mp-MSC and ps-MSC) were isolated using the suitable concentration of puromycin, as determined by the cell death curve. All characterization and confirmatory tests of MSCs, including surface markers, potential of differentiation, and karyotyping, were performed before and after the lentivirus transduction. FIG. 10 illustrates the plot of flow cytometry 1000 in order to evaluate the Cluster of differentiation (CD) marker expression in Mesenchymal stem cells (MSCs) before transduction, consistent with one or more exemplary embodiments of the present disclosure. FIG. 11 illustrates the image of flowcytometry 1100 in order to evaluate differentiation of MSCs into osteocytes (1102) and adipocytes (1104) before transduction, consistent with one or more exemplary embodiments of the present disclosure. FIG. 12 illustrates the information of karyotyping 1200 in MSCs before transduction, consistent with one or more exemplary embodiments of the present disclosure.Ref- 1403008

[0080] FIG. 13 illustrates the image 1300 of fluorescence microscopy of the transduced cells in order to prove the presence of an exemplary mp-eACE2 gene, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 13, the successful transduction was indicated by the expression of GFP. FIG. 14 illustrates the curve 1400 of cell death in MSCs after the process of puromycin selection, consistent with one or more exemplary embodiments of the present disclosure. FIG. 15 illustrates the plot of flow cytometry 1500 carried out after puromycin selection of the transduced MSCs cells, consistent with one or more exemplary embodiments of the present disclosure. The homogeneous transduced population was assessed through flow cytometry and fluorescence microscopy to observe GFP expression. As shown in FIG. 15, the transduced cells were selected by a concentration of 2 pg / ml of puromycin. After using the ideal dose of puromycin, MSC cells became more than 90% GFP positive. FIG. 16 illustrates the image 1600 of osteogenic differentiation 1602 and adipocyte differentiation 1604 in MSCs after the transduction, consistent with one or more exemplary embodiments of the present disclosure. FIG. 17 illustrates the information of karyotyping 1700 in the transduced MSCs, consistent with one or more exemplary embodiments of the present disclosure. FIG. 18 illustrates the plot 1800 of flow cytometry in order to evaluate the CD marker expression in the transduced MSCs, consistent with one or more exemplary embodiments of the present disclosure. As shown in FIG. 18, the chromosome count was consistent with that of normal cells, totalling 46, and there were no observed abnormalities in either the quantity (aneuploidy) or the integrity of the chromosomes.

[0081] To verify the incorporation of an exemplary mp-eACE2 and the ps-ACE2 genes into the genome, genomic DNA was extracted from MSCs utilizing the genomic DNA extraction kit in accordance with the manufacturer's instructions. The polymerase chain reaction (PCR) reaction was conducted using specific primers that were developed based on the optimized sequences of both an exemplary mp-eACE2 and the ps-ACE2 gene. The amplification reaction was notRef- 1403008conducted using the normal MSCs genome, which contains the ACE2 gene, as a template because the primer was specifically designed to target the exon-junction zone of the CDS sequence. Total Ribonucleic acid (RNA) was extracted from mp-MSCs and ps-MSCs, and cDeoxyribonucleic acid (cDNA) synthesis was performed. Real-time quantitative reverse transcription PCR (qRT-PCR) amplification was conducted employing Ampliqon SYBR Green Master Mix, following the Pfaffl method, with normalization against the P-actin gene as the endogenous control.

[0082] The transduced and untransduced MSCs were cultured for 48 hours in a serum-free medium. Subsequently, the culture medium was collected and subjected to centrifugation at 4,000 g for 20 minutes using an Amicon filter with a 50 kDa cutoff. This procedure effectively eliminated proteins smaller than 50 kDa from the total secreted proteins. Following this, the filtrate, which contained either an exemplary mp-eACE2 or the ps-ACE2, was further processed using a 100 kDa filter. The resulting filtrate from the 100 kDa filter was then utilized for quantification via the Bradford protein assay (Fermentas).

[0083] Approximately 40 pg of concentrated proteins were resolved using a 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following separation, the proteins were transferred to nitrocellulose membranes via electrophoresis. The membranes underwent a blocking procedure with 3% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for one hour. Subsequently, they were incubated with a horseradish peroxidase (HRP)-conjugated antibody specific to the human ACE2 polyclonal for an additional hour. The visualization of protein bands was achieved using the enhanced chemiluminescence (ECL) detection kit from Thermo Fisher Scientific.

[0084] FIG. 19 illustrates the result of PCR 1900 by primer of ACE2 on genomic DNA of untransduced MSCs 1902, ps-MSC 1904 (lane 1&2, under lOObp band), and mp-MSC 1904 (lane 3,4,&5, under lOObp band), consistent with one or more exemplary embodiments of the present disclosure. FIG. 20 illustrates the result of qRT-PCR 2000 by primer of ACE2 on cDNA of transduced and un-transduced MSCs, consistent with one or more exemplary embodiments of theRef- 1403008present disclosure. (2002: lane 1-3 by P-actin primers: 131bp band and 73bp band by ACE2 primers for a template from ps-MSC (2004: lane 4, 5), mp-MSC (2004: lane 2, 3), and untransduced MSCs (2004: lane 1)). The detection of PCR products validated the incorporation of exogenous viral DNA into the host genome, and the expression of exemplary ACE2 genes was corroborated at the cDNA level through qRT-PCR analysis. FIG. 21 illustrates the chart 2100 of Real Time qRT-PCR for evaluating the ACE2 expression, consistent with one or more exemplary embodiments of the present disclosure. The expression level of the ACE2 gene was analyzed in both an exemplary mp-MSCs and ps-MSCs groups in comparison to the un-transduced group using Real Time PCR (Pfaffl ratio), with normalization to the expression level of P-actin.

[0085] The culture of mp-MSCs, ps-MSCs, and un-transduced MSCs in serum-free media was collected for analysis. The secreted ACE2 protein was assessed using western blot techniques. Initially, the concentration of the total protein extracted was quantified to be between 200 and 300 pg / ml, utilizing the Bradford method in accordance with the bovine serum albumin standard curve. Subsequently, the presence of both an exemplary mp-eACE2 and ps-ACE2 proteins was confirmed through the coupling of human anti-ACE2 polyclonal antibodies to the proteins expressed by the MSCs, with un-transduced MSCs serving as the control group. FIG. 22 illustrates the level 2200 of the ACE2 in un-transduced 2202, mp-MSC 2204, and ps-MSC 2206 evaluating by western blot, consistent with one or more exemplary embodiments of the present disclosure.Example 3: Evaluation of the affinity to the S protein of COVID-19

[0086] Approximately 50 nanograms of pure S protein (ab290829 from Abeam) were applied to a 96-well enzyme-linked immunosorbent assay (ELISA) plate (high absorbance) and incubated for 24 hours at 4 °C. Following this incubation, the wells were washed with phosphate buffered saline (PBS) and tapped to eliminate excess buffer and any uncoated S protein. A blocking buffer consisting of 5% skimmed milk in PBST was introduced to the wells, where it was allowed to sit for 2 h at 37°C before being gently washed with PBS. Subsequently, a concentrated fractionRef- 1403008containing 2-5 pg of the ps-ACE2 and an exemplary mp-eACE2 was added to the wells and incubated for 6 h at 4°C. After this incubation, an HRP-conjugated human ACE2 polyclonal antibody was introduced to the wells. The 3,3',5,5'-Tetramethylbenzidine (TMB) substrate was utilized to evaluate color development at optical densities of 450 nm and 600 nm. The sandwich ELISA platform was utilized to analyze the characteristics of an exemplary mp-eACE2 in comparison to the ps-ACE2. FIG. 23 illustrates the sandwich ELISA result 2300 for evaluating the affinity to the S protein of COVID-19 in both groups of an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure. Data were mentioned as means ± SD of three separate groups, n = 3, *p<0.05 vs. PBS group. The enhanced binding affinity of an exemplary mp-eACE2 protein to the RBD of the S protein was demonstrated by a 28% increase in emitted light from an exemplary mp-eACE2 well relative to that of ps-ACE2.Example 4: Thermo-stability of an exemplary mp-eACE2 and the ps-ACE2

[0087] The concentrated of an exemplary mp-eACE2 and the ps-ACE2 were subjected to incubation at varying temperatures of 47, 57, and 67° C for a duration of 30 minutes. Subsequently, the incubated enzymes were introduced to a 96-well ELISA plate that had been coated with S protein, and color development was initiated using TMB substrate. A different sandwich ELISA platform was developed utilizing thermal treatment of both an exemplary mp-eACE2 and the ps-ACE2. FIG. 24 illustrates the thermo- stability of an exemplary mp-eACE2 and an the ps-ACE2 2400 evaluated in different temperatures by sandwich ELISA, consistent with one or more exemplary embodiments of the present disclosure. Data were mentioned as means ± SD of three separate groups, n = 3, *p<0.05. The function of the ps-ACE2 as a catalytically active enzyme that exhibits sensitivity to variations in temperature. The results indicated that both an exemplary mp-eACE2 and the ps-ACE2 demonstrated thermal resistance up to 57°C; however, an exemplary mp-eACE2 exhibited a greater overall resistance compared to the ps-ACE2. Consequently, an exemplary mp-eACE2 displayed enhanced strength and superior resistance.Example 5: Enzyme activity of an exemplary mp-eAEC2 and the ps-ACE2Ref- 1403008

[0088] The ACE2-specific quenched fluorescent substrate assay was performed for the assessment of ACE2 activity with specific modifications. The isolated form of an exemplary of mp-eACE2 and the ps-ACE2 were prepared in equal concentrations and subsequently diluted in a 30:70 ratio with enzyme buffer (1 mol / 1, pH 6.5), which contained the following components: NaCl, 75 mmol / 1 Tris-HCl, 5 mmol / 1 ZnCh, along with protease inhibitors including 10 mM Captopril, 5 mM Amastatin-hydrochloride, 10 mM Bestatin-hydrochloride, and 10 mM Z-prolyl-prolinal. The quenched fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-dinitrophenyl)-OH [Mca-APK(Dnp)], was diluted in the enzyme buffer and incubated with the samples at 37 °C, achieving a final concentration of 50 pmol / 1 in a volume of 100 pl. Fluorescence measurements were conducted using a 420-nm cutoff filter, with the maximum fluorescence recorded at excitation and emission wavelengths of Ax = 324 nm and Am = 430 nm, respectively. The initial enzyme activities were calculated based on the linear increase in fluorescence over a time span of 0 to 120 minutes, employing linear regression to analyze the relationship between fluorescence increase and reaction time. The formula used to compute ACE2 activity was mentioned in Formula 2.Formula

[0089] In this context, D denotes the serum dilution, k signifies the variation in fluorescence intensity resulting from the complete cleavage of 0.1 nmol of Mca-APK (Dnp), and S represents the observed rate of increase in fluorescence intensity. One unit of fluorescence (UF) corresponds to the quantity of enzyme necessary to degrade 0.1 nmol of Mca-APK(Dnp) at a temperature of 37° C over the duration of one hour. The specific inhibitor of human ACE2, DX600, was employed to evaluate the specificity of the assay. Consequently, DX600 fully inhibited the cleavage of Mca-APK (Dnp). FIG. 25 illustrates the enzyme activity 2500 of an exemplary mp-eACE2 and the ps-ACE2, consistent with one or more exemplary embodiments of the present disclosure. Data were presented as means ± SD of three separate groups. The findings from the enzyme activity assayRef- 1403008conducted on an exemplary mp-eACE2 and the ps-ACE2 indicated that an exemplary mp-eACE2 has lost its functional activity. This outcome was anticipated given the loss of essential amino acids within the active site and the alteration in the dimensions of the active site pocket. In contrast, the ps-ACE2 maintained its activity, successfully hydrolyzing the Mca-APK (Dnp) substrate to generate fluorescence. The p-values comparing the ps-ACE2 to the ps-ACE2+ inhibitor and an exemplary mp-eACE2 were found to be less than 0.05.Example 6: Comparison between an exemplary mp-eACE2 and the ps-ACE2 in COVID-19 inhibition

[0090] The concentrated of an exemplary mp-eACE2 from the serum-free culture media of mp-MSCs and the ps-ACE2 from the serum-free culture media of ps-MSCs, along with the serum-free culture media of un-transduced MSCs serving as the control group, were prepared alongside a COVID- 19 virus stock (cycle threshold: 12, titer: 106the median tissue culture infectious dose 50 (TCID50) / ml) in three different dilutions (1:1, 1:10, and 1:100). The viral dilutions were combined with the respective protein dilutions and incubated for 2 hours at 37 °C. Vero 76-clone E6 (Vero E6 cells) were cultured in a specific medium consisting of DMEM supplemented with 10% FBS, 100 mg / ml streptomycin, and 100 units / ml penicillin G. These cells were incubated at 37° C in a 5% CO2 environment until they reached 70%-80% confluency. Following the removal of the medium and two washes with PBS, the virus-protein mixtures were introduced to the cells and incubated for an additional 2 hours at 37 °C. Triplicate testing wells were maintained until the appearance of cytopathic effects (CPE). A virus neutralization test (VNT) denotes the capacity to inhibit a virus from replicating or infecting cells. This assay evaluates the impact of an exemplary mp-eACE2 and the-ACE2 by examining cell morphology, which serves as a CPE indicator for viral propagation. Table 6 below illustrates the presentation of the lack of CPE (-) or presence of CPE (+) in different concentrations of virus neutralization at the different dilutions of an exemplary mp-eACE2 and the ps-ACE2 compared with the control group, consistent with one or more exemplary embodiments of the present disclosure. FIG. 26 illustrates the CPE 2600 as a result ofRef- 1403008COVID- 19 infection and proliferation in Vero E6 cell monolayer (cell death and cell fusion), consistent with one or more exemplary embodiments of the present disclosure. The negative result of CPE (2602) and positive result of CPE (2604) were shown in FIG. 26.Table 6: The presentation of the lack of CPE (-) or presence of CPE (+) in different concentrations of virus neutralization at the different dilutions of an exemplary mp-eACE2 and an exemplary the ps-ACE2 compared with the control group, consistent with one or more exemplary embodiments of the present disclosureRef- 1403008

[0091] As mentioned in Table 6, after six days, the results obtained validated the efficacy of an exemplary mp-eACE2 and the ps-ACE2 at the highest concentration (1:1 dilution, equivalent to 30 pg) in neutralizing the target. In contrast, CPE were noted in all control groups. It is noteworthy that the 1:10 concentration of an exemplary mp-eACE2 demonstrated the ability to neutralize viral stock concentrations up to 1:1000 in comparison to the 1:10 concentration of the ps-ACE2.

Claims

Ref- 1403008What is claimed is:

1. A monomer polypeptide comprising an engineered form of Angiotensin-Converting Enzyme 2, the engineered form of Angiotensin-Converting Enzyme 2 comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 2.

2. The monomer polypeptide comprising an engineered form of Angiotensin-Converting Enzyme 2 of claim 1, wherein the monomer polypeptide comprising an engineered form of Angiotensin- Converting Enzyme 2 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 3.

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