WO2024013540A1

WO2024013540A1 - Using genetic manipulation bacteria to deactivate the corona viruses.

Info

Publication number: WO2024013540A1
Application number: PCT/IB2022/056391
Authority: WO
Inventors: Sajad SHABANPOUR HAGHIGHI; Neda BAGHERIAN
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2024-01-18
Anticipated expiration: 2025-01-11

Abstract

The invention using genetic manipulation bacteria in order to deactivate the corona viruses through secretion and surface presentation of virus receptor (ACE2) is a method to control and treat diseases caused by corona viruses, SARS, MERS and influenza and all viruses that enter through the ACE2 receptor cell. In this way, by genetic manipulation, with genetic engineering methods, the digestive and respiratory normal flora bacteria changed in such a way that they produce the ACE2 receptor and then secrete it into the outside ambient or present it on the surface of the bacteria itself (with the help of signal peptide), as a result, since the normal flora lives on the cells with ACE2 receptors (in the respiratory and digestive tracts), the virus binds to these ACE2 receptors before entering these cells, and as a result, they lose their ability to enter to human cells.

Description

TITLE OF INVENTION

USING GENETIC MANIPULATION BACTERIA TO DEACTIVATE THE CORONA VIRUSES.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of biotechnology and especially relates to methods of combating with pathogenic viruses, including corona viruses, as well as to the system and mechanism of operation of ACE2 receptors in this specific species of viruses.

PRIOR ARTS

Currently, mRNA vaccines, or attenuated virus and antigen, are used to prevent corona virus because viruses such as corona virus, influenza, and HIV are constantly changing their surface antigens, the vaccine for these viruses lacks effective and lasting efficacy.

Viral drug groups (including acyclovir, Amantadine, Stavudine, zidovudine, Valacyclovir, etc.) although they have the FDA authentication have severe side effects and have been shown in trials which they are not enough effective against viruses.

A Chinese patent application No. CN111870688 A which is filed dated 09/07/2020 titled “ACE2 protein and IL-6 or TNF alpha antagonist combination and application thereof’ related to the technical field of biomedical engineering and provides an ACE2 protein and IL-6 or TNF alpha antagonist composition and application thereof, wherein the application is specifically the application of the combination of the ACE2 protein and any one or two of an IL-6 antagonist and a TNF alpha antagonist in the preparation of a medicament for treating diseases related to ACE2 expression level or dysfunction-mediated inflammation medium disorder. The medicine is a medicine composition taking ACE2 protein and any one or two of IL-6 antagonist and TNF alpha antagonist as active components, or is a fusion polypeptide formed by ACE2 protein and any one or two of IL-6 antagonist and TNF alpha antagonist. The composition or the formed fusion polypeptide can mediate ACE2 dependent immune cell elimination, reduce ADE effect of ACE2-Ig and reduce immune cell secretion unexpectedly. In animal models, the composition can reduce the over-expression and release of organ inflammation mediators, relieve organ inflammation injury, enhance the anti-stress capability of organs and resist organ injury.

Another Chinese patent No. CN112250763B which is granted dated 26/03/2021 titled “Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use” relates to antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use. In particular to an antibody or an antigen-binding fragment thereof and an antibody pair which specifically bind to coronavirus S protein and a detection product containing the antibody or the antigen-binding fragment thereof and the antibody pair. Furthermore, the invention relates to the prophylactic, therapeutic or diagnostic use of said antibody or antigen-binding fragment thereof, antibody pair. Sequence alignment revealed that SARS-CoV-2 virus and SARS-CoV virus S protein have 75% similarity, and it was reported that amino acid residues at the and sites of the S protein and ACE2 receptor complex interface (distributed mainly in respiratory epithelial cells, lung, heart, kidney and digestive tract in humans) of multiple SARS-CoV coronavirus isolates are highly conserved. Compared with the conventional IgG/IgM detection, the detection method directly detects the RBD protein of the virus in the sample. The antibodies or antibody pairs are useful for diagnosing and/or detecting corona viruses.

An US patent No. US7629443B2 which is granted dated 08/12/2009 titled “Neutralizing monoclonal antibodies against severe acute respiratory syndrome- associated coronavirus” provides an isolated antibody capable of binding to the receptor-binding domain of the spike protein of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) so as to competitively inhibit the binding of the SARS-CoV to host cells. These mAbs or substances can be used: 1) as passive-immunizing agents for prevention of SARS-CoV infection; 2) as biological reagents for diagnosis of SARS-CoV infection; 3) as immunotherapeutics for early treatment of SARS-CoV infection; and 4) as probes for studying the immunogenicity, antigenicity, structure, and function of the SARS-CoV S protein.

Another US patent No. US7888102B2 which is granted dated 15/02/2011 titled “Cell line for producing coronaviruses” relates to the production of coronaviruses. In particular, the invention relates to methods for producing SARS-CoV by using cells expressing a functional SARS-CoV receptor. The invention provides cells suitable for producing coronaviruses. In a preferred embodiment the cells are HER cells expressing the human ACE2 protein. The invention further provides methods for producing coronaviruses, in particular SARS-CoV, making use of the cells. In a first aspect the present invention encompasses cells expressing the human ACE2 protein. The human ACE2 protein may be transiently expressed, but for long-term, high-yield expression of the human ACE2 protein stable expression is preferred. In another US patent application No. US20100172917A1 which is filed dated 22/07/2003 titled “Binding molecules against SARS-coronavirus and uses thereof’ provided binding molecules that specifically bind to SARS-CoV, nucleic acid molecules encoding the binding molecules, compositions comprising the binding molecules, and methods of identifying or producing the binding molecules. The binding molecules are capable of specifically binding to SARS-CoV, and can be used in the diagnosis, prophylaxis, and/or treatment of a condition resulting from SAR.

Another patent with publication No. W02004092360A2 which is filed in WIPO dated 10/04/2003 titled “The severe acute respiratory syndrome coronavirus” relates to nucleic acids and proteins from the SARS coronavirus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations, diagnostic reagents, kits, etc. The invention also provides methods for treating SARS by administering small molecule antiviral compounds, as well as methods of identifying potent small molecules for the treatment of SARS. HR sequences for use in the invention may be produced recombinantly by methods known in the art. The SARS HR sequences may be modified to facilitate bacterial expression. In particular, the HR sequences may be modified to facilitate transport of the recombinant protein to the surface of the bacterial host cell. In this invention, the cells expressing the SARS-CoV receptor, ie, the enzyme of the Angiotensin 2 converter are added to the extra bacterium.

Another patent with publication No. W02005032487A2 which is filed in WIPO dated 06/10/2003 titled “Angiotensin-converting enzyme-2 as a receptor for the sars coronavirus” provides human angiotensin-converting enzyme-2 (ACE-2) as a functional receptor for the SARS coronavirus. Transfection of cells with ACE-2 confers upon them the ability to support viral replication. In addition, assays performed using ACE-2 together with the S protein of the SARS virus or a fragment derived from the S protein can be used to identify inhibitors that block the interaction between virus and host cell, said cells expressing human ACE-2 on their surface are cells that have been transfected with a vector comprising a promoter operably linked to a sequence coding for human ACE-2. These cells are incubated with detectably labeled SARS S protein. This invention also provides a method for blocking the binding of the SARS virus to a host cell, comprising contacting said host cell with an effective amount of an inhibitor of ACE-2.

Another patent with publication No. WO2021196268 Al which filed dated in WIPO dated 30/03/2020 titled “Antibody having neutralizing activity against corona virus, and use thereof’ provides an antibody or an antigen binding fragment specifically binding to a corona virus S protein, a multispecific antibody, and an antibody composition. Further provided are a nucleic acid encoding the antibody or the antigen binding fragment, and the multispecific antibody, a host cell comprising the nucleic acid, and a method for preparing the antibody or the antigen binding fragment, and the multispecific antibody. In addition, further provided are prevention, treatment, and/or diagnosis uses of the antibody or the antigen binding fragment, the multispecific antibody, and the antibody composition. This antibody can effectively block and/or inhibit corona virus infection, and can be used for the prevention and/or treatment of corona virus. First, the binding of S protein to the receptor ACE2 is the first step for corona virus to infect the host, and the SI and S2 subunits of the S protein carry the function of binding receptors and promoting the fusion of the virus envelope and the host cell membrane. The S protein is the ideal target antigen for the development of antibodies with virus-neutralizing activity. The other patent with publication No. WO2021213437A1 which is filed in WIPO dated 23/04/2020 titled “Ace2-fc fusion protein and use thereof’ is a fusion protein formed by means of connecting an ACE2 extracellular region with a polypeptide fragment capable of dimerizing ACE2, preferably by means of connecting an Fc fragment of a human IgGl antibody with a human ACE2 extracellular region. The protein can be combined with RBD, and can simultaneously inhibit the combination between the molecular and cellular levels of RBD and an ACE2 protein. The fusion protein can be used for preventing and treating infection with the novel corona virus (SARS-CoV-2). This invention has a composition comprises a neutralizing antibody against the S protein of the novel corona virus.

And in another patent with publication No. WO2021228854A1 which is filed in WIPO dated 11/05/2020 titled “Virus neutralization by soluble receptor fragments of the ace-2 receptor” refers to a soluble receptor fragment (SRF) of the ACE-2 receptor, wherein the SRF comprises the peptidase domain (PD) of ACE-2 or a fragment and/or derivates thereof. In addition, the present invention provides SRFs for use in a method of treating and/or preventing a virus infection, in particular a virus infection caused by Coronaviridae, more particularly caused by SARS coronavirus, SARS coronavirus-2, human coronavirus NL63 or SARS-CoV-2. Finally, the present invention relates to a method for capturing viral particles, the method comprises the steps of providing immobilized SRFs and contacting a liquid sample or fluid with the SRFs under conditions for allowing the SRFs to bind the viral particles.

Also in the patent with publication No. WO2022037699A1 which is filed in WIPO dated 21/08/2020 titled “ Engineered ACE2 oligomers and uses thereof’ provided the engineered ACE2 oligomers and the compositions resulting there from. In this invention ACE2 oligomer, wherein the ACE2 oligomer is formed by monomers, and each monomer comprises a soluble ACE2, a linker and an oligomerization motif, and wherein the ACE 2 oligomer comprises an ACE2 trimer. Furthermore, ACE2 biological function supports using ACE2 decoy proteins for SARS-CoVs infection treatment. Coronavirus infection or even spike protein binding can cause shedding of ACE2 from cell surface resulting decreased ACE2 expression level and accumulation of plasma angiotensin II and this is closely related with acute lung injury. In fact, it has been shown ACE2 peptidase domain could inhibit SARS-CoVs infection in cell assays and organoids, one clinical trial was also registered to use recombinant ACE2 to treat COVID-19

EXISTING PROBLEMS

Viruses cause irreversible damage to human societies, both as epidemics and as a single infection, and in addition to vaccinations and antiviral drugs (which have many side effects and also they are ineffective against viruses that change their surface antigens frequently) we have no other way to deal with them. In the recent corona epidemic, despite the presence of different vaccines (which are from different groups of mRNA, etc.) since the virus is constantly mutating and changing its surface antigen (because there is an epidemic and proliferation of the virus is very large, so resulting in more mutations); as a result, the vaccines periodically lose their effectiveness or the virus becomes resistant to the vaccine. Also, there is currently no suitable drug that can be effective against the virus and has few side effects as well.

Due to the high cost of producing vaccines against the virus, this epidemic has imposed great financial and human costs on communities. PURPOSE OF THE INVENTION

Making a suitable solution for deactivating and destroying viruses in a way that has the lowest cost and the least side effects and also should not lose its efficiency in case of mutation of the virus and change of its surface antigens.

DESCRIPTION OF THE INVENTION

The entrance of corona to human cells is the respiratory and gastrointestinal tract and the entry of viruses such as (corona, influenza and SARS, etc.) into the human cell is through ACE2 receptor, which are on the cells of the respiratory and gastrointestinal tract [ACE2 is present in kidney cells too, but since kidney cells do not mechanically come into contact with the outside of the body, they naturally cannot be a place for entrance these viruses].

Non-invasive bacteria called normal flora live naturally on the mucosal surface of the respiratory and the gastrointestinal tract. Since the normal flora lives on the surface of the mucosa and in the gastrointestinal tract, the corona virus is in contact with the normal flora before it mechanically contacts with mucosal cells, but as these bacteria do not have a receptor for the virus, no reaction occurs from this contact.

The mechanism of this invention is such that by specific genetic engineering processes, the ACE2 gene is imported into the normal flora of the respiratory and gastrointestinal tract or benign type of Escherichia coli (as well as common non- invasive bacteria colonized on the surface) then normal flora is transferred to the mucosa of healthy and sick people and in this way, we remove viruses mechanically by attaching to these bacteria before they enter to the human cells. If some of these normal floras are destroyed, they will replicate themselves and do not need to be reused as they contain the ACE2 gene.

Even if a small number of viruses cross the barrier, they will remove by the immune system because of their small number [It should be noted that in order to generate pathogenesis by the virus, at least a certain number of cells is needed, and if the number is less, the disease will not occur].

This invention will affect all types of the corona virus [not only all types of the corona but also the Influenza, SARS, and MERS] because the entrance of all of them to the cell is the ACE2 receptor [and only a mutation in the virus will make the invention ineffective, if the target receptor of the virus changes and is not ACE2, in which case the virus also loses its pathogenicity to humans].

Another point is that since these normal flora bacteria are the natural bacteria of the person, they are not pathogenic and dangerous for people, and since they live on the surface of the mucosa and gastrointestinal tract, they do not cause any side effects for the person (Even if they have a risk, it could be treated with antibiotics).

This invention even helps people who are infected with the corona virus in the following ways:

1. Reduce the load and accumulation of the virus by absorbing it into the normal flora

2. Reduce the spread and transmission of the virus to other healthy people

Another point is that in addition to the mentioned probiotics, ACE2 can be purified and injected into sick people (not healthy) and thus reduce the complications of the disease in them. It is possible to increase the number of invented probiotics compared to the previous normal flora of the person, before using an antibiotic course (to reduce the usual normal flora of the person) and then give the invented probiotic orally or by inhalation.

Another point is that the bacteria in a process called conjugation can share the plasmid that we made by genetic engineering (conjugation plasmid) with the previous normal flora of the person, so they also contain the ACE2 gene.

The ACE2 gene transmission process and peptide signal to the target bacterium (normal flora) and expression of hACE2 protein on the bacterial membrane:

For transmission of hACE2 gene of the peptide signal to the bacteria (normal flora) different methods can be used that based on the type of selected bacteria the method of operation will be different. One of the methods below can be selected and stated as an example:

1. The DNA is extracted using QIAGEN kit. Forward and reverse primers for the ACE2 gene are then amplified [PCR product is actually ACE2 amplified genes]. At this point, the hACE2 gene is ready to enter the target vector.

2. Cut the vector PuNol-HACE2 (N5.6kb) with restriction enzymes Nhel and Agel. The hACE2 gene as well as the gene sequence for the peptide signal is inserted (e.g P48 and Srp54) into the vector.

To do this, the hACE2 gene and the cut plasmid must have the complement ending, which is done using the mentioned restriction enzymes.

After using restriction enzymes and cutting the gene and vector plasmid, the ligase enzyme is used to bind the cut fragments of the plasmid and the hACE2 gene and the peptide signal. The gene sequence of the peptide signal enters the plasmid in the same way.

The resulting recombinant plasmid is transformed into the bacterium in the presence of calcium chloride (either electric shock or heat shock).

The resulting recombinant plasmid has an antibiotic resistance gene that allows the bacterium to survive in the presence of a specific antibiotic.

In the environment containing the considered antibiotics, the recombinant plasmid- free bacteria die while the bacteria containing it survive and multiply. Each surviving bacterium forms a colony that all carries the recombinant plasmid.

At this stage, we culture the bacteria with the plasmid vector.

Plasmids utilize the facilities of host bacteria and transcribe from the genes. mRNAs produced from plasmids are translated and synthesized into the target protein in the host bacterium.

Because the peptide signal is attached to the produced protein (the peptide signal gene is located inside the recombinant plasmid along with the hACE2 gene), the resulting hACE2 protein goes to the bacterial membrane under the direction of the peptide signal and presented on the surface of the membrane and does not cause inclusion bodies in the cytoplasm.

Point:

If there is intension for surface presentation of ACE2, one of the signal peptides that can be used is POAGOl (P48, Srp54). One of the signal peptides that can be used is Pel B if there is intension to secrete ACE2 to outside. Sample of signal peptide for ACE2 presentation on bacteria

POAGOl (P48,Srp54) and ...

Sample of signal peptide for ACE2 secretion with bacteria

Pe l B

ADVANTAGES OF THE INVENTION

Covid 19 virus has many mutations due to its multiplication and incidence, and in a short time it creates new types against which previous vaccines are ineffective, but in the current invention, since it targets the virus to enter the cell, the result is effective in addition to all available and predictable types on influenza virus, SARS, MERS, etc. (which ACE2 receptors are the entrance of all). Another advantage over the vaccine is that as it uses the digestive and respiratory normal flora, it does not provoke immune reactions and has no side effects from the vaccine. It also inactivates the virus before it enters the cell. As antiviral drugs mostly excreted by the liver they show severe hepatic side effects and are also not effective and also these drugs are relatively effective after infecting by the disease and are not for prevention.

Recently, research has been done on the use of purified ACE2 protein (in the form of nasal spray) to inactivate corona viruses, but as the durability of this spray (unlike normal flora, which grows and multiplies permanently in the body) is very low and is emitted from the body after each use as a foreign substance by the nasal mucosa as well as the flagella of the respiratory system, so it has not a long time effectiveness and needs to be used frequently and there is a possibility of disease in the intervals between uses. Also, since the structure of this protein is destroyed by digestive acids and enzymes, gastrointestinal use is not possible (it is one of the ways of transmitting gastrointestinal disease), but in the present invention, in addition to respiratory normal flora, it can also be used as gastrointestinal normal flora. These bacteria live permanently in the gastrointestinal tract and respiratory tract, depositing ACE2 on their surface or releasing it into the environment, and this happens permanently and creates a long-term immunity against the virus.

DESCRIPTION OF DRAWINGS

Figure 1 shows the physiology of ACE2.

Figure 2 shows the gene map of the desired plasmid.

Figure 3 shows the steps of making a recombinant plasmid. Figure 4 shows the steps for inserting a plasmid into a bacterium.

Figure 5 shows the normal flora of the various organs of the body.

Figure 6 shows the ACE2 gene sequence.

Figure 7 summarizes the workflow in genetic engineering using bacterial plasmids as vectors. Table 1 shows the different stages of the genetic engineering workflow and related methods and tools.

Claims

What is claimed is:

1. The invention of using genetic manipulation bacteria and equipped with ACE2 receptor through genetic engineering, including identification of normal flora bacteria and at least DNA extraction from the QIAGEN kit, and also identification of forward and reverse primers for the ACE gene and cutting hACE2 vector and sequence extraction of the gene related to peptide signal using restriction enzymes such as Nhel and Agel and using ligase in order to bind the cut fragments of the plasmid and transferring the mentioned DNA with the recombinant plasmid into the bacteria.

2. The method of claim one in which the recombinant bacteria have the hACE2 gene inside and the ACE2 protein receptor can go on the bacterial membrane and be presented on the surface of the membrane or secreted to the outside ambient.

3. The method of claim one which uses non-invasive bacteria of the respiratory system to trap viruses of the corona family.

4. The method of claim one which can use normal flora bacteria or any other type of non-pathogenic bacteria.

5. The method of claim one which uses genetic engineering, especially the introduction of the ACE2 gene into the normal flora of the respiratory tract.

6. The method of claim one in which in case of destroying some of these normal floras, they themselves, which contain the ACE2 gene, are reproduced and there is no need to reuse them.

7. The method of claim one which will be effective against all strains of corona virus and influenza, SARS and MERS.

8. The method of claim one that these normal flora bacteria are a person's natural bacteria and are not pathogenic and dangerous for people.

9. The method of claim one which causes reducing the load and accumulation of the virus by absorbing it into the normal flora.

10. The method of claim one which causes to reduce spreading and transmission of the virus to other healthy people by absorbing it into the normal flora.

11. The method of claim one in which ACE2 can be purified and injected into sick people, thereby reducing the effects of the disease in them.

12. The method of claim one in which it is possible to increase the number of probiotics of the invention compared to the person's previous normal flora, by doing an antibiotic course before using it, and then give the invented probiotic orally or by inhalation.

13. The method of claim one in which the bacteria in a process called conjugation can share the plasmid that made by genetic engineering (conjugation plasmid) with the previous normal flora of the person, so they also contain the ACE2 gene.

14. The method of claim one in which the DNA is extracted using QIAGEN kit and then Forward and reverse primers for the ACE2 gene are amplified and then the prepared hACE2 gene is entered to the target vector.

15. The method of claim one which cut the vector PuNol-HACE2 (N5.6kb) with restriction enzymes Nhel and Agel and the hACE2 gene and also the gene sequence for the peptide signal is inserted into it.

16. The method of claim one in which the hACE2 gene and the cut plasmid must have the complement ending, which is done using the mentioned restriction enzymes.

17. The method of claim one in which the ligase enzyme is used to bind the cut fragments of the plasmid and the hACE2 gene and the peptide signal.

18. The method of claim one in which the gene sequence of the peptide signal enters the plasmid. The method of claim one the recombinant plasmid is transformed into the target bacterium in the presence of calcium chloride or electric shock or heat shock. The method of claim one in which the plasmids utilize the facilities of host bacteria and transcribe from the genes. The method of claim one the peptide signal is attached to the produced protein and the resulting hACE2 protein goes to the bacterial membrane under the direction of the peptide signal, located inside it and presented on the surface of the membrane.