WO2022070193A1

WO2022070193A1 - Theranostic aptamer constructs

Info

Publication number: WO2022070193A1
Application number: PCT/IL2021/051181
Authority: WO
Inventors: Roy FARFARA
Original assignee: Trioxnano Ltd
Priority date: 2020-10-01
Filing date: 2021-09-30
Publication date: 2022-04-07

Abstract

The invention provides constructs comprising at least one biological binding moiety for use in the treatment, prevention, or diagnosis of diseases, including methods of treatment using the same and processes for their preparation.

Description

THERANOSTIC APTAMER CONSTRUCTS

BACKGROUND OF THE INVENTION

[001] The ability of antibodies to neutralize toxins, viruses, fungi or bacteria can protect against infection but does not, on its own, solve the problem of how to remove the pathogens and their products from the body. This neutralizing ability is based on the affinity created by the antibody and its antigen which is often decreased by glycosylation and other mechanisms developed by pathogens to avoid humoral immunity. For example, binding of the antibody to its antigen leads to the formation of antigen-antibody complexes that initiates an immune response, which leads to ingestion of antibody-coated bacteria by macrophages and other phagocytic cells that destroy them (Janeway 2001). Different isotypes of antibodies have abilities to bind 2-5 antigens (for example viruses) to each other per antibody (IgM which is a pentamer can bind 5 antigens to each other while IgG can bind only 2 for example).

[002] The protective effect of intravenous immunoglobulin (IVIG) treatment against infectious diseases has been attributed to the ability of specific antibodies to neutralize pathogens, microbial toxins and superantigens. IVIG preparations are pooled IgG antibodies from the serum of multiple donors that contain antibodies against a specific pathogen and a variety of pathogens. Therefore, IVIG treatment might trigger a potentially harmful immunological response by the recipient, such as thrombosis, severe hypersensitivity, anaphylactic reactions, and renal disfunction, etc.

[003] By comparison, aptamers can have significantly greater affinity for pathogenic antigens compared to antibodies and do not induce a potentially detrimental immune response.

[004] During infection, the host recognizes pathogen-associated molecular pattern (PAMP) possessed by the invading organism, which leads to cellular recruitment and a proinflammatory cytokine response including IL-6 and TNF-a. Various pathogenic viruses (e.g., influenza A, COVID-19) and bacteria (e.g., Francisella tularensis) have been found to induce ’’cytokine storm” or hypercytokinemia, that leads to tissue damage and potentially death (D'Elia, Harrison et al. 2013). Though understanding of the processes that trigger cytokine storms has made dramatic progress over the last decades, these findings have not yet been translated into effective and safe treatments (Chousterman, Swirski et al. 2017). The use of aptamers to bind PAMP may result in immune modulation. It is clear that lowering the inoculum of pathogens may prevent initiation of such harmful immunological cascade.

One of the hallmark roles of respiratory mucus of the respiratory tract is to act as a protective barrier against the external environment by trapping particulate matter, including pathogens. Trapped matter can then be expelled from the airways by muco-ciliary clearance, the rhythmic beating of cilia bundles on the airway epithelium. Cilia provide the force necessary to transport foreign materials in the respiratory tract toward the mouth where they can be expectorated (Zanin, Baviskar et al. 2016, Bustamante-Marin and Ostrowski 2017). Another hallmark is the production of IgA, IgG, and IgM (which cause inflammatory responses).

[005] Viral entry into target cells is often a multiple-step process that involves complicated viral-host interactions that can serve as targets for inhibitors, such as the use of common cellular components, such as lipids and lipid rafts, or cellular functionalities, such as endocytosis (Zhou and Simmons 2012). Binding of aptamers to viral targets and generation of viral aggregates in some cases inhibits the virus ability to enter host cells through endocytosis and therefore decreases their infectious potential.

[006] In another aspect, manufactured antibodies are often used for diagnosis of infectious agents in patients. Sometimes, binding of antibodies to the infectious agents is measured, and sometimes commercial antibodies are used as reagents for detecting the presence of infectious agent-specific antibodies. The sensitivity of methods which employ such antibodies is often dependent on the detection means, which can be based on reagents such as colorimetric (e.g. ELISA), fluorescent, and radioactive reagents. Polymerase chain reaction analysis (RT-PCR), that is used to measure DNA or RNA, has also been used to identify infectious agents in patients. However, these methods only identify and/ or quantify the presence of an infectious agent and do not diagnose the functional status of the agent in a patient (i.e. whether he/ she is sick, positive PCR, but not sick and not infective, positive PCR, and sick and infective). In the cases of epidemics, pandemics, and outbreaks, it is imperative to quickly and effectively identify the status of many individuals in order to isolate and/ or treat them as necessary in order to limit the spread of disease, mutation of the infectious agents, and death. The current COVID-19 pandemic exemplifies such a situation in which the health status (sick, positive PCR, but not sick and not infective, positive PCR, and sick and infective) of individuals is not effectively determined, thus would benefit from effective means for both effective diagnosis and treatment as contemplated for the present invention.

References:

Bayry, J., S. Lacroix-Desmazes, M. D. Kazatchkine and S. V. Kaveri (2004). "Intravenous immunoglobulin for infectious diseases: back to the pre-antibiotic and passive prophylaxis era?" Trends Pharmacol Sci 25(6): 306-310.

Bustamante-Marin, X. M. and L. E. Ostrowski (2017). "Cilia and Mucociliary Clearance." Cold Spring Harb Perspect Biol 9(4).

Chousterman, B. G., F. K. Swirski and G. F. Weber (2017). "Cytokine storm and sepsis disease pathogenesis." Semin Immunopathol 39(5): 517-528.

D'Elia, R. V., K. Harrison, P. C. Oyston, R. A. Lukaszewski and G. C. Clark (2013). "Targeting the "cytokine storm" for therapeutic benefit." Clin Vaccine Immunol 20(3): 319-327.

Deyev, S. M. and E. N. Lebedenko (2009). "Modern Technologies for Creating Synthetic

Antibodies for Clinical application." Acta Naturae 1(1): 32-50. Janeway, C. J. T., P; Walport, M; (2001). The destruction of antibody-coated pathogens via Fc receptors. Immunobiology: The Immune System in Health and Disease. New York, Garland Science.

Song Y, Song J, Wei X, Huang M, Sun M, Zhu L, Lin B, Shen H, Zhu Z, Yang C. Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein. Anal Chem. 2020 Jul 21 ;92(14):9895-9900.

Zanin, M., P. Baviskar, R. Webster and R. Webby (2016). "The Interaction between Respiratory Pathogens and Mucus." Cell Host Microbe 19(2): 159-168.

Zhou, Y. and G. Simmons (2012). "Development of novel entry inhibitors targeting emerging viruses." Expert Rev Anti Infect Ther 10(10): 1129-1138.

SUMMARY OF THE INVENTION

[007] The invention thus provides a construct comprising at least one biological binding moiety, wherein said at least one biological binding moiety is at least 0.1% of said construct, for use in the treatment, prevention, or diagnosis of a disease. The construct described above uses its biologic binding moiety to bind to each other said targets described below.

[008] Diseases which may be treated using the above-mentioned constructs include an infectious disease, an inflammatory disease or disorder, an oncologic disease, a neurologic disease or disorder, a hematologic disease, an endocrine disease, fibrosis, a proliferative disease or disorder, and inflammatory disease, a disease or disorder of the immune system, a disease or disorder of the cardiovascular system, a metabolic disease or disorder, a disease or disorder of the skeletal system, or a disease or disorder of the skin or eyes.

[009] Non limiting examples of diseases and disorder that may be treated with the constructs of the invention include, a proliferative disorder (e.g., a cancer, such as hematological cancers (e.g., AML, CML, CLL and multiple myeloma) and solid tumors (e.g., melanoma, renal cancer, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, NSCLC), immune (e.g., ulcerative colitis, Crohn's disease, IBD, psoriasis, asthma, autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and SLE) and inflammatory diseases, neurologic diseases (e.g., diabetic retinopathy, Duchenne's muscular dystrophy, myotinic dystrophy, Huntington's disease and spinal muscular atrophy and other neurodegenerative diseases), metabolic diseases (e.g., type II diabetes, homocystinurea, obesity), cardiovascular diseases (e.g., clotting disorders, thrombosis, coronary artery disease, restenosis, amyloidosis, hemophilia, anemia, hemoglobulinopathies, atherosclerosis, high cholesterol, high tryglycerides), endocrine related diseases and disorders (e.g., NASH, diabetes mellitus, diabetes insipidus, Addison's disease, Turner syndrome, Cushing's syndrome, osteoporosis,) and infectious disease.

[0010] In some embodiments, at least one biological binding moiety is selected from ligand, nucleotide, amino acid, peptide, protein, RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof.

In some embodiments, said at least one biological binding moiety is at least one DNA aptamer. In some embodiments, said at least one biological binding moiety is at least one PNA aptamer.

In some embodiments, said at least one biological binding moiety is at least one RNA aptamer.

[0011] The invention further provides a method of treating an infectious disease comprising the step of administering to a subject in need thereof a composition comprising at least one biological binding moiety selected from RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof. [0012] In some embodiments, said at least one infectious disease is selected from bacterial infection, virus infection, parasite infection, fungal infection and any combinations thereof. In some embodiments, said at least one infectious disease is a viral infection. In some embodiments, said viral infection is corona virus infection (SARS-COVID 19 infection).

[0013] In some embodiments, said at least one infectious disease is selected from respiratory (upper and/or lower) infection, vaginal infection, rectal infection, ear infection, eye infection, systemic infection or any internal organ infection and any combinations thereof.

[0014] The invention further provides a method of treating a hematologic disease comprising the step of administering to a subject in need thereof a composition comprising at least one biological binding moiety selected from RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof.

[0015] The invention further provides a method of treating an oncologic disease comprising the step of administering to a subject in need thereof a composition comprising at least one biological binding moiety selected from RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof.

[0016] The invention further provides a method of treating a neurologic disease comprising the step of administering to a subject in need thereof a composition comprising at least one biological binding moiety selected from RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), exosome, and any combination thereof.

[0017] The invention further provides a method of treating a metabolic disease comprising the step of administering to a subject in need thereof a composition comprising at least one biological binding moiety selected from RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof.

[0018] The invention further provides a method of treating an immunologic disease comprising the step of administering to a subject in need thereof a composition comprising at least one biological binding moiety selected from RNA (ss and ds), DNA (ss and ds), PNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof.

[0019] In some embodiments, said construct further comprises at least one agent selected from at least one labeling agent, at least one contrast agent, at least one dye, and any combinations thereof.

[0020] In some embodiments, said at least one biological binding moiety is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% of the surface of said construct.

[0021] The invention further provides a physical barrier, filtration system, or ventilation system to which particles are applied or attached.

[0022] The invention further provides a pharmaceutical composition comprising at least one construct as disclosed herein above for use in the treatment of at least one infectious disease.

[0023] In some embodiments said pharmaceutical composition is a mucosal membrane delivery composition.

[0024] In some embodiments said pharmaceutical composition is an intranasal composition. In some embodiments said composition is an intranasal spray, intranasal cream, intranasal gel, intranasal delivery device and any combinations thereof.

[0025] In some embodiments, said pharmaceutical composition is administered to the upper respiratory tract of a subject in need thereof (in some embodiments oral wash/rinse, nasal spray, nasal wash and so forth). [0026] In some embodiments, said pharmaceutical composition is administered to the lower respiratory tract of a subject in need thereof (in some embodiments inhalers, nebulizers, powder administration via tubus). In some embodiments said pharmaceutical composition is an intravaginal composition. In some embodiments said composition is a vaginal tablet, vaginal cream, vaginal gel, vaginal suppository vaginal ring, vaginal foam and any combination thereof.

[0027] In some embodiments said pharmaceutical composition is an intrarectal composition. In some embodiments said composition is a rectal tablet, rectal cream, rectal gel, rectal suppository rectal ring and any combinations thereof.

[0028] In some embodiments said pharmaceutical composition is an ointment, gel or a cream applied to the skin and/ or mucosal membrane. In some embodiments, said pharmaceutical composition is patch applied to the skin and/ or mucosal membrane.

[0029] In some embodiments said pharmaceutical composition is an enteral treatment including a tablet, a capsule, microcapsule, suspension, foam, powder, sustained, slow, or timerelease system and any combination thereof.

[0030] In some embodiments said pharmaceutical composition is a parenteral treatment including IV, IM, or SC treatment.

[0031] In some embodiments said pharmaceutical composition is administered via an implant loaded with the treatment.

[0032] The invention further provides a method of treating or preventing an infectious disease in a subject in need thereof, said method comprises administering to said subject at least one construct, nanoparticle or microparticle. In the first aspect, the invention provides a construct, nanoparticle or microparticle (in some embodiments mesoporous silica MSNP) comprising at least one biological binding moiety (in some embodiments DNA aptamer) covering at least 0.1% of the surface of said construct, nanoparticle, or microparticle for use in the treatment and/or prevention of at least one infectious disease.

[0033] In another aspect, the invention provides a construct, nanoparticle or microparticle comprising a plurality of (specific or non-specific) biological binding moieties, (DNA, PNA, RNA, peptide, protein, antibody, FAB), covering at least 0.1% of said nanoparticle surface.

[0034] In another aspect, the invention provides a construct, nanoparticle or microparticle comprising a plurality of organic and inorganic scaffolds (polymers, metals, ceramics), with various pore sizes and diameters.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0036] FIG. 1 shows Sl-specific aptamer-conjugated construct reduction of lentivirus-induced lysis of HEK-293 cells. When the aptamer sequence was scrambled, there was no significant effect.

[0037] FIGS. 2A-2H shows micrographs of Gold bead-labeled pseudo-virus aggregation due to DNA-pentameric aptamer binding. The labeled viruses were incubated with (2A, 2B) pentameric aptamer 1, (2C, 2D) pentameric aptamer 2, (2E, 2F) pentameric scrambled aptamer, and (2G, 2H) pentameric aptamer 1 and target beads without the SI protein. Micrographs of representative fields (of x40, x80 magnifications) are presented.

[0038] FIG. 3 shows an electron micrograph of a Sl-specific pentameric aptamer that specifically complexes with a pseudo-virus South African COVID-19 variant. The large oval is an inserted object having the size of a bacteria, for comparing with the size of the pseudo viruses. [0039] FIGS. 4A-4B shows micrographs of Gold bead-labeled pseudo-virus aggregation due to DNA-pentameric aptamer binding. The labeled viruses were incubated with (4A) scrambled (nonspecific) pentameric aptamer and (4B) pentameric COVID-19 delta SI -specific aptamer.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0040] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0041] The term “ nanoparticle” in the context of the present invention should be understood to relate to a particle with the size of between 1 and 1000 nanometers (nm). In some embodiments, said nanoparticle is a solid particle (for example a silica nanoparticle). In other embodiments, said nanoparticle creates a gel. In other embodiments, said nanoparticle is a core shell nanoparticle having a shell surrounding a core material. In some embodiments the nanoparticle has an organic scaffold (for example Silica). In some embodiments the nanoparticle has an inorganic scaffold (for example Gold). In some embodiments the nanoparticle is coated. In some embodiments the nanoparticle is porous with the pore size between 0.1 and 50 nanometers (nm).

[0042] The term microparticle in the context of the present invention should be understood to relate to a particle with the size of between 1 and 10 microns. In some embodiments, said nanoparticle is a solid particle (for example a silica nanoparticle). In other embodiments, said microparticle creates a gel. In other embodiments, said microparticle is a core shell microparticle having a shell surrounding a core material. In some embodiments the microparticle has an organic scaffold (for example Silica). In some embodiments the microparticle has an inorganic scaffold (for example Gold). In some embodiments the microparticle is coated. In some embodiments the microparticle is porous with the pore size between 50 and 1000 nanometers (nm). [0043] When referring to said nanoparticle or microparticle "surface it should be understood to encompass any part of the surface area of said particle or microparticle.

[0044] The term “construct in the context of the present invention should be understood to relate to at least one biological binding moiety as defined herein, either alone or attached by covalent bonds or via at least one noncovalent bond to other binding moi eties, nanoparticle or microparticle surfaces, chemically- or immunologically inert compounds or theranostic compounds.

[0045] The term “binding targets " in the context of the present invention should be understood to include receptors, outer membrane/ surface proteins, channels, transporters, phospholipids, specific binding domain and any other component on the cell surface. The in some embodiments the binding target is specific to a particular pathogen. In some embodiments the binding target is none- specific to a particular pathogen.

[0046] The term “biological binding moiety” in the context of the present invention should be understood to encompass an agent that is capable or is known for its ability to specifically or none- specifically bind a biological target being an infective agent/pathogen. In some embodiments said at least one binding moiety is selected from a ligand, nucleotide, amino acid, peptide, protein, RNA, DNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv) or any combination thereof.

[0047] The term “pathogen” in the context of the present invention should be understood to include any infectious disease-producing agent including viruses, bacteria, fungi, virions, parasites, and other microorganisms that can cause a disease in either human, animal, or plant subjects.

[0048] The term "treatment" as used herein means the management and care of a patient for the purpose of combating an infectious disease or condition associated therewith. The term is intended to include prophylaxis, the delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition. The patient to be treated is preferably a mammal, particularly a human being. In some embodiments the application of treatment can also extended to veterinary or agricultural purposes.

[0049] In some embodiments, said at least one biological binding moiety is targeting at least one specific pathogen.

[0050] When referring to “construct, nanoparticle or microparticle comprising at least one binding moiety” it should be understood that said binding moiety is attached to said construct, nanoparticle or microparticle in any possible form, such as for example: chemical bond, hydrophobic bonding, electrostatic bonds, ligands and any combination thereof.In some embodiments, a construct of the invention further comprises at least one agent selected from at least one labeling agent, at least one contrast agent, at least one dye, and any combinations thereof. Said further agent may be included in said construct in any of the forms suggested herein above: construct encompasses said further agent within (entrapped, capped or encapsulated) said construct, construct encompasses said further agent on the surface of said construct, said construct is chemically attached to said further agent (within or on the surface of said construct), said construct is electrostatically/coordinatively attached to said further agent (within or on the surface of said construct) and any combination thereof.

[0051] In some embodiments, a construct of the invention further comprises at least one agent selected from at least one anti-microbiol, at least one anti-viral, at least one anti-fungal, at least on anti -parasitic and any combinations thereof. Said further agent may be included in said construct in any of the forms suggested herein above: construct encompasses said further agent within (entrapped, capped or encapsulated) said nanoparticle, construct encompasses said further agent on the surface of said construct, said construct is chemically attached to said further agent (within or on the surface of said nanoparticle or microparticle), said construct is electrostatically/coordinatively attached to said further agent (within or on the surface of said construct) and any combination thereof.

[0052] In some embodiments said pharmaceutical composition is an intranasal composition, a mouthwash composition or an inhalation composition.

[0053 ] The phrase “covers at least 0.1% of said nanoparticle or microparticle surface” relates to the percentage of coverage of said at least one binding moiety of the surface area of said nanoparticle or microparticle.

[0054] In other embodiments, binding moiety is 0.1% 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of said construct.

[0055] In a further aspect, the invention provides a nanoparticle or microparticle comprising a plurality of binding moieties covering at least 0.1% of said nanoparticle or microparticle surface.

[0056] In a further aspect, the invention provides a method of treating and preventing infectious diseases in a subject in need thereof, said method comprises administering to said subject at least one nanoparticle or microparticle comprising at least one binding moiety and covering at least 0.1% of said nanoparticle surface.

[0057] The present invention also relates to pharmaceutical compositions comprising at least one nanoparticle as disclosed herein above and below for the use defined for the subject invention in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxiliaries must be “ acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.

[0058] Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration or administration via an implant. The compositions may be prepared by any method well known in the art of pharmacy. [0059] Such methods include the step of bringing in association compounds used in the invention or combinations thereof with any auxiliary agent. The auxiliary agent(s), also named accessory ingredient(s), include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavouring agents, anti-oxidants, and wetting agents.

[0060] Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets, dragees or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.

[0061] The invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material, including instructions for the use of the composition for a use as hereinbefore described.

[0062] For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injection. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example water, prior to use. For transdermal administration, e.g. gels, patches or sprays can be contemplated. Compositions or formulations suitable for pulmonary administration e.g. by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators.

[0063] The exact dose and regimen of administration of the composition will necessarily be dependent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. [0064] The invention further includes a method of diagnosing an infectious disease in a subject, wherein a sample is taken from the subject, and the sample is contacted with a construct. The construct may be selected from an aptamer, a pentameric aptamer, and an aptamer attached to a nanoparticle. The method can be used to determine whether the subject is sick, positive PCR, but not sick and not infective, or positive PCR, and sick and infective.

EXAMPLES

EXAMPLE 1 Preparation of pentameric aptamers

[0065] Pentameric aptamers are prepared using the following complexation steps:

1). Incubate oligonucleotide 1 to oligonucleotide 2, for 5 min at 65C.

2). Transfer the annealed complex and PBS. Let cool for 5 min at 45C.

3). Sequentially add oligonucleotide 3 and oligonucleotide 4 as describe in step #2.

4). Transfer to another tube and add Origami buffer with 15mM Mg, at 45C (about 1 :6 dilution).

5). Add oligonucleotide 5, at 65C.

6). Let cool for 5 min at 45C.

7). Cool slowly to RT and let 60 min at RT and add PBS.

EXAMPLE 2 Preparation of DNA aptamer-conjugated beads

[0066] In order to prepare silica bead-conjugated aptamer pentamers, one micrometer silica beads were thiol activated with EMCS and reacted in PBS, overnight at room temperature, with DNA aptamers described in Song, et al. (2020). A control aptamer was prepared with a scrambled sequence for nonspecific binding comparison. Following the reaction, the beads were thoroughly washed with PBS. Attachment of the aptamers to the beads was verified by monitoring the unbound aptamers (ext. 638 nm/em 660 nm).

[0067] In order to prepare gold bead-conjugated aptamer pentamers, protein G was adsorbed to gold particles having a 30 nm diameter. “Gold 60nm Protein-G”, about 10^el° beads, in 150pl, were incubated with 15pl of anti-His Ab 200pg/ml (3pg, 20pmol, about 10^e12 particles) and 5pl 200pg/ml Slm-His, ( I pg, lOpmo, about 5xl0^e11 particles). After overnight incubation, at room temperature, the gold particle-pentamer complexes were ready.

EXAMPLE 3 DNA aptamer binding to Spike (SARS-CoV-2) Pseudo-typed Lentivirus [0068] DNA aptamer-conjugated beads were gently mixed for 30 minutes, at room temperature, with Pseudo-typed Lentivirus in 2 mL. Unbound virus was isolated in supernatant from the beads following centrifugation.

EXAMPLE 4 Luciferase assay for quantifying the presence of Pseudo-typed Lentivirus [0069] Human embryonic kidney cells derived from HEK-293/ACE2 cell line were the model for coronavirus infection. Angiotensin-Converting Enzyme 2 (ACE2) is the receptor that is recognized by coronavirus. Luciferase-expressing HEK-293 cells transfected with hACE-2 plasmid were infected with the remaining spike Pseudotyped Lentivirus (Luciferase Reporter) (Almog diagnostics, 79942) using the following protocol:

Day 1- cells were cultured in a 96-well flat plate at different concentrations starting from 2xl05/well.

Day 2- medium was removed, 20 pl beads and 5 pl virus was added + polybrene (1 : 100) per well.

Day 3- medium was added to a volume of 100 pl.

Day 4- luciferase assay, according to the kit manufacture’s protocol.

Spike (SARS-CoV-2) pseudo-typed Lentivirus (Luciferase Reporter), 79942 https://bpsbioscience.com/spike-sars-cov-2-pseudotyped-lentivirus-luc-reporter-79942

[0070] The SARS-CoV-2 Spike pseudo-typed Lentivirus was produced with SARS-CoV-2 Spike (Genbank Accession #QHD43416.1) as the envelope glycoproteins instead of the commonly used VSV-G. These pseudovirions also contain the firefly luciferase gene driven by a CMV promoter, therefore, the spike-mediated cell entry can be conveniently measured via luciferase reporter activity.

EXAMPLE 5. Luciferase assay

[0071] Human embryonic kidney cells derived from HEK-293/ACE2 cell line are a recognized model for coronavirus infection. Angiotensin-Converting Enzyme 2 (ACE2) is the receptor that is recognized by coronavirus. We transfected HEK-293 cell line with hACE-2 plasmid in order to express it on their cell surface. Cells were transfected and stained with anti ACE-2 and analyzed by flow cytometry. 293T ‘expressing’ hACE-2 were infected with Spike (SARS-CoV- 2) Pseudotyped Lentivirus (Luciferase Reporter) (Almog diagnostics, 79942) by the following protocol:

Day 3- medium was added to a volume of 100 pl.

Day 4- luciferase assay, according to manufacture protocol.

Spike (SARS-CoV-2) Pseudo-typed Lentivirus (Luciferase Reporter), 79942 https://bpsbioscience.com/spike-sars-cov-2-pseudotyped-lentivirus-luc-reporter-79942 The SARS-CoV-2 Spike Pseudo-typed Lentivirus was produced with SARS-CoV-2 Spike (Genbank Accession #QHD43416.1) as the envelope glycoproteins instead of the commonly used VSV-G. These pseudovirions also contain the firefly luciferase gene driven by a CMV promoter, therefore, the spike-mediated cell entry can be conveniently measured via luciferase reporter activity.

EXAMPLE 6. Pseudo-virus particle reagent preparation [0072] The reagent for detection of SI -specific binding was prepared by coating 30 nm gold particles with protein G. These beads were complexed with anti-His tag antibodies. These beads were either used as is as a negative control. Alternatively, SI protein with a His tag was further bound to the beads via the anti-His tag antibodies.

EXAMPLE 7. Aptamers block infection

[0073] Silica beads loaded with three different CoV2-RBD-specific aptamers developed to bind the COVTD-19 S protein. Control beads were loaded with an aptamer having the same bases as aptamer 5, which were arranged in a scrambled order.In order to determine the neutralizing capacity of the aptamers, the loaded beads were incubated with a preparation of the pseudo-virus for 30 minutes, at room temperature. The beads binding pseudo-virus were separated from the free virus by centrifugation. The free virus-containing sup was assessed for lytic capacity by incubating with the luciferase-expressing HEK-293/ACE2 cell line as described above. As presented in Fig. 1, removal of pseudo-virus by three SI -specific aptamers significantly reduced HEK cell lysis. The lysis due to supernatant following incubation with the scrambled aptamerbeads was similar to that of the control value. Therefore, SI -specific aptamers can protect individuals from COVID-19 infection.

EXAMPLE 8. Pentameric aptamers can enable sensitive detection of COVID-19 virus

[0074] Complexes of pentameric S 1 -specific aptamers with pseudo- virus gold particles were detected following 4-hour incubations, in PBS, at room temperature (Fig. 2A, 2B, 2C, 2D). When the scrambled. Aptamers were incubated with the pseudo-virus gold particles, essentially no complexes were present (Fig 2E, 2F). Additionally, when the SI protein was not included in the target particles, essentially no complexes are observed. Therefore, pentameric SI -specific aptamers specifically complex SI -presenting particles and may be a useful reagent for detecting

COVID-19 viruses. [0075] Complexes of pentameric Sl-specific aptamers with a South African mutated pseudovirus were also portrayed by scanning electron microscopy (Fig. 3) after placement on electron microscope sample grids. These complexes were formed by 4-hour incubations, in PBS, at room temperature, As is shown in Fig. 3, the dark gold-labeled penta-aptamer complexes with the mutated pseudo-viruses were significantly smaller than bacteria.

EXAMPLE 9. ELISA-like (aptamer instead of antibody enables sensitive differentiation between mutations) analysis with pentameric aptamers can enable sensitive COVID-19 detection.

[0076] An ELISA-like method using the above-mentioned pentameric Sl-specific aptamers was established. Serial dilutions of the SI antigen in PBS buffer containing 0.25% BSA were first added to 96-well plates such that the amount of SI antigen in four replicate wells ranged from 10 fg (femtogram) to 1 pg. The four replicate wells for determining background binding contained 0.25% BSA in PBS. The plate was incubated at room temperature for 2 hours. Then, the plate was washed with buffer and 200 pmol of either biotin-conjugated pentameric Sl- specific aptamers or biotin-conjugated pentameric aptamers, either no aptamer or scrambled aptamers were added per well. The plate was incubated at room temperature for 1 hour. Unbound aptamer was removed by washing with PBS buffer, and streptavidin-horseraddish peroxidase (HRP, Merck) reagent (1 :250), in PBS buffer containing BSA 0.5%, was added to each well for incubation at room temperature for 1 hour. The wells were washed five times with PBS buffer containing BSA 0.5% before the HRP substrate (Merck) was added, After 15 minutes, the reaction was terminated with stopper reagent (Merck) and the optical density (OD), at 450 nm, was read. The results are presented in Table 1. These results indicate background value of approximately 0.1 OD. Statistically greater OD values were found in wells which contained 10 pg SI protein. This result indicates the sensitivity of the assay. In general, ELISA methods are much less sensitive. In contrast to the results with the Sl-specific aptamer, the results with scrambled aptamer and those with no aptamer were similar to the background results. Therefore, this method is highly specific and sensitive.

Table 1. Sensitivity of the ELISA-like Method for COVID-19 SI Protein

EXAMPLE 10. Pentameric aptamers can enable sensitive detection of the Delta Variant COVID-19 virus

[0077] Complexes of pentameric SI -specific aptamers with a Delta variant virus were also portrayed by scanning electron microscopy (Fig. 4) after placement on electron microscope sample grids. These complexes were formed by 4-hour incubations, in PBS, at room temperature, As is shown in Fig. 4, while no complexes formed in the presence of a scrambled (nonspecific) aptamer (FIG. 4A), the dark gold-labeled penta-aptamer complexes with the mutated viruses were identified with the Delta variant-specific aptamer (FIG. 4B).

EXAMPLE 11. Pentameric aptamers can enable detection of the functionally-active Delta

Variant COVID-19 virus. [0078] In order to quantify the presence of the Delta variant COVID-19 RNA, including the portions for expressing the E (envelop), S (S protein), and N (nucleocapsid) proteins, a sample was taken from a Delta variant -positive COVID-19 patient for real time reverse transcription- polymerase chain reaction analysis (RT-PCR). The RNA of the sample was either directly analyzed or analyzed following precipitation with either a Delta variant COVID-19 pentameric aptamer (1c & New) or a nonspecific (scrambled) aptamer. The results presented in Table 2 demonstrate that the scrambled aptamer precipitated background levels of Delta variant COVID- 19 RNA, while the Delta variant COVID-19 pentameric aptamer precipitated the same amount of viral RNA as that measured in the original sample (i.e. 10-fold greater than background level for PCR). This aptamer precipitated RNA for expressing the E (envelop), S (S protein), and N (nucleocapsid) proteins. Therefore, these results confirmed that the patient is indeed sick and infectious. As such, this method not only identifies the presence of the virus, it also provides a means for functionally assessing the patient’s status. Since it appears that viral RNA was not fully precipitated, it is foreseen that greater sensitivity will be achieved when the procedure has been optimized.

Table 2. COVID-19 Delta Variant-specific Pentameric Aptamers Enable Specific, Sensitive Precipitation and Identification of Infective Viruses

* E (envelop), S (S protein), N (nucleocapsid)

Claims

22 CLAIMS

1. A construct comprising at least one biological binding moiety, wherein said at least one biological binding moiety is at least 0.1% of said construct, for use in the treatment, prevention, or diagnosis of a disease.

2. A construct according to claim 1, wherein said at least one biological binding moiety is selected from ligand, nucleotide, amino acid, peptide, protein, RNA, DNA, metal, antibody ion, antigen-binding fragment (Fab), single-chain variable fragment (scFv), and any combination thereof.

3. A construct according to claim 1, wherein said at least one biological binding moiety is at least one DNA aptamer.

4. A construct according to claim 1, wherein said at least one biological binding moiety is at least one PNA aptamer.

5. A construct according to claim 1, wherein said at least one biological binding moiety is at least one RNA aptamer.

6. A construct according to claim 1 for the treatment of a disease.

7. A construct according to claim 1 for prevention of a disease.

8. A construct according to claim 1 for diagnosis of a disease.

9. A construct according to claim 1, wherein said disease is selected from an infectious disease, an inflammatory disease or disorder, an oncologic disease, a neurologic disease or disorder, a hematologic disease, an endocrine disease, fibrosis, a proliferative disease or disorder, and inflammatory disease, a disease or disorder of the immune system, a disease or disorder of the cardiovascular system, a metabolic disease or disorder, a disease or disorder of the skeletal system, or a disease or disorder of the skin or eyes.

10. A construct according to claim 9, wherein said infectious disease is selected from bacterial infection, viral infection, parasite infection, fungal infection and any combinations thereof.

11. A construct according to claim 10, wherein said infectious disease is a viral infection.

12. A construct according to claim 11, wherein said viral infection is corona virus infection (SARS-COVID 19 infection).

13. A construct according to claim 9, wherein said infectious disease is selected from respiratory (upper and/or lower) infection, vaginal infection, rectal infection, ear infection, eye infection, systemic infection, internal organ infection, skin infection and any combinations thereof.

14. A construct of claim 1, further comprising at least one agent selected from at least one antibacterial, antiviral, antifungal, antiparasitic, labeling agent, at least one contrast agent, at least one dye, and any combinations thereof.

15. A construct of claim 1, wherein said at least one biological binding moiety is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of said construct.

16. A composition comprising at least one construct of claims 1 to 12, for use in the treatment of at least one infectious disease.

17. A composition according to claim 16, being a mucosal membrane delivery composition.

18. A composition according to claim 16, being a respiratory delivery composition.

19. A method of treating or preventing an infectious disease in a subject in need thereof, said method comprises administering to said subject at least one construct comprising at least one biological binding moiety, wherein said at least one biological binding moiety that is at least 0.1% of said construct.

20. A method of diagnosing an infectious disease in a subject, said method comprises taking a sample from said subject, contacting the sample with least one construct comprising at least one biological binding moiety, wherein said at least one biological binding moiety that is at least 0.1% of said construct.

21. The method of claim 20 wherein the construct is selected from an aptamer, a pentameric aptamer, and an aptamer attached to a nanoparticle.

22. The method of claims 20 or 21, wherein the construct enables determination of the subject’s functional status.

23. The method of claim 21, wherein the functional status is selected from sick, positive PCR, but not sick and not infective, positive PCR, and sick and infective.