Biomarkers and their use in the diagnosis and treatment of neurological post-acute sequelae of COVID-19 (NPASC)

Abstract

Described herein are methods and related compositions for determining the likelihood of neurological acute sequelae of COVID-19 (NPASC) in a subject based on the levels of biomarkers in a combination of biomarkers from a biological sample from the subject. Also disclosed herein are methods for treating a subject identified as likely to be suffering from NPASC, and methods for treating a subject by modulating the level or activity of an NPASC therapeutic target identified herein.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 398,363, filed August 16, 2022, and U.S. Provisional Patent Application No. AU2023 / 900376, filed February 16, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] The present application relates generally to the field of diagnostics, and in particular to methods and kits for the diagnosis and / or treatment of Neural Post Acute Sequelae of COVID ("NPASC"). [Background technology]

[0003] At the end of this specification, bibliographic details of the references in this specification are also listed.

[0004] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] It is understood that suboptimal levels or function of biomolecules in the human system can dysregulate homeostasis and severely impact essential biological functions such as gene expression regulation, cell division and differentiation, development, and repair. Ultimately, such structural dysfunction can lead to a variety of diseases and pathologies, including nervous system disorders.

[0006] The coronavirus disease pandemic caused by SARS-CoV-2 infection has dramatically affected more than 500 million people worldwide since 2019. SARS-CoV-2 causes COVID-19, often defined as an acute illness lasting up to four weeks from the onset of initial symptoms. A significant portion of infected individuals develop a variety of symptoms long after the viral infection and acute symptoms of COVID-19, which appear to persist after such acute COVID-19 symptoms or complications. This "long COVID" has been termed post-acute sequelae of COVID-19 (PASC), and symptoms can persist and / or last for six weeks, up to 12 weeks, and / or beyond, up to 26 weeks, and / or one and two years. PASC can affect the musculoskeletal, gastrointestinal, pulmonary, nervous, and other systems. Neurological PASC (NPASC) is a subset of PASC in subjects with neurological symptoms, including cognitive impairment (reported, for example, as confusion, difficulty thinking, decreased attention, impaired executive function and memory), headache, paresthesia (tingling and numbness), taste disorders, smell disorders, muscle pain, post-traumatic stress disorder (PTSD), sleep disorders, anxiety and depression, with fatigue believed to be the most common neurological symptom (Pinzon et al. J. Infection and Public Health 2022;15:856-860).

[0007] A study by Hampshire et al. (EClinical Medicine 2021 Sept;39:10144) supports the hypothesis that individuals infected with SARS-CoV-2 may have persistent, objectively measurable cognitive impairment, even after carefully controlling for pre-illness IQ, pre-existing medical conditions, sociodemographic factors, and mental health symptoms. Individuals who recovered from COVID-19, including those who no longer reported symptoms, showed significant cognitive impairment compared with controls, controlling for age, sex, education level, income, race / ethnicity, pre-existing medical disorders, fatigue, depression, and anxiety. This impairment was substantial in both hospitalized individuals (who represent a small proportion of COVID-19 subjects) and non-hospitalized cases (who represent the majority of SARS-CoV-2-infected subjects with biologically confirmed COVID-19). NPASC subjects have been observed to have symptoms, as assessed by a number of methods.

[0008] These methods may have been established while searching for symptoms of neurological disorders frequently seen in subjects with other nervous system disorders, such as neurodegenerative, neuropsychiatric, neurodevelopmental, or neuropathological conditions. Diagnosis and monitoring of NPASC involves standardized cognitive tests designed to measure aspects such as processing speed (e.g., pattern comparison processing speed test), attention and executive memory (e.g., inhibitory control and attention test), executive function (e.g., size-change card sort test), and working memory (e.g., list-sorting working memory test). These aspects of the condition can be assessed through detailed interviews and questionnaires, such as the NIH Toolbox v2.1 instrument administered by researchers and clinicians, expressed as T-scores adjusted for age, education, sex, and race / ethnicity, with a score of 50 representing the normative mean / median for the U.S. population and a standard deviation of 10. Diagnosis may also be via the PROMIS-57, a questionnaire for self-reporting different aspects of physical, mental, and social health. Diagnosis of NPASC is currently limited by clinical assessments, such as those using the criteria described above. In some conditions, scans and biopsies may also inform diagnosis and treatment. NPASC treatment or therapy may include counseling, memory, and olfactory training.

[0009] Regarding causal mechanisms, immunological and virological studies, metabolomics, genomics, proteomics, and pathway analysis have yet to fully characterize the myriad processes involved at various levels, from molecular biology and cellular immune responses to the tissues and organs involved in PASC or NPASC. The key endogenous molecules that influence nervous system pathology are poorly understood. Multimodal neuroimaging methods, including magnetic resonance imaging techniques, have been used to investigate nervous system disorders at the structural-functional level, but research is in a relatively early stage, and the key molecular mechanisms and pathways underlying the observed pathology remain largely elusive.

[0010] Given the large number of people worldwide affected by NPASC, the duration of NPASC, and the lack of established treatments for this condition, there is an urgent need to identify biomarkers for NPASC, preferably noninvasive ones, that would facilitate diagnostic and therapeutic development, treatment testing, and treatment monitoring. This would be useful for subjects with PASC after demonstrating viral infection with SARS-CoV-2, as well as for many NPASC subjects who have not been tested to confirm COVID-19 but who nevertheless exhibit neurological symptoms assessed by currently available methods, and for NPASCs unrelated to SARS-CoV-2. This testing could be useful for subjects who have tested positive for SARS-CoV and MERS-CoV, as well as for such subjects without confirmed infection. Summary of the Invention

[0011] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as explicitly endorsing both meanings or either meaning. As used herein, the term "about," unless otherwise specified, refers to + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% of the specified value.

[0012] Throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0013] As used herein, the singular forms "a," "an," and "the" include singular and plural references unless the context dictates otherwise.

[0014] "About" means a measurement, quantity, level, activity, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference measurement, quantity, level, activity, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0015] Each embodiment in this specification applies mutatis mutandis to all other embodiments unless otherwise specified.

[0016] Although the present invention is described and exemplified with respect to the long-term neurological sequelae observed in viral infection with SARS-CoV-2, the invention also extends to neurological post-acute sequelae (NPASV) of viral infection with pathogenic viruses other than SARS-CoV-2, such as other coronavirus infections with long-term neurological sequelae or others known in the art.

[0017] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). SEQ ID NO: Sequence Identifier <400> 1 (SEQ ID NO: 1), <400> 2 (SEQ ID NO: 1). The sequence identifiers for the biomarkers are set forth in the Table. A sequence listing may be provided after the claims for any sequence described herein.

[0018] In accordance with the present invention, the inventors have considered that while gene expression studies provide an indication of canonical proteins expressed in a subject, protein studies such as 2D electrophoresis and proteomics increasingly provide a more forensic investigation of the amount and form of a given protein in a given biological niche of a subject, and in combination with further analytical techniques may provide the skilled artisan with insight into diagnostic and therapeutic targets.

[0019] The present invention is based, at least in part, on the discovery of several endogenous circulating proteins whose levels are surprisingly altered in NPASCs and which, individually or in combination, can serve as (i) non-invasive biomarkers for subject and sample assessment, or, in some cases, (ii) therapeutic targets for the treatment of NPASCs as disclosed herein.

[0020] Thus, in one aspect, provided herein is a method for determining the likelihood of neurological acute sequelae of COVID-19 (NPASC) in a subject, the method comprising measuring or having measured a level of at least one of C5a anaphylatoxin (C5a) or gliomedin in a biological sample from the subject. In some preferred embodiments, the method comprises measuring or having measured a level of C5a and a level of gliomedin. In some embodiments, the method further comprises determining or having determined that the likelihood of NPASC in the subject is higher if the measured level of C5a, gliomedin, or both is above a threshold level of C5a, gliomedin, or both.

[0021] In some embodiments, the method also measures, or has measured, the level of at least one of the biomarkers selected from the list consisting of transforming growth factor beta 1 (TGF beta 1), galactosylceramide sulfotransferase (Gal3ST1), interferon (IFN) lambda-1, growth hormone-releasing hormone (GHRH), lymphocyte function-associated antigen 3 (LFA-3), Fas ligand (FASLG), transgelin, immunoglobulin heavy chain constant gamma 1 (IgGH1), glycoprotein NMB (GPNMB), and antithrombin-III.

[0022] In some embodiments, the method includes measuring, or having measured, levels of C5a, gliomedin, and TGFβ 1. In some embodiments, the method also includes determining, or having determined, that the subject is more likely to be an NPASC if the measured level of one or more of C5a, gliomedin, or TGFβ 1 is above a threshold level of C5a, gliomedin, TGFβ 1, or any combination thereof.

[0023] In some embodiments, the method includes measuring, or having measured, levels of C5a, gliomedin, TGFβ1, and Gal3ST1. In some embodiments, the method also includes determining, or having determined, that the subject is more likely to have NPASC if the measured level of one or more of C5a, gliomedin, or TGFβ1 is above a threshold level of C5a, gliomedin, TGFβ1, or any combination thereof, and the level of Gal3ST1 is below a threshold level of Gal3ST1.

[0024] In some embodiments, the method includes measuring or having measured levels of C5a, gliomedin, Gal3ST1, IFNlambda-1, and GHRH. In some embodiments, the method also includes determining or having determined that the subject is more likely to have NPASC if (i) the measured level of one or more of C5a, gliomedin, or GHRH is above a threshold level of C5a, gliomedin, GHRH, or any combination thereof, and (ii) the level of Gal3ST1 or IFNlambda-1 is below a threshold level of Gal3ST1, IFNlambda-1, or any combination thereof.

[0025] In some embodiments, the method includes measuring, or having measured, levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFNlambda-1, and LFA-3. In some embodiments, the method also includes determining, or having determined, that the subject is more likely to be an NPASC if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, or LFA-3 is above a threshold level of C5a, gliomedin, TGFβ1, LFA-3, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1 or IFNlambda-1 is below a threshold level of Gal3ST1, IFNlambda-1, or any combination thereof.

[0026] In some embodiments, the method includes measuring, or having measured, levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFNlambda-1, GHRH, and LFA-3. In some embodiments, the method also includes determining, or having determined, that the subject is more likely to have NPASC if (i) the measured level of one or more of C5a, gliomedin, GHRH, or LFA-3 is above a threshold level of C5a, gliomedin, GHRH, LFA-3, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1 or IFNlambda-1 is below a threshold level of Gal3ST1, IFNlambda-1, or any combination thereof.

[0027] In some embodiments, the method includes measuring, or having measured, levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFNlambda-1, FASLG, and transgelin. In some embodiments, the method also includes determining, or having determined, that the subject is more likely to have NPASC if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, FASLG, or transgelin is above a threshold level of C5a, gliomedin, TGFβ1, FASLG, transgelin, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1 or IFNlambda-1 is below a threshold level of Gal3ST1, IFNlambda-1, or any combination thereof.

[0028] In some embodiments, the methods measure or include having measured levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFN lambda-1, GHRH, GPNB, and IgGH1. In some embodiments, the method also determines, or includes having determined, that the likelihood of NPASC in the subject is higher if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, GHRH, or GPNB is above a threshold level of C5a, gliomedin, TGFβ1, GHRH, GPNB, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1, IFN lambda-1, or IgGH1 is below a threshold level of Gal3ST1, IFN lambda-1, IgGH1, or any combination thereof.

[0029] In some embodiments, the method includes measuring or having measured levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFNlambda-1, GHRH, LFA-3, IgGH1, and GPNMB. In some embodiments, the method also includes determining or having determined that the subject is more likely to have NPASC if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, GHRH, LFA-3, or GPNMB is above a threshold level of C5a, gliomedin, TGFβ1, LFA-3, GPNMB, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1, IFNlambda-1, or IgGH1 is below a threshold level of IFNlambda-1, IgGH1, or any combination thereof.

[0030] In some embodiments, the biological sample in any of the foregoing methods is a whole blood sample, a plasma sample, or a serum sample. In some embodiments, the plasma is EDTA plasma or heparinized plasma.

[0031] In some preferred embodiments, at least one biomarker is measured by one or more immunoassays. In some embodiments, the one or more immunoassays comprise a multiplexed immunoassay. In some embodiments, the one or more immunoassays or multiplexed immunoassays comprise an ELISA immunoassay.

[0032] In other embodiments, the level of at least one biomarker is measured through the use of a slow off-rate modified aptamer that specifically binds to the protein whose level is being measured.

[0033] In some embodiments, any of the foregoing methods does not include measuring the level of more than 12 biomarkers in the biological sample.

[0034] In other embodiments, the level of at least one biomarker is determined by mass spectrometry.

[0035] In some embodiments, the subject has or has had COVID-19.

[0036] In a related aspect, provided herein is a method for treating NPASC in a subject, the method comprising identifying the subject as likely to be affected with NPASC based on any of the aforementioned methods, and providing or having provided to the subject an NPASC therapy.

[0037] In some embodiments, a subject is diagnosed as likely to have NPASC based on the methods disclosed herein, and if the level of TGFβ1 in the subject is above a threshold, the NPASC therapy comprises administering or having administered to the subject a therapeutically effective amount of an antisense oligonucleotide against α4 integrin. In some preferred embodiments, the subject to whom the antisense oligonucleotide is administered is not a subject identified as having multiple sclerosis or Duchenne muscular dystrophy (DMD). In some embodiments, the antisense oligonucleotide is ATL-1102, the nucleotide sequence of which is set forth in SEQ ID NO:1: 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3' (SEQ ID NO: 1), a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides at positions 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; e) All cytosines are 5-methylcytosines ( Me C) is ATL-1102, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0038] A related aspect is the use of an antisense oligonucleotide against integrin α4 integrin in the manufacture of a medicament for the treatment of NPASC in a subject identified as likely to be affected with NPASC based on the methods disclosed herein and in which the level of TGFβ1 in the subject is above a threshold.

[0039] In a further aspect, a method for treating a human subject identified as suffering from or at risk for suffering from neurological acute sequelae of COVID-19 (NPASC) comprises administering a therapeutically effective amount of: (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), (xii) complement component 5a (C5a) / complement 5 anaphylotoxin; (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) a growth hormone-releasing hormone (GHRH);

[0040] In some embodiments, the modulator of a therapeutic target is an inhibitor of a therapeutic target. In some embodiments, when the administered modulator is an inhibitor, the inhibitor is (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), and (xii) directed against a therapeutic target selected from the list consisting of complement component 5a (C5a) / complement 5 anaphylotoxin.

[0041] In other embodiments, the modulator of a therapeutic target is an activator of a therapeutic target. In some embodiments, when the administered modulator is an activator, the activator is (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xiii) IgG, (xix) palmitoyl protein thioesterase, and (xx) directed against a therapeutic target selected from the list consisting of growth hormone-releasing hormone (GHRH).

[0042] In some embodiments, the activator of a therapeutic target comprises a nucleic acid encoding the therapeutic target.

[0043] In another aspect, a method for treating a human subject identified as suffering from or at risk for suffering from neurological acute sequelae of COVID-19 (NPASC) comprises administering to a subject a therapeutically effective amount of: (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), and (xii) a method is provided herein that comprises administering to a subject, or having administered to a subject, an inhibitor of a therapeutic target selected from the list consisting of complement component 5a (C5a) / complement 5 anaphylotoxin.

[0044] In some embodiments, the methods comprise administering a therapeutically effective amount of a TGFβ1 inhibitor.

[0045] In some embodiments, the methods comprise administering a therapeutically effective amount of a complement C5b-C6 complex inhibitor.

[0046] In some embodiments, the methods comprise administering a therapeutically effective amount of a complement lymphocyte function-associated antigen 3 CD58 inhibitor.

[0047] In some embodiments, the methods comprise administering a therapeutically effective amount of a vascular endothelial growth factor D (VEGF-D) inhibitor.

[0048] In some embodiments, the methods comprise administering a therapeutically effective amount of a myeloid cell surface antigen CD33 inhibitor.

[0049] In some embodiments, the methods comprise administering a therapeutically effective amount of an alcohol dehydrogenase 1B inhibitor.

[0050] In some embodiments, the methods comprise administering a therapeutically effective amount of a tumor necrosis factor receptor superfamily member 10B inhibitor.

[0051] In some embodiments, the methods comprise administering a therapeutically effective amount of a tumor necrosis factor receptor superfamily member 1A inhibitor.

[0052] In some embodiments, the methods comprise administering a therapeutically effective amount of a Cathepsin S inhibitor.

[0053] In some embodiments, the methods comprise administering a therapeutically effective amount of a TLR4:lymphocyte antigen 96 complex inhibitor.

[0054] In some embodiments, the methods comprise administering a therapeutically effective amount of a BcL2-like 1 (Bcl2L1) inhibitor.

[0055] In some embodiments, the methods comprise administering a therapeutically effective amount of a C5a inhibitor.

[0056] In some embodiments, the administered therapeutic target inhibitor is a small molecule inhibitor. In some embodiments, the method comprises administering the small molecule TGF-β1 inhibitor pirfenidone (CAS 53179-13-8). In other embodiments, the method comprises administering the small molecule alcohol dehydrogenase 1B inhibitor fonepizole (CAS 7554-65-6). In some embodiments, the method comprises administering the small molecule cathepsin S inhibitor petesicatib (CAS 1252637-35-6). In some embodiments, the method comprises administering the small molecule TLR4:lymphocyte antigen 96 complex inhibitor resatorbid (CAS 243984-11-4) or eritoran (CAS 185954-98-7). In some embodiments, the Bcl2L1 inhibitor is navitoclax / ABT-263 (CAS 923564-51-6) or obatoclax (CAS 803712-67-6).

[0057] In other embodiments, the administered therapeutic target inhibitor is an antibody inhibitor. In some embodiments, the method comprises administering the antibody complement C5b-C6 complex inhibitor ravulizumab. In some embodiments, the method comprises administering the antibody myeloid cell surface antigen CD33 inhibitor gemtuzumab. In some embodiments, the method comprises administering the antibody tumor necrosis factor receptor superfamily member 10B inhibitor conatumumab or lexatumumab. In some embodiments, the method comprises administering the antibody tumor necrosis factor receptor superfamily member 1A inhibitor atrocimab or TNF receptor 1 silencer (TROS).

[0058] In other embodiments, the administered therapeutic target inhibitor is a fusion protein inhibitor. In some embodiments, the method comprises administering the protein fusion lymphocyte function-associated antigen 3 CD58 inhibitor alefacept. In some embodiments, the method comprises administering the protein fusion VEGF-D inhibitor OPT-302.

[0059] In other embodiments, the administered therapeutic target inhibitor comprises a nucleic acid inhibitor. In some embodiments, the nucleic acid comprises an antisense oligonucleotide (ASO), siRNA, miRNA, aptamer, or sgRNA. In some embodiments, the therapeutic method comprises administering the antisense oligonucleotide TGFβ1 inhibitor ATL1102 (comprising a nucleotide sequence corresponding to SEQ ID NO: 1). In some embodiments, the growth hormone-releasing hormone (GHRH) inhibitor or C5a inhibitor is an siRNA. In some embodiments, the siRNA C5a inhibitor is semdisirane.

[0060] In some embodiments, the administered therapeutic target inhibitor comprises an expression construct, hi some embodiments, the expression construct is provided in a recombinant virus.

[0061] In some embodiments, any of the foregoing methods of treatment comprise administering therapeutically effective amounts of inhibitors of at least two of the therapeutic targets disclosed herein in combination.

[0062] In some embodiments of any of the aforementioned methods, the therapeutically effective amount of the inhibitor administered is based on the level of the therapeutic target or therapeutic target activity in the subject, or the level of activity of the therapeutic target in the subject.

[0063] In some embodiments, any of the foregoing methods includes determining the level of the therapeutic target or the level of activity of the therapeutic target in the human subject after administration.

[0064] In some embodiments, any of the foregoing methods also include a step of diagnosing the human subject, or diagnosing the human subject as having or at high risk of having NPASC, prior to the administering step.

[0065] In a further aspect, there is provided the use of a modulator of a therapeutic target in the manufacture of a medicament for the treatment of neurological acute sequelae (NPASC) of COVID-19, wherein the therapeutic target is (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), (xii) complement component 5a (C5a) / complement 5 anaphylotoxin; (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroididase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) growth hormone-releasing hormone (GHRH).

[0066] In some embodiments, the modulator in the pharmaceutical product is an inhibitor of a therapeutic target. In some embodiments, when the modulator in the pharmaceutical product is an inhibitor, the therapeutic target is (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), and (xii) complement component 5a (C5a) / complement 5 anaphylotoxin.

[0067] In other embodiments, the modulator included in the pharmaceutical product is an activator of the therapeutic target. In some embodiments, when the modulator included is an activator, the therapeutic target is (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) growth hormone-releasing hormone (GHRH).

[0068] In a further aspect, there is provided a method for treating neurological acute sequelae (NPASC) of COVID-19, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of: (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), and (xii) Complement component 5a (C5a) / complement 5 anaphylotoxin Provided herein is an inhibitor of a therapeutic target selected from the list consisting of:

[0069] In another aspect, for use in treating neurological acute sequelae (NPASC) of COVID-19, (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) Growth hormone-releasing hormone (GHRH) Provided herein is an activator of a therapeutic target selected from the list consisting of:

[0070] In some embodiments of any of the foregoing methods of treatment, in a biological sample obtained from the subject: Prostaglandin-H2D-isomerase, TNF receptor superfamily member 1A, SLIT and NTRK-like protein 2, carbohydrate sulfotransferase 15, ribonuclease T2, alpha-1,6-mannose glycoprotein 6-beta-N-acetylglucosamine transferase A, ephrin type B receptor 2, thrombospondin-4, Alanyl-tRNA editing protein A, galactosylceramide sulfotransferase, C5a anaphylatoxin (complement component), Protein phosphatase 1F (PPM1F), and Subjects were identified as having NPASC based on the level of at least one, two, or more NPASC-associated circulating biomarkers selected from the group consisting of neutrophil cytoplasmic factor 2 (NCF2);

[0071] In some embodiments, the method of treatment comprises measuring, in a biological sample from the subject, (i) the level of protein phosphatase 1F (PPM1F), and (ii) the level of C5a anaphylatoxin and / or neutrophil cytoplasmic factor 2 (NCF2). In other embodiments, the method of treatment comprises measuring, in a biological sample from the subject, (i) the level of protein phosphatase 1F (PPM1F), (ii) the level of C5a anaphylatoxin and / or neutrophil cytoplasmic factor 2 (NCF2), and (iii) the level of galactosylceramide sulfotransferase and / or the level of prostaglandin D synthase (PDGS).

[0072] In a further aspect, provided herein is a kit or panel for determining likelihood of neurological acute sequelae of COVID-19 (NPASC) in a subject, the kit or panel comprising a specific binding agent for at least each of the biomarkers C5a anaphylatoxin (C5a) and gliomedin.

[0073] In some embodiments, the kit or panel comprises a specific binding agent for at least each of the biomarkers C5a anaphylatoxin (C5a) and gliomedin and transforming growth factor β1 (TGFβ1).

[0074] In some embodiments, the kit or panel also comprises one or more specific binding agents for at least one protein selected from the group consisting of transforming growth factor beta 1 (TGF beta 1), galactosylceramide sulfotransferase (Gal3ST1), interferon (IFN) lambda-1, growth hormone-releasing hormone (GHRH), lymphocyte function-associated antigen 3 (LFA-3), Fas ligand (FASLG), transgelin, immunoglobulin heavy chain constant gamma 1 (IgGH1), glycoprotein NMB (GPNMB), and antithrombin III.

[0075] Symptoms associated with NPASC are known in the art and include brain-related symptoms, including anxiety / depression, sleep disorders, PTSD, cognitive impairment, and headaches. In some embodiments, the subject may have olfactory dysfunction. For example, to identify a subject suffering from a significant neurological disorder, the subject may complete a cognitive assessment using the National Institutes of Health (NIH) Toolbox v2.1 instrument, including assessments of processing speed (Pattern Comparison Processing Speed ​​Test), attention and executive memory (Inhibitory Control and Attention Test), executive function (Dimensional Change Card Sort Test), and working memory (List Sorting Working Memory Test). Thus, in some embodiments, clinical or self-assessment of NPASC includes assessment of one or more of processing speed, attention and executive memory, executive function working memory, "clouding of consciousness," numbness / tingling, headache, dizziness, fatigue, sleep disorders, depression, and anxiety. Self-assessment may, for example, employ a suitable symptom checker app.

[0076] The above summary is not, nor should it be viewed as, an exhaustive list of all embodiments of the present disclosure. [Brief explanation of the drawings]

[0077] [Figure 1] Two boxplots showing the difference between C5a anaphylatoxin levels within and between groups: (i) Comparing NPASC subjects (NP) in Groups 1+2 with healthy controls (HC) in Group 4, showing a 100% median increase in NP vs. HC; (ii) Comparing NP in Groups 1+2 with COVID convalescent subjects (CC) in Group 3, showing an 88% median increase in NP vs. CC. [Figure 2] Boxplot showing the difference between gliomedin levels comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between gliomedin: (i) Comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), there was a 31% median increase in NP vs. HC. (ii) Comparing NP in Groups 1+2 with 20 subjects in CC in Group 3, there was a 41% median increase in NP vs. CC. [Figure 3]Boxplot showing the difference between levels of TGF beta1 comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between TGF beta1: (i) Comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), with a median increase of 27% for NP vs. HC. (ii) Comparing NP in Groups 1+2 with 20 subjects in CC in Group 3, with a median increase of 17% for NP vs. CC. [Figure 4] Boxplots showing the difference between levels of galactose-3-O-sulfotransferase (Gal3ST1) (also known as "galactosylceramide sulfotransferase") are shown, comparing NP Groups 1+2 and 3 CC with Group 4 HC. The figure shows the difference between Gal3ST1: (i) comparing NP from Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), with a median decrease of 23% for NP vs. HC; (ii) comparing NP from Groups 1+2 with 20 subjects in Group 3 CC, with a median decrease of 22% for NP vs. CC. [Figure 5] Boxplot showing the difference between interferon lambda-1 (IFN lambda-1) levels comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between IFN lambda-1: (i) comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), with a median reduction of 29% in NP vs. HC; (ii) comparing NP in Groups 1+2 with 20 subjects in CC in Group 3, with a median reduction of 14% in NP vs. CC. [Figure 6] Boxplot showing the difference between growth hormone-releasing hormone (GHRH) (also known as "somatoliberin") levels comparing NP Groups 1+2 and CC Group 3 with HC Group 4. The figure shows the difference between GHRH: (i) Comparing NP Groups 1+2 and 24 subjects in Group 4, healthy controls (HC), with a median increase of 34% for NP vs. HC; (ii) Comparing NP Groups 1+2 and 20 subjects in Group 3, CC, with a median increase of 16% for NP vs. CC. [Figure 7]Boxplot showing the difference between lymphocyte function-associated antigen 3 (LFA-3) levels comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between LFA-3: (i) comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), with a median increase of 17% for NP vs. HC; (ii) comparing NP in Groups 1+2 with 20 subjects in Group 3 CC, with a median increase of 5% for NP vs. CC. [Figure 8] Boxplot showing the difference between levels of Fas ligand (FASLG) comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between FASLG: (i) Comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), a median increase of 46% for NP vs. HC; (ii) Comparing NP in Groups 1+2 with 20 subjects in CC in Group 3, a median increase of 16% for NP vs. CC. [Figure 9] Boxplot showing the difference between transgelin levels comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between transgelin: (i) Comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), a median increase of 39% for NP vs. HC; (ii) Comparing NP in Groups 1+2 with 20 subjects in CC in Group 3, a median increase of 53% for NP vs. CC. [Figure 10] Boxplot showing the difference between levels of glycoprotein NMB (GPNMB) comparing NP Groups 1+2 and CC in Group 3 with HC in Group 4. The figure shows the difference between GPNMB: (i) Comparing NP in Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), a median increase of 16% for NP vs. HC; (ii) Comparing NP in Groups 1+2 with 20 subjects in CC in Group 3, a median increase of 25% for NP vs. CC. [Figure 11] Boxplot showing the difference between levels of immunoglobulin heavy constant gamma 1 (IgGH1) comparing NP Groups 1+2 and CC Group 3 with HC Group 4. The figure shows the difference between IgGH1: (i) comparing NP Groups 1+2 with 24 subjects in Group 4 healthy controls (HC), with a median decrease of 18% for NP vs. HC; (ii) comparing NP Groups 1+2 with 20 subjects in Group 3 CC, with a median decrease of 23% for NP vs. CC. [Figure 12]Exemplary identified therapeutic targets, CD33 and VEGFD, are higher in subjects with NPASC. Scatter plots showing plasma levels of CD33 (upper panel) and VEGFD (lower panel) in NPASC subjects (groups 1+2), COVID-convalescent subjects without NPASC (group 3), and healthy control subjects who never had COVID (group 4). Protein levels were measured by SomaScan® proteomics analysis (y-axis represents relative fluorescence units). CD33 and VEGFD levels were significantly higher in NPASC subjects (false discovery rate - FDR = 0.018 and 0.017, respectively). [Figure 13] Exemplary identified therapeutic targets, thyroid peroxidase and amyloid A4, are lower in subjects with NPASC. Scatter plots showing plasma levels of thyroid peroxidase (upper panel) and amyloid A4 (lower panel) in NPASC subjects (Groups 1+2), COVID-convalescent subjects without NPASC (Group 3), and healthy control subjects who never had COVID (Group 4). Protein levels were measured by SomaScan® proteomics analysis (Y-axis represents relative fluorescence units). Thyroid peroxidase and amyloid A4 levels were significantly lower in NPASC subjects (false discovery rate - FDR = 0.014 and 0.014, respectively). [Figure 14] Transforming growth factor-β1, an exemplary identified therapeutic target, is higher in subjects with NPASC. Scatter plot showing plasma levels of TGF-β1 in NPASC subjects (Groups 1+2), COVID-convalescent subjects without NPASC (Group 3), and healthy control subjects who never had COVID (Group 4). Protein levels were measured by SomaScan® proteomics analysis (Y-axis represents relative fluorescence units). TGF-β1 levels are significantly higher in NPASC subjects (false discovery rate - FDR=0.011).

[0078] Table 1. List of biomarkers used in the diagnostic methods described herein.

[0079] Table 2. Statistical tests and statistics showing the biomarkers used in the methods described herein.

[0080] Table 3. Exemplary biomarker combinations for use in the methods described herein, and diagnostic sensitivity and specificity characteristics.

[0081] Table 4 provides subject information from the study of Visvabharathy et al.: http: / / www.medrxiv.org / content / 10.1101 / 2021.08.08.21261763 (2021). The subject sample of the present disclosure includes 48 non-hospitalized NP subjects in Groups 1+2, 42 of whom were from Visvabharathy et al.'s non-hospitalized group of 48 subjects. The subject sample herein includes 20 non-hospitalized CC subjects in Group 3, consisting of 17 of Visvabharathy et al.'s 24 subjects, and 24 of Visvabharathy et al.'s 31 HC subjects.

[0082] Table 5. Summary of pathway analyses performed / pathways identified to aid in the identification of NPASC therapeutic targets.

[0083] Table 6. Summary of pathways of interest identified by statistical analysis of hit frequencies (FDR<0.02).

[0084] Table 7. Treatment targets identified by ANOVA FDR<0.02 (Group 1 vs. Group 3). DETAILED DESCRIPTION OF THE INVENTION

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice or test the present disclosure. Practitioners should refer to, inter alia, Wild D. "The Immunoassay Handbook" Nature Publishing Group, 4th Edition, 2013, and Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Sousa-Pereira et al., (2019) Antibodies 8(4):57, Remington's Pharmaceutical Sciences, 1990; The Proteomics Protocols Handbook Ed. John M. Walker Humana Press Inc., 2005; Proteomic and Metabolomic Approaches to Biomarker Discovery 2nd Edition, Eds. Haleem Issaq, Timothy Veenstra, 2019 Academic Press ISBN: 9780128186077, Chapter 17. Top-down mass spectrometry for protein molecular diagnostics, structure analysis, and See biomarker discovery;Chapter 19.Imaging mass spectrometry of intact biomolecules in tissue section;Chapter 22:Analytical methods and biomarker validation,Chapter 23 Multivariate analysis for metabolomics and proteomics data.The method may be carried out in any convenient format known in the art.

[0086] The term "coronavirus" or "coronavirus family" refers to the viruses commonly known as "coronaviruses" or "CoVs," which are enveloped, positive-sense, single-stranded RNA viruses. The coronavirus family includes two subfamilies: the Letovirinae and the Orthocoronavirus. In some embodiments, the CoV is selected from the genus Alphacoronavirus (alphaCoV), the genus Betacoronavirus (betaCoV), the genus Gammacoronavirus (gammaCoV), and the genus Deltacoronavirus (deltaCoV). In some embodiments, the alphaCoV is selected from coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), feline infectious peritonitis virus (FIPV), and canine coronavirus (CCoV). In some embodiments, the beta-CoV is selected from human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome-associated coronavirus (SARS-CoV), severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), Middle East respiratory syndrome-associated coronavirus (MERS-CoV), mouse hepatitis virus (MHV), and / or bovine coronavirus (BCoV). In some embodiments, the CoV is capable of infecting humans. In some embodiments, the CoV capable of infecting humans is selected from the following: SARS-CoV-2, HCoV-OC43, HCoV-HKU1, HCoV-229E, HCoV-NL63, SARS-CoV, and MERS-CoV, or a subtype thereof. In some embodiments, the CoV is SARS-CoV-2 or a subtype or variant thereof. In some embodiments, the SARS-CoV-2 is SARS-CoV-2 subtype L. In some embodiments, the SARS-CoV-2 is SARS-CoV-2 subtype S. In one embodiment, the SARS-CoV-2 is SARS-CoV-2 hCoV-19 / Australia / VIC01 / 2020.In some embodiments, the SARS-CoV-2 comprises a sequence set forth in NCBI Reference Sequence: NC_045512.2 (ancestral Hu-1). In some embodiments, the SARS-CoV-2 comprises a sequence set forth in GenBank: MN908947.3 or a pathogenic variant thereof. The World Health Organization (WHO) labeling includes alpha, beta, gamma, delta, and omicron, along with their phylogenetic groups and clades, as outlined and updated continuously on Wikipedia at https: / / en.wikipedia.org / wiki / Variants_of_SARS-CoV-2.

[0087] The present invention is based, in part, on the inventors' determination that the levels of several specific proteins are significantly regulated in the circulatory blood of subjects with neural PASCs. Therefore, the measured levels of one or more of these NPASC-associated biomarkers can be used to identify the likelihood of NPASC in a subject, the risk of developing NPASC in a subject infected with a pathogenic coronavirus (e.g., COVID-19), or a healthy subject. Furthermore, the measured levels of biomarkers can be used to determine whether a subject is likely to respond to treatment with one or more modulators of these specific biological molecules, or in combination with other NPASC therapies. For one or more of these specific biological molecules, their levels are significantly reduced in the circulatory system of subjects with NPASCs. Therefore, treatments that increase the levels of the specific biological molecules identified herein are contemplated.

[0088] In particular, the present specification provides a set of PASC-associated circulating biomarkers that are proposed for use in determining the likelihood that a subject has PASC.

[0089] Proteomic plasma profiles were determined for three subject groups: Group 1 was subjects with NPASC, Group 3 was COVID-recovered subjects without PASC or NPASC, and Group 4 was healthy control subjects, as described in the Examples.

[0090] Level data can be measured or expressed in any convenient parameter, including mass concentration and luminescence, relative fluorescence units (RFU). Using a particular assay and sample, level data can be compared to the normalized RFU of healthy adult controls (e.g., SomaScan® EDTA plasma level data for NPASC subjects), or the healthy control values ​​can be used to compare the nRFU of healthy adult controls for each SomaScan. Healthy control values ​​are robust point estimates generated during assay validation of the aptamer; values ​​are the median of 1,000 individuals from the adult US population (both males and females) aged 18-80 years; no healthy data set exists that matches CC controls with EDTA plasma.

[0091] Comparing two or more levels includes determining or obtaining a measure of difference or similarity between the levels (eg, visual or machine / computer-based comparison).

[0092] Level data from subjects can be summarized or represented graphically to facilitate comparison and interpretation of the data. As one skilled in the art will appreciate, graphical representations of data can take many different forms and can represent or focus different aspects of the data, depending on the analysis and the requirements of the user, which can also be modified. For example, the user can be a computer program or a human. Graphical representations of data include tables, line plots, charts (e.g., pie charts, heat maps, bubble charts, zoom charts, chord diagrams, graphs, signatures, bar codes, box plots, and combinations thereof.

[0093] Box plots conveniently summarize comparative data derived from subjects or populations or from databases, and represent levels by highlighting factors such as distribution, spread, and skewness in a data set, as well as outliers / unusual observations / extreme values, through summary data such as the median, lower and upper quartiles, minimum and maximum values, first and third quartiles, lower and third quartiles, whiskers (thin vertical lines connecting the minimum and maximum (non-outliers) to the box), and bee swarm data (showing individual levels, median, mode, and mean). The median (or interquartile range) is considered a particularly useful representative value (especially for skewed distributions) and is the point at which there are equal numbers of data points with values ​​above and below the median. In addition to showing the median, first and third quartiles, and maximum and minimum values, box plots are also used to show the mean, standard deviation, average deviation, and interquartile deviation. In the example employed, DataViz, the upper whisker is also known as the upper fence, and the lower whisker is also known as the minimum (or lower fence).

[0094] As further described herein, state-of-the-art proteomic assays were used to identify, from approximately 7,300 plasma proteins, differentially present plasma proteins indicative of disease (NPASC) between groups of plasma samples: COVID convalescent controls and healthy controls; from different groups. Among the dozens of plasma proteins identified by Bonferroni analysis using different parametric and nonparametric tests, and the hundreds identified as differentially present by false discovery rate analysis using parametric and nonparametric tests, the inventors conducted a series of studies, including pathway analysis of statistically significant proteins, to identify proteins that may serve as biomarkers for assessing the likelihood of NPASC in a sample or subject.

[0095] In some embodiments, NPASC-associated circulating biomarkers have been identified as set forth in a table selected from Table 1. The levels of each of these proteins have been determined to be substantially elevated or decreased in NPASC subjects compared to reference values ​​from non-NPASC subjects, as described and illustrated herein in Tables 1 and 2. [Table 1] [Table 2-1] [Table 2-2]

[0096] As used herein, the term "sensitivity" with respect to the diagnostic or prognostic methods disclosed herein refers to the ability of the method to correctly identify a subject as having NPASC, i.e., the total proportion of individuals within a group of tested subjects who are correctly identified as having NPASC.

[0097] As used herein, the term "specificity" with respect to the diagnostic or prognostic methods disclosed herein refers to the ability of the method to identify a subject as not suffering from NPASC, i.e., the total proportion of individuals within a group of tested subjects who are correctly identified as not suffering from NPASC.

[0098] Together, sensitivity and specificity define the accuracy of a diagnostic test; 100% sensitivity would detect all subjects in a given subject group who have NPASC (i.e., no false negatives for NPASC), and 100% specificity would detect all subjects who do not have NPASC (i.e., no false positives for NPASC). A test with 100% sensitivity and 100% specificity provides 100% accuracy. In some embodiments of the present methods, the test identifies subjects with at least 70% accuracy, e.g., 75%, 80%, 85%, 90%, 95%, or another level of accuracy ranging from at least 70% accuracy to 100% accuracy, depending on the subject subpopulation and / or assay. Those skilled in the art will understand that diagnostic assays with the same level of accuracy can achieve different combinations of this base sensitivity. For example, a diagnostic assay with 90% accuracy may include 95% sensitivity and 85% specificity, or 85% sensitivity and 95% specificity, or different combinations of sensitivity and specificity levels as would be understood by one of skill in the art. 90% accuracy in a group of subjects may be 70%, 75%, 80%, 85%, 90%, or higher accuracy in different independent groups of subjects and / or in small or large studies, or depending on the assay.

[0099] In some embodiments of the methods disclosed herein, measuring levels of either or both of C5a and / or gliomedin above a threshold level identifies subjects with NPASC with a sensitivity of about 88%. In other embodiments, where a third marker, TGFβ1 (above a threshold), is included, the test provides a sensitivity of about 98%. Alternatively, if Gal3ST1 is included in the three-biomarker panel rather than TGFβ1, the sensitivity is about 92%. When both TGFβ1 and Gal3ST1 are measured, in addition to C5a and gliomedin, the sensitivity is about 98%, as outlined in Table 3.

[0100] In some embodiments of the methods disclosed herein, measuring either or both levels of C5a and / or gliomedin within the normal range threshold level identifies subjects not suffering from NPASC with a specificity of about 80% if they are convalescent control (CC) subjects, or 88% if they are healthy control (HC) subjects and are not infected, with an average specificity of 84% for these two control subjects. Thus, in such embodiments of the methods disclosed herein, measuring levels of C5a and / or gliomedin provides an accuracy of about 86%.

[0101] In other embodiments, where a third marker, TGFβ1 (normal) or a surrogate third marker, Gal3ST1 (normal), is included, the test still provides an average specificity of about 84% for the two controls. Alternatively, if interferon lambda-1 (normal levels) is included in the biomarker panel along with GHRH (normal levels), the specificity for healthy controls (HC) increases to about 100%. Furthermore, if interferon lambda-1 (normal levels) is included along with LFA-3 (normal levels), the specificity for convalescent controls (CC) increases to about 85%. When using these targets for these two control subject groups, and also measuring levels of C5a and / or gliomedin, a potential average specificity of about 92.5% can be obtained, as outlined in Table 3. Up to four additional markers, FASLG (normal) and transgelin (normal), GPNMB (normal) and IgGH1 (normal levels), in addition to C5a and / or gliomedin, in combination with at least interferon lambda-1 (normal levels), each potentially provide up to 100% specificity, as outlined in Table 3.

[0102] In other embodiments, where a third marker, TGFβ1 (normal) or a surrogate third marker, Gal3ST1 (normal), is included, the test still provides an average specificity of about 84% for the two controls. Alternatively, if interferon lambda-1 (normal levels) is included in the biomarker panel along with GHRH (normal levels), the specificity for healthy controls (HC) increases to about 100%. Furthermore, if interferon lambda-1 (normal levels) is included along with LFA-3 (normal levels), the specificity for convalescent controls (CC) increases to about 85%. When using these targets for these two control subject groups, and also measuring levels of C5a and / or gliomedin, a potential average specificity of about 92.5% can be obtained, as outlined in Table 3. Up to four additional markers, FASLG (normal) and transgelin (normal), GPNMB (normal) and IgGH1 (normal levels), in addition to C5a and / or gliomedin, in combination with at least interferon lambda-1 (normal levels), each potentially provide up to 100% specificity, as outlined in Table 3. [Table 3-1] [Table 3-2]

[0103] In some embodiments, the level of a biomarker is detected using a specific binding agent, such as an antibody or an antigen-binding fragment of an antibody, that binds to the biomarker and directly or indirectly provides a detectable signal that can be quantified visually or instrumentally. The term "level" or "levels" also encompasses ratios of biomarker levels determined using a binding agent detection protocol and quantification.

[0104] As used herein, "immunoassay" refers to an immunoassay, typically, but by no means exclusively, a sandwich assay, capable of detecting and quantifying a desired biomarker. The immunoassay can be one of a range of immunoassay formats known to those skilled in the art. In some preferred embodiments, the immunoassay used is an ELISA-format assay.

[0105] While microdrops of proteins delivered to flat surfaces are widely used, related alternative architectures include microfluidics (e.g., available from Gyros) and specialized chip designs, such as CD centrifugation devices based on engineered microchannels in plates (e.g., The Living Chip™ available from Biotrove) and tiny 3D posts on silicon surfaces (e.g., available from Zyomyx).

[0106] Particles in suspension can also be used as the basis for arrays if they are coded for specificity. Systems include color-coding microbeads (e.g., available from Luminex, Bio-Rad, Nanomics Biosystems) and semiconductor nanocrystals (e.g., available from QDots™, Quantum Dots), as well as barcoding beads (UltraPlex™, available from Smartbeads) and multimetal microrods (NanoBarcode™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). When particles are used, individual protein capture agents are typically attached to each particle to provide spatial definition or separation of the array. The particles can then be assayed separately but in parallel in a compartmentalized manner, for example, in wells of a microtiter plate or in separate test tubes.

[0107] In operation, a protein sample (e.g., U.S. Patent Application Publication No. 2002 / 0055186) is delivered to a protein capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. The presence or amount of protein or peptide bound to each feature of the array is then detected using a suitable detection system. The amount of protein bound to a feature of the array can be determined by comparison with the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second or subsequent protein in the sample is already known or is known to be constant.

[0108] In an illustrative example, fluorescent labels can be used to detect proteins bound to the array. The same instrumentation used to read DNA microarrays is applicable to protein capture arrays. For differential display, capture arrays (e.g., antibody arrays) can be probed with fluorescently labeled proteins from or labeled with different fluorophores (e.g., Cy-3 and Cy-5) and mixed, allowing color to serve as a readout of changes in target abundance. Fluorescence readout sensitivity can be amplified 10-100-fold by tyramide signal amplification (TSA) (e.g., available from PerkinElmer Lifesciences). Planar waveguiding technology (e.g., available from Zeptosens) allows for ultrasensitive fluorescence detection with the added advantage of no washing steps. High sensitivity can also be achieved with suspension beads and particles using the properties of phycoerythrin (e.g., available from Luminex) or semiconductor nanocrystals (e.g., available from Quantum Dot) as labels. Fluorescence resonance energy transfer has been adapted to detect binding of unlabeled ligands, which may be useful in arrays (e.g., available from Affibody). Several alternative readouts have been developed, including adaptations of surface plasmon resonance (e.g., available from HTS Biosystems and Intrinsic Bioprobes), rolling circle DNA amplification (e.g., available from Molecular Staging), mass spectrometry (e.g., available from Sense Proteomic, Ciphergen, Intrinsic, and Bioprobes), resonant light scattering (e.g., available from Genicon Sciences), and atomic force microscopy (e.g., available from BioForce Laboratories). A microfluidic system for automated sample incubation and washing with arrays on glass slides was jointly developed by NextGen and Perkin Elmer Life Sciences.

[0109] Electrochemiluminescence (ELICA), enzyme-linked immunosorbent assay (ELISA), and Luminex, eg, LabMAP, magnetic Luminex, immunoassays are examples of suitable assays for detecting levels of biomarkers.

[0110] Bead-based immunoassays can utilize the Luminex LabMAP system. The LabMAP system incorporates polystyrene microspheres internally dyed with two spectrally distinct fluorescent dyes. Using precise ratios of these fluorescent dyes, arrays consisting of different sets of microspheres with specific spectral addresses are created. Each microsphere set can have a different reactant on its surface. Because the sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorescent dye attached to a reporter molecule quantifies biomolecular interactions occurring on the microsphere surface. The microspheres are individually interrogated in a rapidly flowing fluid stream as they pass two separate lasers in the Luminex analyzer. High-speed digital signal processing classifies the microspheres based on their spectral addresses and quantifies the surface reactions within seconds for each sample.

[0111] Lateral flow assays, and more recently non-lateral flow and microfluidics, provide useful settings for biomarker screening. Such assays can be qualitative, quantitative, or semi-quantitative. In microfluidic devices, small volumes of liquid move through microchannels generated, for example, in a chip or cartridge. Detection reagents containing metal nanoparticles, colored materials, or luminescent materials are widely available. Resonance-enhanced adsorption (REA) of bioconjugated metal nanoparticles offers rapid processing times and other advantages. These devices have been combined with barcode technology to identify the patient and analyte being tested. Computer software and hardware for evaluating input data are encompassed by this disclosure.

[0112] Methods for detecting antibodies against specific antigens are also widely known. For example, enzyme-linked immunosorbent assays (ELISAs), Western blot and dot blot assays, and radioimmunoassays (RIAs) are routinely used in laboratories. Arrays and high-throughput screening methods are also used. General formats and protocols for performing various forms of ELISAs are disclosed in the art and are known to those skilled in the diagnostic field. See, for example, Chapter 11 of Ausubel (Ed) Current Protocols in Molecular Biology, 5th Edition, John Wiley & Sons, Inc., NY, 2002. Rundstrom, G. et al., Lateral Flow Immunoassay using Europium(III) Chelate Microparticles and Time-Resolved A Wide Range of Immunoassays. Fluorescence for eosinophils and neutrophils in Whole Blood. Clinical Chemistry 53, 342-348 (2007) (incorporated herein).

[0113] Qualitative assays that provide intermediate or definitive diagnoses require an integrated cutoff, gate, or window that allows for scoring of samples that are likely to have or not have the condition. Instrument readers and software are often used to collate the data and process it through a diagnostic algorithm or decision tree.

[0114] A wide range of detection reagents are available, including metal nanoparticles, colored materials, or luminescent materials. Resonance-enhanced adsorption (REA) of bioconjugated metal nanoparticles offers rapid processing times and other advantages.

[0115] These biomarker assays may be combined with barcode or ledger technology to identify the patient and biomarker being tested. Computer software and hardware for evaluating input data are encompassed by this disclosure. Point-of-care devices and arrays, as well as high-throughput screening methods, are also contemplated. In some embodiments, the capture moiety is a test line.

[0116] Qualitative assays that provide intermediate or definitive diagnoses require integrated thresholds, gates, or windows that allow for scoring of samples that are likely to have or not have the condition. Instrument readers and software are often used to collate the data and process it through diagnostic algorithms or decision trees.

[0117] The presence of the complex can be assessed using an ELISA-type procedure. A wide variety of immunoassay techniques are available, as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These include non-competitive single-site and two-site or "sandwich" assays, as well as traditional competitive binding assays. Those skilled in the art will understand that the selection and implementation of known labeling systems involves no more than routine experimentation.

[0118] Sandwich assays are among the most useful and commonly used assays. Several variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, a binding agent is immobilized on a solid or semi-solid substrate, and the sample to be tested is contacted with the binding molecule. After a suitable incubation period (sufficient to allow for the formation of a binding agent-antigen complex), a second binding agent specific for the antigen, labeled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing sufficient time for the formation of another binding agent-antigen-labeled binding agent complex. Any unreacted material is washed away, and the presence of the marker is determined by observation of a signal produced by the detectable marker (reporter molecule). Results can be qualitative or quantitative by simple observation of the visible signal, or can be quantified by comparison with a control sample containing a known amount of marker. Variations of the forward assay include simultaneous assays, in which both the sample and the labeled binding agent are added simultaneously to the bound binding agent. These techniques, including minor variations that will be readily apparent, are well known to those skilled in the art. In accordance with the present invention, the sample is generally a biological sample comprising a biological fluid, most conveniently a whole blood sample such as capillary or venous blood, which may be treated with an anticoagulant.

[0119] "Solid substrate" refers to a material with a rigid or semi-rigid surface that is suitable for some assays and can take the form of, for example, beads, resins, gels, spheres, microspheres, particles, fibers, or other geometric configurations or physical forms that can be regular or irregular. Solid substrates typically include materials applicable to medical, biochemical, or biological assays, such as apheresis, column chromatography for the purification or separation of biological or organic molecules, and substrates used in ELISA assays. Solid substrates can be porous or non-porous.

[0120] Solid substrates or surfaces for immobilizing biomarkers or binding agents. Non-limiting examples of solid substrates include polymers such as polysaccharides, particularly polysaccharides with a molecular weight of 100 kDa or greater, such as agarose. The agarose may be in particulate form, which may optionally be cross-linked. Specific examples of agarose are Sepharose or cellulose, which may be cross-linked. Other polymers suitable as substrates include, for example, carboxylated polystyrene. The solid substrate may also be provided in the form of magnetic beads. Glass is also a suitable substrate material. Any suitable blood or plasma filtration column or system can be adapted for this process. Non-limiting examples include the columns described in U.S. Pat. No. 4,619,639, membrane filtration systems (e.g., MDF) used with suitable particles, surfaces, or substrates, and PlasmaFlo® OP-05(W)L and RheoFilter® AR2000 blood filters manufactured by Asahi Medical Company, Ltd. (Japan).

[0121] In a typical forward sandwich assay, a first binding agent with specificity for the marker is covalently or passively bound to a solid or semi-solid support. The support is typically glass or a polymer; the most commonly used polymers are nitrocellulose, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene, or mixtures or derivatives thereof. The solid support can be in the form of a tube, bead, disk, or microplate, or any other surface suitable for conducting an immunoassay. The binding process is well known in the art and generally consists of cross-linking the polymer-binding agent complex to covalently or physically adsorb it to a solid surface, which is then washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid-phase complex and incubated for a period sufficient to allow binding of any subunits present to the binding agent (e.g., 2-40 minutes, or more conveniently, overnight) and under suitable conditions (e.g., room temperature, including 25°C, up to about 37°C). After the incubation period, the solid phase of the binder subunits is washed and incubated with a second binder specific for a portion of the antigen, which is linked to a detectable marker, which is used to indicate binding of the second binder to the antigen.

[0122] An alternative method involves immobilizing target molecules in a biological sample and then exposing the immobilized target to a specific binding agent (which may or may not be labeled with a detectable marker). Depending on the amount of target and the strength of the signal from the detectable marker, the bound target may be detectable by directly labeling it with the binding agent. Alternatively, a second, labeled binding agent specific to the first binding agent is exposed to the target-first binding agent complex, forming a target-first binding agent-second binding agent tertiary complex. The complex is detected by the signal emitted by the reporter molecule. A significant improvement over bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide or electrophoretic tag, to facilitate identification of the amino acid sequence of each library member. These improved bead-based methods are described in WO 93 / 06121.

[0123] In other embodiments, the method is a liquid-phase method. In one example of a liquid-phase immunoassay (see, e.g., U.S. Pat. No. 6,632,603), the sample is contacted with a detection agent, which includes an agent capable of binding to a marker and a visually detectable agent, such as labeled colloidal gold or silver. The test sample is applied by flowing it through a defined area of ​​an insoluble porous support film having pores that prevent the passage of complexes formed between the marker and its target, and, if present, the complex formed between the binding substance and the detection substance, but allows the binding substance and detection substance to pass without forming a complex if the desired target is not present. If the target is present in the test sample, the detection substance binds to the target and the binding substance to form a visually inspectable complex on the surface of the porous support film. After applying the test sample to the porous support, the color of the surface of the porous support is visually inspected to determine the presence and amount or absence of the marker being assayed.

[0124] Another assay uses a magnetic antibody that binds to the marker to tag it, and a high-Tc superconducting quantum interference device to measure the amount of target protein. Liposome immunotransfer, a liquid-phase competitive strip immunoassay, is described, for example, in Glorio-Paulet et al. J Agric Food Chem 48(5):1678-1682, 2000.

[0125] Antibody-based methods are more sensitive than 2D gels or MS, capable of detecting analytes in the sub-nM range due to the high affinity (typically nM to pM) of antibodies for their targets. They are widely used for single-analyte testing and are proposed herein for multiple assay types. However, in the case of multiplex or mini-multiplex assays, such as bead-based assays, cross-reactivity caused by antibodies reduces sensitivity and specificity. 2D electrophoresis provides forensic analysis of proteins in samples, while mass spectrometry-based approaches are used by themselves or in combination with other steps. Proteomics assays must be sensitive and specific enough to quantify multiple proteins in the presence of other proteins that may be present at much higher concentrations. Affinity molecule-based proteomics assays, including an additional calibration step, offer the best combination of sensitivity and specificity and are preferred.

[0126] Proximity ligation assay (PLA) is a highly sensitive technique for the multiplexed detection of biomarkers in plasma with little or no interfering background signal. Antibodies, along with proximity ligation and SOMAmers, provide highly sensitive and specific multiplexed assays for proteomics. PLA was originally demonstrated with aptamers, which are easier to implement and offer much greater calibration potential than antibodies. DNA aptamers belong to a group of molecules that are cheaper alternatives to antibodies. These alternatives include peptide aptamers isolated by phage and ribosome display technologies, but DNA aptamers stand out as the simplest and most economical to generate. One of the reasons why Gold et al. (Gold L, Ayers D, Bertino J et al. Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5(12), e15004 (2010)) were successful in developing highly multiplexed assays is because their slow off-rate aptamers (SOMAmers) contain amino acid-like functional groups. Gold L, Walker JJ, Wilcox SK, Williams S. Advances in human proteomics at high scale with the SOMAscan proteomics platform. N. Biotechnol. 29(5), 543-549 (2012). This chemistry is similar to that described by Eaton et al. (Eaton BE. The joys of in vitro selection: chemically dressing oligonucleotides to satiate protein targets. Curr. Opin. Chem. Biol. 1(1), 10-16 (1997) and Vaught JD, Bock C, Carter Developed by J et al. Expanding the chemistry of DNA for in vitro selection. J. Am. Chem. Soc. 132(12), 4141-4151(2010).This forms part of an approach moving towards peptide-like hybrid molecules that are compatible with enzymatic (eg PCR) amplification.

[0127] One proteomic method for biomarker evaluation is the proximity extension assay (PEA) (Olink Proteomics, Uppsala, Sweden), which allows for the simultaneous analysis of multiple protein biomarkers on separate panels for different pathways. Here, a small amount of plasma is incubated overnight and bound to an oligonucleotide-labeled antibody pair to form a specific DNA duplex. The template is then extended, pre-amplified, and individual protein markers are measured using high-throughput microfluidic real-time PCR. The resulting Ct values ​​are normalized to extension controls, inter-plate controls, and adjusted with a correction factor according to the manufacturer's instructions to calculate normalized protein expression values ​​(NPX) on a log2 scale. Raw expression values ​​are then batch-corrected by normalizing to duplicate reference samples within each plate.

[0128] In one example of a proteomic affinity capture assay (Gold et al., PLos ONE 5(12), e15004 (2010)), SOMAmers are mixed with a target sample (purified protein or plasma) and incubated to allow binding to equilibrium. Catch-1 bound SOMAmer (S)-protein (P) complexes are captured on streptavidin beads (SA), and proteins are tagged with biotin (NHS-biotin) and a fluorescent label (F) (NHS-Alexa647). Unbound protein is washed away. Bound complexes are released from the beads by cleaving a photocleavable linker with UV light. In Catch-2, SOMAmer-protein complexes are captured on monomeric avidin beads, washed, and eluted from the beads with 2 mM biotin. At this stage, the SOMAmer-protein complexes are subjected to a kinetic challenge. Specific complexes survive the challenge, while nonspecific complexes dissociate. In the final step, Catch-3, the bound complex is captured onto primer beads by a DNA primer complementary to a portion of the SOMAmer, and the remaining unbound protein resulting from the kinetic challenge is washed away. Finally, the captured complex is dissociated with 20 mM NaOH, and the target protein is eluted for analysis by PAGE.

[0129] A "biological sample" suitable for the methods and kits described herein includes any sample containing or suspected of containing a biomarker for detection, including, but not limited to, a biological fluid (e.g., whole blood or a portion thereof), a blood product, plasma, a mucosal surface sample (e.g., serum, saliva, nasal swab, throat swab, respiratory swab, nasal scraping, nasal wash, respiratory wash, lung lavage, intestinal sample, feces, or gingival crevicular fluid. In a particularly advantageous embodiment, the sample is whole blood or a portion or derivative thereof, including circulating plasma proteins. In some embodiments, the sample is purified or partially purified. In one embodiment, the sample is plasma. In one embodiment, the sample is serum. Blood, plasma, or serum samples are, in some embodiments, treated with anticoagulants such as citrate, EDTA, and heparin. In one embodiment, the biological sample is collected from the subject at two additional time points. In one embodiment, the sample is stored at about 4°C, about 15°C, or at room temperature prior to use. or about 24°C for a period of time. In one embodiment, the sample is dried, lyophilized, or flash-frozen. Non-limiting examples of blood products include whole blood, serum, plasma, etc., and combinations thereof. Blood products may be devoid or depleted of cells, or may contain cells (e.g., red blood cells, platelets, and / or lymphocytes). Reference to a "blood sample" includes a plasma sample or serum sample, or other blood fraction containing proteins derived from a blood sample. Whole blood refers to an essentially unprocessed sample of blood from a subject; generally, anticoagulants are added when the blood is obtained by venous collection, or may or may not be omitted if the blood is obtained by fingerprick or fingerstick collection and used at the point of care without delay. In some embodiments, subject samples are analyzed using EDTA plasma. In some embodiments, different subject groups may be on one type of anticoagulant.

[0130] A "subject" as contemplated herein generally refers to a human subject and may also be referred to as a patient, individual, or recipient. A human subject may be a male or female newborn or infant, child, adolescent, teenager, young adult, adult, or elderly. A reference subject is a selected group of subjects, often referred to as a subject / patient population. In some embodiments, the biological sample for testing is from a reference subject, such as a subject who has recovered from the "infectious" phase of a viral infection, e.g., a convalescent control (CC) or healthy control subject (HC) in this study. In some embodiments, the biological sample for testing is from a subject who has recovered from the "infectious" phase of a viral infection, although samples may also be collected from subjects in individual or general population screening to rule out the possibility or risk of NPASC. Reference samples may reflect different subgroups and populations, as known in the art. Any subject group can be usefully used as a reference population, including "healthy" or "unhealthy" subjects, such as immunocompromised, or age-, ethnicity-, or gender-specific populations.

[0131] In some embodiments, the subject is a human, including different human populations. However, the invention extends to non-mammals such as primates, livestock animals, laboratory test animals including fish models, companion animals, and birds, as well as fish and reptiles. The assay thus has therapeutic and diagnostic applications in humans, livestock, veterinary animals, and wildlife.

[0132] Thus, the term includes humans and a wide range of mammals, non-mammals, birds, or other animals, including wild and domesticated animals, horses, camelids, rabbits, rodents, guinea pigs, dogs, pets, pests, and potential vehicles of emerging infectious diseases. In some embodiments, the subject is a mammal or avian species. In one embodiment, the mammal is a human. With respect to subjects, the subject may be at risk for, suspected of, or diagnosed with an infection or pathogen, condition, COVID / COVID-19, or NPASC resulting from SARs-CoV-2 infection, including any emerging coronavirus variant or disease.

[0133] In some embodiments, a biomarker-specific binding agent for use in the methods and kits is one or more of an antibody or antigen-binding derivative thereof, an antibody mimetic scaffold, a peptide, a receptor / ligand or receptor / ligand derivative, an aptamer or modified aptamer, a nucleic acid, a molecularly imprinted polymer (MIP), a PLA, an adnectin, or an ankyrin. Such molecules are known in the art and to those skilled in the art.

[0134] In some embodiments, the aptamer comprises biotin or other members of a binding pair, a photocleavable group, and a fluorescent or luminescent tag. Modified aptamers with proteinaceous binding sites are advantageously used in some embodiments.

[0135] In some embodiments, the binding agent is conjugated to a detectable marker or a microparticle containing a detectable marker that can provide a detectable signal.

[0136] The biomarker-binding agent complex may directly or indirectly provide a detectable signal that can be quantified visually or photometrically (by an instrument reader), including by fluorescence measurement.

[0137] In some embodiments, the binding agent may be conjugated to a detectable marker or a microparticle containing a detectable marker that provides a detectable signal. Exemplary particles and methods are described in Rundstrom, G et al.

[0138] The term "binding agent" and similar terms refer to any compound, composition, or molecule capable of binding specifically or substantially specifically (i.e., with limited cross-reactivity) to a biomarker. A "binding agent" generally has a single specificity. Nevertheless, binders with multiple specificities for two or more biomarkers are also contemplated herein. Alternatively, "combibodies," comprising a non-covalent association of VH and VL domains, can be produced in a matrix format generated from the combination of diabody-producing bacterial clones (e.g., available from Domantis). Exemplary antigen-binding molecules for use as protein capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab', and F(ab')2 immunoglobulin fragments, synthetic stabilized Fv fragments, e.g., single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), single variable domain (dAb) minibodies, combibodies, and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids, or engineered human equivalents.

[0139] Individual, spatially distinct protein capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

[0140] Methods for making specific binding agents, including antibodies and their derivatives and analogs, binding proteins, nucleic acid ligands, and aptamers, including modified aptamers and other slow off-rate aptamers, are well known in the art.

[0141] Polyclonal antibodies can be generated by immunization of animals. Monoclonal antibodies can be prepared according to standard (hybridoma) methodologies. Antibody derivatives and analogs, including humanized antibodies, can be prepared recombinantly by isolating DNA fragments from the DNA encoding the monoclonal antibody and subcloning the appropriate V regions into suitable expression vectors according to standard methods. Phage display and aptamer technologies have been described in the literature and allow for the in vitro clonal amplification of antigen-specific binding reagents with very low affinity cross-reactivity. Phage display reagents and systems are commercially available and include the Recombinant Phage Antibody System (RPAS) available from Amersham Pharmacia Biotech, Inc. (Piscataway, New Jersey) and the pSKAN phagemid display system available from MoBiTec, LLC (Marco Island, Florida). Aptamer technology is described, for example but not limited to, US Pat. Nos. 5,270,163, 5,475,096, 5,840,867, and 6,544,776.

[0142] Antibodies are often used in immunoassays because they can be produced in large quantities and the products are homogeneous. Hybridoma cell lines for monoclonal antibody production can be prepared by fusing immortal cell lines and lymphocytes sensitized to the antigen of interest, or by techniques well known to those skilled in the art. (See, for example, Douillard and Hoffman, "Basic Facts about Hybridomas," Compendium of Immunology Vol. II, ed. Schwartz, 1981; Kohler and Milstein, "Nature" 256:495-499, 1975; "European Journal of Immunology" 6:511-519, 1976; or more recent references, such as Sambrook, "Molecular Cloning: A Laboratory Manual," 3rd Edition, CSHLP, CSH, NY, 2001.) Antibody-encoding DNA can be manipulated in vitro and then returned to lymphoid cell lines, thus enabling the production of genetically engineered antibodies. Over the past decade, the use of transient mammalian expression systems for the production of native complex proteins has been facilitated by the publication of efficient transfection protocols and the availability of suspension cell lines that grow at high densities (see S. Geisse, B. Voedisch, Methods Mol. Biol., 899 (2012), pp. 203-219). For transient expression, HEK-293 and CHO-K1 cell lines have been primarily used. Both cell lines can be adapted for suspension culture, and subclones that grow at high cell densities in chemically defined media are available. The human embryonic kidney 293 cell line may also be used due to its ease of transfection, high expression yields, and native human glycosylation.

[0143] Antibodies (and their smaller forms, e.g., scFv and Fab fragments) can be produced in any cell type known in the art. Cell-free protein synthesis, also known as in vitro translation, facilitates the production of a given target protein by utilizing the translation machinery without the use of living cells. Cell-free systems have been successfully used for the high-throughput production of protein libraries and the high-yield synthesis of selected target proteins. In particular, the use of linear DNA templates contributes to the ease and speed of cell-free translation systems, as no time-consuming cloning steps are required prior to protein production. While antibody production of a given target protein takes approximately 1–2 days, cell-based expression, including cloning procedures and cell transformation, can take up to 2 weeks. Phage display technology is the most commonly used technique for the in vitro selection and evolution of antibody fragments.

[0144] Alternatives to antibodies as specific binding agents have been described in the literature and are recognized in the art as potentially improving, inter alia, the reproducibility and stability of assays. A review of antibody alternatives is provided by McLeod et al., The Scientist, February 2016, and includes aptamers and affimers. Such binding agents can be used in the present assays and kits without undue experimentation.

[0145] Exemplary binding agents include antigen-binding constructs comprising a protein scaffold linked to one or more epitope-binding domains, the antigen-binding construct having at least two antigen-binding sites, at least one of which is derived from the epitope-binding domain and at least one of which is derived from the paired VH / VL domain.

[0146] In one broad embodiment, the method comprises (i) measuring or having measured the level of at least one of the biomarkers C5a or gliomedin, and preferably measuring or having measured the level of both of these biomarkers. In other embodiments, the method comprises measuring the level of at least one or more biomarker levels selected from: 1. Gliomedin (UniProt Q6ZMI3) 2. C5a anaphylatoxin (UniProt P01031) 3. Transforming growth factor β1 (UniProt P01137) 4. Galactosylceramide sulfotransferase (UniProt C9JIS3) 5. Inteferon lambda-1 (UniProt G9C945) 6. Growth hormone-releasing hormone (also known as somatoliberin) (UniProt P01286) 7. Lymphocyte function-associated antigen 3 (UniProt P19256) 8. FASLG (UniProt P48023) 9. Transgelin (UniProt Q7Z517) 10. Immunoglobulin heavy chain constant gamma 1 (UniProt P01857) 11. Glycoprotein NMB (UniProt Q14956)

[0147] In some embodiments, the methods comprise measuring levels of gliomedin, C5a, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of the biomarkers 3-11 listed above, in any combination.

[0148] In some preferred embodiments, the methods measure or include measuring the levels of each of the following: C5a, gliomedin, and TGFβ1. In some embodiments, the biomarkers included are: C5a, gliomedin, TGFβ1, and Gal3ST1. In some embodiments, the biomarkers included are: C5a, gliomedin, Gal3ST1, IFNlambda-1, and GHRH. In some embodiments, the biomarkers included are: C5a, gliomedin, TGFβ1, Gal3ST1, IFNlambda-1, and LFA-3. In some embodiments, the biomarkers included are: C5a, gliomedin, TGFβ1, Gal3ST1, IFNlambda-1, GHRH, and LFA-3. In some embodiments, the biomarkers included are as follows: C5a, gliomedin, TGFβ1, Gal3ST1, IFN lambda-1, GHRH, FASLG, and transgelin. In some embodiments, the biomarkers included are as follows: C5a, gliomedin, TGFβ1, Gal3ST1, IFN lambda-1, GHRH, GPNB, and IgGH1. In some embodiments, the biomarkers included are as follows: C5a, gliomedin, TGFβ1, IFN lambda-1, LFA-3, IgGH1, and GPNMB. In some embodiments, the biomarkers included are as follows: C5a, gliomedin, TGFβ1, Gal3ST1, IFN lambda-1, GHRH, LFA-3, FASLG, transgelin, IgGH1, and GPNMB.

[0149] In some embodiments, (iii) a further step is to analyze the levels from (i) and / or (ii) against a preselected threshold or gate derived from levels determined from a reference subject / population to derive a score indicating whether the subject is likely to have NPASC. Those skilled in the art will be able to design various gating strategies to optimize testing and reporting.

[0150] The level of linear or reproducible nonlinear correlation required for a marker to function as an accurate indication risk will vary depending on the marker and the assay used. Generally, a correlation of about 70% or greater will be valid. Levels of 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%, or 99% are contemplated. Correlation analyses can be performed within NP patients with and without various neurological conditions, such as those in Table 1 below, as well as other neurological and other conditions, such as diabetes, obesity, and gender.

[0151] Terms such as "differentially present" are used herein to describe the level of a biomarker and refer to an increase or decrease in the amount of the biomarker detected in a subject sample relative to the amount detected in a reference subject or population. In some embodiments, the amount is expressed as the mean, median, or mode amount from a reference population or subpopulation. The term encompasses higher or lower levels of the biomarker in a test sample compared to the reference value. In certain embodiments, a biomarker is differentially present if the level of the biomarker in a biological sample subject is at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001% of the amount or activity of the corresponding biomarker in a reference sample obtained from a reference subject or population. In certain embodiments, the biomarkers are differentially present as determined by application of an algorithm and, optionally, assignment of a score.

[0152] The term "reference sample" includes any sample that can be used to establish reference data and predetermined control values ​​from subjects determined to be "healthy" or to have a known disease state. In some embodiments, the reference sample is a healthy control sample or a healthy control sample from a selected reference population of healthy subjects. In some embodiments, the reference sample is a sample from a COVID-19 patient who recovered without symptoms of COVID-19 during or shortly after the acute phase of COVID-19, such as a convalescent control, or a sample from a subject who recovered from NPASC or a population. Ideally, the severity of COVID-19 will be comparable, and currently, non-hospitalized patients; however, if NPASC is being studied in hospitalized patients, the reference sample from COVID-19-recovered subjects should be comparable. In some embodiments, the reference sample is a population or subpopulation of healthy subjects. In some embodiments, the reference value is determined from a previous sample from the same subject undergoing testing or is predetermined. In some embodiments, the subject has NPASC as determined from established diagnostic tools / protocols, such as questionnaires or neurological evaluations.

[0153] The reference value may also be a value determined in the practice of the method, or may be a predetermined value. In either case, the value may be a level, combination of levels, a score, a threshold, or a range to which the level in the sample is compared, and the method includes assigning a score to the sample based on the level of at least one biomarker.

[0154] Reference to a protein biomarker includes modified forms thereof, e.g., mutants, isoforms, post-transcriptional forms, post-translationally modified forms, etc. Modified forms include derivatives, polymorphic variants, truncated forms (truncations), and aggregated or multimeric forms, or forms with extension elements (e.g., amino acid extension elements).

[0155] An "altered" level refers to an increase or elevation, or a decrease or reduction, in the level or ratio of one or more biomarkers. Determining the level of a biomarker allows for the establishment of a diagnostic rule based on the level or ratio, which may be relative to a reference level. Alternatively, the diagnostic rule is based on the application of statistical, variance analysis, and / or machine learning procedures. Such algorithms use the relationship between biomarkers and disease or health states observed in reference subjects to infer a relationship that is then used to predict the status of a subject with an unknown condition. The algorithm can be used to provide a visually detectable score or index of the probability that a patient is not or is not an indication for NPASC. In some embodiments, the algorithm performs a multivariate or univariate analytical function.

[0156] A positive score for NPASC likelihood or risk according to the present disclosure allows for the prompt administration of NPASC therapy. Thus, the present disclosure extends to methods of treatment and prevention that involve screening subjects according to the methods disclosed herein and administering NPASC therapy to the subject depending on the results of the assay. Thus, in other words, the present disclosure teaches the use of the assays, kits, and algorithms disclosed herein in the diagnosis and treatment and / or prevention of NPASC.

[0157] The methods and kits and diagnostics are for use in the treatment and / or prevention of conditions associated with NPASCs, including monitoring of subjects before and / or after treatment.

[0158] Thus, in some embodiments, the present disclosure provides a method for assessing in a biological sample from a subject having or at risk for a neurological post-viral condition, NPASV, NPASC, Long COVID, COVID-19, the method comprising: (i) contacting the sample with a binder that specifically binds to one of biomarkers 1-11 in the sample to form a biomarker-binder complex a; (ii) simultaneously or sequentially with (i) and / or (ii), contacting the sample with a second binding agent that specifically binds to a in the sample and forms one of complexes b of biomarkers 1-11; (iii) using the amounts of complex a and complex b to determine the likelihood that the subject has NPASC or NPASCoV.

[0159] In some embodiments, the methods involve directly or indirectly scoring the sample to include healthy control levels, convalescent COVID levels, or other reference levels of one or two or more of biomarkers 1-11.

[0160] In some embodiments, the method comprises determining a ratio of a plurality of biomarkers selected from biomarkers 1-11 from a sample from the subject.

[0161] In some embodiments, the method comprises scoring the likelihood of NPASC when the level of a biomarker in a subject sample is altered compared to a predetermined mean plus two or more standard deviations of a healthy reference population.

[0162] In some embodiments, the method comprises scoring the likelihood of NPASC when the biomarker level in the subject sample is differentially present when compared to a predetermined mean plus two standard deviations or more of a healthy reference population.

[0163] In some embodiments, the method comprises scoring the likelihood of NPASC when the level of a biomarker in the subject's sample is above a predetermined mean plus two standard deviations or more of a healthy or convalescent control population. In some embodiments, the biomarker is one or more of C5a or gliomedin. In other embodiments, the biomarker measured is (i) C5a and / or gliomedin, and (ii) one or more of transforming growth factor β1 (TGFβ1), galactosylceramide sulfotransferase (Gal3ST1), interferon (IFN) lambda-1, growth hormone-releasing hormone (GHRH), lymphocyte function-associated antigen 3 (LFA-3), Fas ligand (FASLG), transgelin, immunoglobulin heavy chain constant gamma 1 (IgGH1), and glycoprotein NMB (GPNMB).

[0164] In some embodiments, the method comprises scoring the likelihood of NPASC if the biomarker level is below or above a predetermined mean plus two or more standard deviations from a healthy control or COVID convalescent control population value.

[0165] In some embodiments, the required level is 3 standard deviations or more of a reference population value (eg, the median).

[0166] The assays described herein allow for integration into existing or newly developed pathology architectures or platform systems. For example, the methods described herein allow a user to determine a subject's status relative to a neuropathophysiological condition associated with, (a) receiving data via a communications network in the form of levels of any one or more of biomarkers 1-11 or one or more of the biomarkers set forth in Table 1 in a test sample from a subject; (b) processing the subject's data through an algorithm that provides a score or disease index value by comparing the levels and / or ratios of the corresponding biomarkers to levels from predetermined reference levels.

[0167] In some embodiments, the display of the object's status to the user is transferred over a communications network. It will also be appreciated that in one example, the end station may be a handheld device, such as a PDA or cell phone, that can transfer object data to a base station and receive reports over a communications network, such as the Internet. If a server is used, it is generally a client-server, or more specifically, a Simple Object Application Protocol (SOAP). This analysis may use principal component analysis.

[0168] Aspects of the present disclosure provide various ranges of values. Slight variations above or below the stated ranges can be used to achieve substantially the same results as values ​​within the ranges. These ranges are also intended as continuous ranges, including all values ​​between the minimum and maximum values. Additionally, the present disclosure covers ratios of biomarker levels that provide values ​​related to NPASC status, development, or monitoring.

[0169] Treatment methods for NPASC As disclosed herein, the levels of several proteins in subjects identified as suffering from NPASCs are significantly altered from COVID-recovered subjects without NPASCs and / or from subjects who have never been infected with COVID. Without being bound by theory, it is believed that such proteins may underlie the aberrant immune response to COVID infection that persists after the acute phase. Through data mining and pathway analysis, a subset of such proteins has been identified as therapeutic targets NPASCs in the treatment methods disclosed herein.

[0170] Accordingly, the methods described herein include treating a subject identified as suffering from or at risk of suffering from neurological acute sequelae of COVID-19 (NPASC), the methods comprising: (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), (xii) complement component 5a (C5a) / complement 5 anaphylotoxin; (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) administering to a subject or having administered to a subject a therapeutically effective amount of a modulator of a therapeutic target selected from the list consisting of growth hormone-releasing hormone (GHRH).

[0171] In some embodiments, where there is a need to reduce the activity or expression level of a therapeutic target, the therapeutic modulating agent: (i) the complement C5b-C6 complex, (ii) lymphocyte function-associated antigen 3 CD58; (iii) transforming growth factor-beta-1 (TGF-β1); (iv) vascular endothelial growth factor D; (v) myeloid cell surface antigen CD33; (vi) alcohol dehydrogenase 1B; (vii) tumor necrosis factor receptor superfamily member 1A; (viii) tumor necrosis factor receptor superfamily (TNFRSF) 10b; (ix) cathepsin S, (x) TLR4:lymphocyte antigen 96 complex, (xi) BCL2-like 1 (Bcl2L1), and (xii) an inhibitor of a therapeutic target selected from complement component 5a (C5a) / complement 5 anaphylotoxin.

[0172] In other embodiments, when an increase in the activity or expression level of a therapeutic target is required, the therapeutic modulating agent is (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) activators of therapeutic targets selected from growth hormone-releasing hormone (GHRH).

[0173] In some embodiments, a therapeutic target from among (i) to (xii) listed above is activated. In some embodiments, a therapeutic target from among (xiii) to (xx) listed above is inhibited.

[0174] Symptoms and diagnostic tests for identifying subjects suffering from NPASC are known in the art. See, for example, Pinzon et al. (2022). For example, to identify subjects suffering from significant neurological disorders, subjects can complete a cognitive function assessment using the National Institutes of Health (NIH) Toolbox v2.1 instrument, including assessments of processing speed (Pattern Comparison Processing Speed ​​Test), attention and executive memory (Inhibitory Control and Attention Test), executive function (Dimensional Change Card Sort Test), and working memory (List Sorting Working Memory Test) (Weintraub et al. 2013, Lai et al., 2011). Preferably, diagnostic tests for NPASC include methods for identifying subjects as suffering from NPASC based on the levels of one or more NPASC biomarkers or combinations of NPASC biomarkers disclosed herein.

[0175] Modulation of NPASC therapeutic targets The methods for treating and / or preventing NPASC disclosed herein comprise administering to a subject identified in need thereof a modulator of an NPASC therapeutic target disclosed herein. In some embodiments, the modulator is an inhibitor of the therapeutic target. In other embodiments, the administered modulator is an activator of the therapeutic target.

[0176] As used herein, inhibition of a therapeutic target refers to a target-specific or selective decrease in one or more of the net gene expression, net protein levels, or function / activity (e.g., protein-protein interaction or enzymatic activity) of a therapeutic target disclosed herein. In some preferred embodiments, the therapeutic target that is inhibited is selected from among the following:

[0177] Exemplary NPASC therapeutic targets to be inhibited (i) Complement C5b-C6 complex (GenBank No. NP_001108603), (ii) lymphocyte function-associated antigen 3 CD58 (GenBank No. CAA01784); (iii) transforming growth factor-beta-1 (TGF-β1) (GenBank No. KAI4042861); (iv) Vascular endothelial growth factor D (GenBank No.CAA03942), (v) myeloid cell surface antigen CD33 (GenBank No. AAK83654); (vi) alcohol dehydrogenase 1B (GenBank No. AAH33009); (vii) tumor necrosis factor receptor superfamily member 1A (GenBank No. AAH10140); (viii) tumor necrosis factor receptor superfamily member 10B (GenBank No. NP_003833); (ix) cathepsin S (GenBank No. AAC37592); (x) TLR4:lymphocyte antigen 96 complex (GenBank No. AAF05316); (xi) BCL2-like 1 (Bcl2L1) (GenBank No. KAI4005058), and (xii) Complement component 5a (C5a) (GenBank No. NP_001726.2)

[0178] As used herein, activation of a therapeutic target refers to a target-specific or selective increase in one or more of the net gene expression, net protein levels, or function / activity (e.g., protein-protein interaction or enzymatic activity) of a therapeutic target disclosed herein. In some preferred embodiments, the therapeutic target that is activated is selected from among the following:

[0179] Exemplary NPASC Therapeutic Targets to be Activated (xiii) B cell receptor CD22, (xiv) antithrombin-III (GenBank No. NP_000479.1 ); (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit alpha (GenBank No. P49354); (xvi) amyloid A4 protein (GenBank No. CAA30050.1), (xvii) thyroid peroxidase (GenBank No. AAA61217); (xviii) IgG (GenBank No.AAA02914), (xix) palmitoyl protein thioesterase (GenBank No. AAB06236), and (xx) Growth hormone-releasing hormone (GHRH) (GenBank No. KAI4005470).

[0180] Inhibition of a therapeutic target can include at least about a 10% to 100% decrease in the activity level or expression of a therapeutic target in the presence of or resulting from a given dose of a therapeutic target inhibitor, compared to the activity level of the therapeutic target in its absence, e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or another percent decrease of about 10% to about 100% of the activity or level of the therapeutic target.

[0181] Activation of a therapeutic target can include at least about a 10% to 200% increase in the activity level or expression of a therapeutic target in or attributable to the presence of a given dose of a therapeutic target inhibitor, compared to the activity level of the therapeutic target in its absence, e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 110%, 120%, 150%, 175%, 190%, or another percent increase of about 10% to about 200% in the activity level or expression of a therapeutic target.

[0182] Examples of types of NPASC therapeutic target modulators useful in the present invention include, but are not limited to, small molecule modulators, antibody modulators, polypeptide / protein modulators, nucleic acid modulators, peptide modulators, and peptidomimetic modulators.

[0183] small molecule In some embodiments, the administered therapeutic target modulator is a small molecule therapeutic target modulator, hi some preferred embodiments, the small molecule therapeutic target modulator is a therapeutic target inhibitor.

[0184] In some embodiments, small molecule therapeutic target inhibitors specifically bind to and reduce the activity of a therapeutic target protein, for example, by reducing the interaction of the therapeutic target with a binding partner or receptor, or by interfering with enzymatic activity.

[0185] In some embodiments, the therapeutic target inhibited with the small molecule inhibitor is TGF-β 1. In some embodiments, the small molecule TGF-β 1 inhibitor administered is pirfenidone (CAS 53179-13-8).

[0186] In other embodiments, the therapeutic target inhibited is alcohol dehydrogenase 1B. In some embodiments, the small molecule alcohol dehydrogenase 1B inhibitor is honepizole (CAS 7554-65-6).

[0187] In other embodiments, the therapeutic target inhibited is cathepsin S. In some embodiments, the small molecule cathepsin S inhibitor administered is petesicatib (CAS 1252637-35-6).

[0188] In other embodiments, the therapeutic target inhibited is the TLR4:lymphocyte antigen 96 complex. In some embodiments, the small molecule TLR4:lymphocyte antigen 96 complex inhibitor administered is resatorbid (CAS 243984-11-4) or eritoran (CAS 185954-98-7).

[0189] In further embodiments, the therapeutic target inhibited is Bcl2L1. In some embodiments, the small molecule Bcl2L1 inhibitor administered is navitoclax (CAS 923564-51-6) or obatoclax (CAS 803712-67-6).

[0190] Suitable small molecule inhibitors (and activators) for the corresponding therapeutic targets disclosed herein for use in the disclosed therapeutic methods can be identified using standard procedures, such as screening a library of candidate compounds for binding to the NPASC therapeutic targets disclosed herein, and then determining whether any of the binding compounds reduce the activity of the therapeutic target. In some embodiments, screening for small molecule inhibitors or activators of the therapeutic targets disclosed herein involves assessing whether the compounds inhibit or activate the therapeutic target of interest in a cell. Small molecules useful in the present invention can also be identified using in silico screening procedures, which may include screening known library compounds to identify candidates that reduce or activate the activity of the therapeutic target. In some embodiments, the small molecule therapeutic target inhibitor is an irreversible therapeutic target inhibitor. In other embodiments, the small molecule therapeutic target inhibitor is a reversible therapeutic target inhibitor.

[0191] In some embodiments, the administered small molecule therapeutic target inhibitor or activator is a precursor compound, commonly referred to as a "prodrug," which has no activity or relatively little activity, but is converted (i.e., metabolized) to an active therapeutic target inhibitor or activator after administration. In these embodiments, the administered small molecule compound may be referred to as a prodrug. Alternatively, or additionally, the administered modulator compound may be metabolized to produce an active metabolite that inhibits or activates the therapeutic target in the treated subject's cell population compared to the cell population in the absence of administration of the small modulator compound. Use of such active metabolites is also within the scope of the present disclosure.

[0192] Depending on the substituents present in the modulator compound, the compound may optionally exist in the form of a salt. Salts of compounds suitable for use in the described methods are those in which the counterion is pharmaceutically acceptable. Suitable salts include salts formed with organic or inorganic acids or bases. In particular, suitable salts formed with acids include those formed with mineral acids, strong organic carboxylic acids (e.g., alkanecarboxylic acids of 1 to 4 carbon atoms, unsubstituted or substituted (e.g., by halogens), e.g., saturated or unsaturated dicarboxylic acids, e.g., hydroxycarboxylic acids, e.g., amino acids), or organic sulfonic acids, substituted or unsubstituted (e.g., by halogens) (C 1-4 Pharmaceutically acceptable acid addition salts include those formed with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, isethionic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid, glutamic acid, lysine, and arginine. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts (e.g., potassium and sodium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and salts with organic bases (e.g., dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-, or tri-lower alkylamines (e.g., ethyl-, t-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl-, or dimethyl-propylamine), or mono-, di-, or trihydroxy lower alkylamines (e.g., mono-, di-, or triethanolamine)). Corresponding internal salts can also be formed.

[0193] Those skilled in the art of organic chemistry and / or medicinal chemistry will understand that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates." For example, complexes with water are known as "hydrates." Solvates, such as hydrates, exist when a drug substance incorporates a solvent, such as water, into the crystal lattice in either stoichiometric or non-stoichiometric amounts. Because solvates, such as hydrates, can be encountered at any stage, drug substances are routinely screened for their presence. Therefore, it is understood that compounds useful in the present invention can exist in the form of solvates, such as hydrates. Solvated forms of compounds suitable for use in the present invention are those in which the associated solvent is pharmaceutically acceptable. For example, a hydrate is an example of a pharmaceutically acceptable solvate.

[0194] The compounds useful in the present invention may exist in amorphous or crystalline form. Many compounds exist in multiple polymorphic forms, and the use of all such forms of the compounds is encompassed by the present disclosure.

[0195] antibody In some embodiments, the administered therapeutic target modulator is an antibody therapeutic target modulator. In some preferred embodiments, the antibody target modulator is a therapeutic target inhibitor, whereby the antibody therapeutic target inhibitor specifically binds to the therapeutic target and reduces the activity of the therapeutic target protein, for example, by reducing the interaction of the therapeutic target with a binding partner or receptor, or by interfering with enzymatic activity.

[0196] In some embodiments, the therapeutic target inhibited with an antibody inhibitor is the complement C5b-C6 complex. In some embodiments, the administered antibody complement C5b-C6 complex inhibitor is ravulizumab or a monoclonal antibody that competes for binding to the same epitope as ravulizumab.

[0197] In other embodiments, the therapeutic target inhibited with an antibody inhibitor is the myeloid cell surface antigen CD33. In some embodiments, the administered antibody myeloid cell surface antigen CD33 inhibitor is gemtuzumab or a monoclonal antibody that competes for binding to the same epitope as gemtuzumab.

[0198] In other embodiments, the therapeutic target inhibited with the antibody inhibitor is tumor necrosis factor receptor superfamily member 10B. In some embodiments, the administered antibody tumor necrosis factor receptor superfamily member 10B inhibitor is conatumumab, lexatumumab, or a monoclonal antibody that competes for binding to the same epitope as either conatumumab or lexatumumab. In other embodiments, the therapeutic target inhibited with the antibody inhibitor is tumor necrosis factor receptor superfamily member 1A. In some embodiments, the administered antibody tumor necrosis factor receptor superfamily member 1A inhibitor is atrocimab, TNF receptor 1 silencer (TROS) (see Fischer et al., 2020), or a monoclonal antibody that competes for binding to the same epitope as either of these.

[0199] In some embodiments, the inhibitors of NPASC therapeutic targets disclosed herein are antibodies or binding fragments thereof that bind to the therapeutic target and inhibit its interaction with a binding partner, target protein, and / or receptor.

[0200] The term "antibody" as used herein includes polyclonal antibodies, monoclonal antibodies, bispecific antibodies, fusion diabodies, triabodies, heteroconjugate antibodies, and chimeric antibodies. Also contemplated are antibody fragments that retain at least substantial (about 10%) antigen binding relative to the corresponding full-length antibody. Such antibody fragments are referred to herein as "antigen-binding fragments." Antibodies include various forms of modifications, including, but not limited to, domain antibodies containing either a VH or VL domain, a dimer of heavy chain variable regions (VHH, as described for Camelidae), a dimer of light chain variable regions (VLL), an Fv fragment containing only the light chain (VL) and heavy chain (VH) variable regions, which may be linked directly or via a linker, or an Fd fragment containing the heavy chain variable region and CH1 domain.

[0201] The term "antibody" also encompasses scFvs, which consist of heavy and light chain variable regions linked together to form a single-chain antibody, as well as oligomers of scFvs, such as diabodies and triabodies. Also encompassed are antibody fragments, such as Fab, (Fab')2, and FabFc2 fragments, which contain portions of the variable and constant regions. Also encompassed are complementarity-determining region (CDR)-grafted antibody fragments and oligomers of antibody fragments. The heavy and light chain components of an Fv can be derived from the same or different antibodies, thereby generating a chimeric Fv region. Antibodies can be derived from animals (e.g., mice, rabbits, or rats) or humans, or can be chimeric or humanized.

[0202] As used herein, the term "antibody" encompasses these various forms. Antibodies for use in the methods of the invention can be readily generated using the guidelines provided herein and methods well known to those of skill in the art, as described in the references cited above and in publications such as Harlow & Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0203] The antibody may be an Fv region comprising a variable light chain (VL) and a variable heavy chain (VH), in which the light and heavy chains can be linked directly or via a linker. As used herein, a linker refers to a molecule that covalently links the light and heavy chains and provides sufficient spacing and flexibility between the two chains so that they can achieve a conformation in which they can specifically bind to their intended epitopes. Protein linkers are particularly preferred because they can be expressed as an inherent component of the Ig portion of the fusion polypeptide.

[0204] In another embodiment, recombinantly produced single chain scFv antibodies, preferably humanized scFvs, are used in the methods of the invention.

[0205] In one embodiment, the antibody is capable of intracellular delivery. Antibodies capable of intracellular delivery include antibodies such as camelid and llama antibodies, shark antibodies (IgNAR), scFv antibodies, intrabodies, or nanobodies (e.g., scFv intrabodies and VHH intrabodies). The yeast SPLINT antibody library is available for testing intrabodies capable of disrupting protein-protein interactions. Such agents may include cell-penetrating or nuclear-localizing peptide sequences, such as those disclosed in Constantini et al. (2008). Vectocell or Diato peptide vectors, such as those disclosed in De Coupade et al. (2005), are also useful for in vivo delivery.

[0206] In addition, antibodies can be fused to cell-penetrating agents, such as cell-penetrating peptides. Cell-penetrating peptides include short amphipathic peptides such as those from the Tat peptide, penetratin, Pep, and MPG families, oligoarginines, and oligolysines. In one example, cell-penetrating peptides are also conjugated to lipid (C6-C18 fatty acid) domains to improve intracellular delivery (Koppelhus et al., 2008). Examples of cell membrane-penetrating peptides can be found in Howl et al. (2021) and Deshayes et al. (2008). Thus, the present invention also provides therapeutic uses of antibodies optionally fused to a cell-penetrating peptide sequence via a covalent bond (e.g., a peptide bond) at the N- or C-terminus.

[0207] protein In some embodiments, the administered therapeutic target modulator is a protein therapeutic target modulator. In some preferred embodiments, the protein therapeutic target modulator is a therapeutic target inhibitor, whereby the protein therapeutic target inhibitor specifically binds to the therapeutic target and reduces the activity of the therapeutic target protein, for example, by reducing the interaction of the therapeutic target with a binding partner or receptor, or by interfering with enzymatic activity.

[0208] In some preferred embodiments, the protein therapeutic target inhibitor is a fusion protein. In some embodiments, the therapeutic target inhibited with the fusion protein inhibitor is lymphocyte function-associated antigen 3 (LFA3) CD58. In some embodiments, the fusion protein lymphocyte function-associated antigen 3 CD58 inhibitor administered is alefacept, a fusion protein comprising the first extracellular domain of human LFA3 fused to a hinge segment and the constant region of human IgG1.

[0209] peptide In some embodiments, the therapeutic target inhibitor or therapeutic activator used in the therapeutic methods disclosed herein is a peptide. In some embodiments, the peptide inhibits the activity of one or more of the NPASC therapeutic targets disclosed herein. In other embodiments, the peptide is solely active as an NPASC therapeutic target disclosed herein, or has similar activity. In some embodiments, when the therapeutic target to be activated is growth hormone-releasing hormone, the peptide is synthetic growth hormone analog-modified peptide MR409 (CAS 1445155-39-4).

[0210] Peptides suitable for use in the methods of the present invention can be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues according to conventional peptide synthesis methods. Preferably, peptides are synthesized according to standard solid-phase methods, such as those performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.), following the manufacturer's instructions. Other methods of synthesizing peptides, either by solid-phase or solution-phase methods, are well known to those skilled in the art. When solid-phase synthesis is utilized, the C-terminal amino acid is linked to an insoluble resin support that can produce a severable bond by reacting with the carboxyl group in the C-terminal amino acid. For example, in some cases, the insoluble resin support used is p-hydroxymethylphenoxymethylpolystyrene (HMP) resin. Other useful resins include, but are not limited to, phenylacetamidomethyl (PAM) resin for the synthesis of some N-methyl-containing peptides (this resin is used with the Boc method of solid-phase synthesis) and MBHA (p-methylbenzhydrylamine) resin for producing peptides with a C-terminal amide group. During peptide synthesis, branched-chain amino and carboxyl groups can be protected / deprotected as needed using commonly known protecting groups. In some embodiments, N-I-amino groups are protected with the base-labile 9-fluorenylmethyloxycarbonyl (Fmoc) group or t-butyloxycarbonyl (Boc) group. Side chain functional groups consistent with Fmoc synthesis can be protected with the following protecting groups: arginine (2,2,5,7,8-pentamethylchroman-6-sulfonyl), asparagine (Ot-butyl ester), cysteine, glutamine, and histidine (trityl), lysine (t-butyloxycarbonyl), serine, and tyrosine (t-butyl). Modifications utilizing alternative protecting groups for peptides and peptide derivatives will be apparent to those skilled in the art.

[0211] Peptide mimetics In some embodiments, the therapeutic target inhibitor is a peptidomimetic. All peptides are susceptible to enzymatic degradation in vivo. Thus, peptidomimetics that retain or even enhance the biological activity of basic peptides but have longer circulating half-lives are particularly advantageous for use in the therapeutic methods of the invention.

[0212] A peptide backbone is characterized by one or more internal peptide bonds, whereas a peptide has peptide bonds connecting each amino acid residue. Thus, a compound in which one or more amide bonds have been replaced by an alternative linker, but at least one amide bond remains, is considered a peptidomimetic.

[0213] The peptidomimetic backbone will generally be a linear or linear chain of fused cyclic groups that mimics a peptide backbone.

[0214] Peptide mimetics are typically characterized by retaining the polarity, three-dimensional size, and functionality (biological activity) of their peptide equivalents, but the peptide bond is often replaced by a more stable bond. "Stable" means more resistant to enzymatic degradation by hydrolases. Generally, bonds that replace amide bonds (amide bond surrogates) preserve many of the properties of amide bonds, such as conformation, steric bulk, electrostatic properties, and hydrogen-bonding potential. Chapter 14 of "Drug Design and Development," Krogsgaard, Larsen, Liljefors, and Madsen (Eds.) 1996, Horwood Acad. Pub., provides a general discussion of the prior art for the design and synthesis of peptidomimetics. Suitable amide bond surrogates include the following groups: N-alkylated, retro-reverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, vinyl, methyleneamino, methylenetethio, alkane, and sulfonamide.

[0215] Peptides and peptidomimetics generally have backbones between 4 and 20 atoms in length, preferably between 7 and 16. Molecules with backbones at the higher end of these ranges generally contain beta and / or gamma amino acids or their equivalents.

[0216] nucleic acid In some embodiments, the administered therapeutic target modulator comprises a nucleic acid therapeutic target modulator. In some embodiments, the nucleic acid therapeutic target modulator is a therapeutic target inhibitor, whereby the nucleic acid therapeutic target inhibitor specifically or selectively targets the nucleic acid encoding the therapeutic target protein to be inhibited, thereby reducing the expression level, and thereby the activity level, of the therapeutic target in the treated subject. In other embodiments, the nucleic acid (e.g., therapeutic aptamer) therapeutic target inhibitor binds directly to the therapeutic target and reduces the activity of the therapeutic target protein, for example, by reducing the interaction of the therapeutic target with a binding partner or by interfering with enzymatic activity.

[0217] In some embodiments, when the administered therapeutic target inhibitor comprises a nucleic acid therapeutic target inhibitor, the nucleic acid therapeutic target inhibitor is selected from the group consisting of an antisense oligonucleotide (ASO), an aptamer, an siRNA, an miRNA, or an sgRNA disclosed herein.

[0218] antisense oligonucleotides In some embodiments, when the administered therapeutic target inhibitor comprises a nucleic acid therapeutic target inhibitor, the nucleic acid therapeutic target inhibitor is an antisense oligonucleotide (ASO) that targets an mRNA encoding an NPASC therapeutic target disclosed herein. In other embodiments, the ASO targets an mRNA encoding a non-therapeutic target protein, the level of which may indirectly affect the level of the therapeutic target protein.

[0219] In some embodiments, if a subject is diagnosed as possibly suffering from NPASC based on the disclosed methods and the subject is identified as having a level of TGFβ1 above the threshold level disclosed herein, treating the NPASC comprises administering a therapeutically effective amount of an antisense oligonucleotide against α4 integrin. Targeting α4 integrin has previously been shown to inhibit TGFβ1 activity or signaling by reducing thrombospondin-1 (TSP-1), increasing latent transforming growth factor beta-binding protein 4 (LTBP-4), and / or increasing C-X-C motif chemokine ligand 16 (CXCL16), as disclosed in WO 2023 / 039643. In some preferred embodiments, the subject being treated is not a subject identified as suffering from multiple sclerosis (MSC) or Duchenne muscular dystrophy (DMD). In some preferred embodiments, the antisense oligonucleotide is 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CA Me U Me U Me C Me U-3' (SEQ ID NO: 1), a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; c) the nucleotides at positions 4 to 12 from the 5' end are 2'-deoxyribonucleosides; d) the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides; e) All cytosines are 5-methylcytosines ( Me C) or or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0220] Antisense oligonucleotides against α4 integrin The present disclosure provides antisense oligonucleotides for inhibiting the expression of α4 integrin. Such antisense oligonucleotides target nucleic acids encoding the α4 integrin chain of VLA-4 or α4β7 integrin. As used herein, the term "inhibit" refers to any measurable decrease (e.g., 10%, 20%, 50%, 90%, or 100%) in α4 integrin expression.

[0221] In some embodiments, ASOs are administered as activators of the therapeutic targets disclosed herein. Those skilled in the art recognize that in many, if not all, "background" mis-splicing events in wild-type gene mRNA transcripts result in portions of untranslatable (e.g., frameshifted and / or truncated) mRNAs that are removed by nonsense-mediated decay (NMD), resulting in a decrease in the net level of the corresponding protein isoform derived from the corresponding gene. To reduce the background of such mis-splicing events and increase the level of full-length wild-type proteins, one or more ASOs are directed to hybridize with sequences on pre-mRNA transcripts that contain cryptic splice sites that tend to induce mis-splicing. Without wishing to be bound by theory, it is believed that upon hybridization with pre-mRNA, ASOs sterically hinder the access of splicing complexes to the cryptic splice sites, thereby avoiding mis-splicing and NMD of the mis-spliced ​​transcripts. For a review, see Lim et al. (2020). Thus, in some embodiments, an ASO activator of an NPASC therapeutic target is administered, where the therapeutic target is selected from among the following: (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) Growth hormone-releasing hormone (GHRH)

[0222] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of RNA or DNA, or mimetics, chimeras, analogs, and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to desirable properties such as, for example, enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0223] When forming oligonucleotides, phosphate groups covalently link adjacent nucleosides to each other to form linear polymeric compounds.Then, each end of this linear polymeric compound can be further linked to form a circular compound, but generally, linear compounds are preferred.In addition, linear compounds may have internal nucleobase complementarity, and therefore may fold in a manner that produces a completely or partially double-stranded compound.With respect to oligonucleotides, phosphate groups are generally referred to as forming the internucleoside backbone of oligonucleotides.The usual bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond.

[0224] Antisense oligonucleotides useful in the methods of the disclosure include, for example, ribozymes, siRNAs, external guide sequence (EGS) oligonucleotides, alternative splicers, primers, probes, and other oligonucleotides that hybridize to at least a portion of a target nucleic acid.

[0225] Antisense oligonucleotides can be administered in single-stranded, double-stranded, circular, or hairpin form and may contain structural elements such as internal or terminal bulges or loops. Upon administration, antisense oligonucleotides can induce the action of one or more enzymes or structural proteins, resulting in modification of the target nucleic acid.

[0226] A non-limiting example of such an enzyme is RNAse H, a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds that are "DNA-like" induce RNAse H. Thus, activation of RNase H leads to cleavage of the RNA target, thereby greatly improving the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases, such as enzymes from the RNase III and ribonuclease L families.

[0227] As used herein, the term "oligonucleotide" refers to an oligomer or polymer of RNA or DNA, or mimetics, chimeras, analogs, and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to desirable properties such as, for example, enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0228] Oligonucleotides may contain chiral (asymmetric) centers, or the entire molecule may be chiral. Individual stereoisomers (enantiomers and diastereomers) and their mixtures are within the scope of the present disclosure. For the disclosure of antisense oligonucleotides containing chiral phosphorothioate bonds, see Wan et al. Nucleic Acids Research 42 (22:13456-13468, 2014).

[0229] When forming an oligonucleotide, phosphate groups covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound can then be further linked to form a circular compound, but linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and therefore may fold in a manner that produces a fully or partially double-stranded compound. With respect to oligonucleotides, phosphate groups are generally referred to as forming the internucleoside backbone of the oligonucleotide. The usual bond or backbone of RNA and DNA is a 3' to 5' phosphodiester bond. Those skilled in the art can identify antisense oligonucleotides useful for the methods of the present disclosure without undue experimentation.

[0230] Modified internucleoside linkage (backbone) Antisense compounds of the present disclosure include oligonucleotides having modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0231] Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates), phosphinates, phosphophosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having their normal 3'-5' and 2'-5' linked analogs, as well as those having inverted polarity in which one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linked. Oligonucleotides with inverted polarity contain a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleoside residue that may be abasic (either lacking a nucleobase or having a hydroxyl group instead). Various salts, mixed salts, and free acid forms are also included.

[0232] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455 ,233, US 5,466,677, US 5,476,925, US 5,519,126, US 5,536,821, US 5,541,306, US 5,550,111, US 5,563,253, US 5,571,799, US 5,587,361, US 5,194,599, US 5,565,555, US 5,527,899, US 5,721,218, US 5,672,697, and US 5,625,050.

[0233] Modified oligonucleotide backbones do not contain a phosphorus atom therein and include backbones formed by, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH moieties.

[0234] Representative U.S. patents that teach the preparation of the above oligonucleotides include U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307 , US5,561,225, US5,596,086, US5,602,240, US5,610,289, US5,602,240, US5,608,046, US5,610,289, US5,618,704, US5,623,070, US5,663,312, US5,633,360, US5,677,437, US5,792,608, US5,646,269, and US5,677,439.

[0235] Modified sugars and internucleoside linkages The antisense compounds of the present disclosure include oligonucleotide mimetics in which both the sugar and the internucleoside linkage (i.e., backbone) of the nucleotide units are replaced with novel groups while the nucleobase units are maintained for hybridization with a target nucleic acid.

[0236] Oligonucleotide mimics that have been shown to have excellent hybridization properties are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of oligonucleotides is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US 5,539,082, US 5,714,331, and US 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al., 1991.

[0237] The antisense compounds of the present disclosure also include oligonucleotides with phosphorothioate backbones and oligonucleotides with heteroatom backbones, such as -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (known as methylene(methylimino) or MMI backbones), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (wherein the natural phosphodiester backbone is represented as -OPO-CH2-) of US 5,489,677, and the amide backbones of US 5,602,240. The antisense compounds of the present disclosure also include oligonucleotides with morpholino backbone structures of US 5,034,506 (which is incorporated herein by reference for its disclosure of the oligonucleotides just described).

[0238] modified sugar Antisense compounds of the present disclosure include oligonucleotides with one or more substituted sugar moieties.

[0239] Examples include oligonucleotides containing one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl (wherein alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl, or C2-C10 alkenyl and alkynyl).

[0240] In one embodiment, the oligonucleotide comprises one of the following at the 2' position: O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2 (wherein n and m are from 1 to about 10). Further examples of modified oligonucleotides include oligonucleotides containing one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties.

[0241] In one embodiment, the modification comprises a 2'-methoxyethoxy (2'-O-CH2CHOCH3 (also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995), i.e., an alkoxyalkoxy group. In a further embodiment, the modification comprises a 2'-dimethylaminooxyethoxy, i.e., an O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), or a 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0242] Other modifications include 2'-methoxy (2'-O-CH), 2'-aminopropoxy (2'-OCHCHCHNH), 2'-allyl (2'-CH-CH=CH), 2'-O-allyl (2'-O-CH-CH=CH), and 2'-fluoro (2'-F). The 2'-modification can be in the arabino (up) position or the ribo (down) position. In one embodiment, the 2'-arabino modification is 2'-F.

[0243] Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.

[0244] Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0245] Representative U.S. patents that teach the preparation of such modified sugar structures include U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,567,811, 5,567,812, 5,567,813, 5,567,814, 5,567,815, 5,567,816, 5,567,817, 5,567,818, 5,567,819 ... US 5,576,427, US 5,591,722, US 5,597,909, US 5,610,300, US 5,627,053, US 5,639,873, US 5,646,265, US 5,658,873, US 5,670,633, US 5,792,747, and US 5,700,920.

[0246] Further sugar modifications include locked nucleic acids (LNAs), in which a 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In one embodiment, the linkage is a methylene (-CH2-)n group (where n is 1 or 2) bridging the 2' oxygen atom and the 4' carbon atom. LNAs and their preparation are described in WO98 / 39352 and WO99 / 14226.

[0247] Natural and Modified Nucleobases Antisense compounds of the present disclosure include oligonucleotides having nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0248] Modified nucleobases include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CC-CH3) uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil, Other synthetic and natural nucleobases include uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0249] Further modified nucleobases include tricyclic pyrimidines (e.g., phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one)), G-clamps (e.g., substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one)), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).

[0250] Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles (e.g., 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone). Additional nucleobases include those disclosed in US 3,687,808, JI Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), Englisch et al. (1991), and YS Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, ST Crooke, B. Lebleu (editors), CRC Press (1993).

[0251] Certain of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine). 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C. In one embodiment, these nucleobase substitutions are combined with 2'-O-methoxyethyl sugar modifications.

[0252] Representative U.S. patents that teach the preparation of certain of the above modified nucleobases, as well as other modified nucleobases, include U.S. Pat. Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,512,102, 5,516,104, and 5,522,106. ,502,177, US 5,525,711, US 5,552,540, US 5,587,469, US 5,594,121, US 5,596,091, US 5,614,617, US 5,645,985, US 5,830,653, US 5,763,588, US 6,005,096, US 5,681,941, and US 5,750,692.

[0253] Conjugates The antisense compounds of the present disclosure may be conjugated to one or more moieties or groups that enhance the activity, cellular distribution, or cellular uptake of the antisense compounds. These moieties or groups may be covalently attached to functional groups such as primary or secondary hydroxyl groups.

[0254] Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of the oligomer, and groups that enhance the pharmacokinetic properties of the oligomer. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0255] Moieties or groups that enhance the pharmacodynamic properties include moieties or groups that improve uptake, increase resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid.

[0256] Moieties or groups that enhance the pharmacokinetic properties include moieties or groups that improve uptake, distribution, metabolism, or excretion of the compounds of the present disclosure. Representative moieties or groups are disclosed in PCT / US92 / 09196 and US 6,287,860. Moieties or groups include, but are not limited to, cholesterol moieties, cholic acid, thioethers (e.g., hexyl-S-tritylthiol), thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids (e.g., di-hexadecyl-lac-glycerol or triethylammonium 1,2-di-O-hexadecyl-lac-glycero-3-H-phosphonate), polyamine or polyethylene glycol chains, or lipid moieties such as adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0257] Chimeric Compounds It will be understood by those of skill in the art that not all positions in a given compound need be uniformly modified, and in fact more than one of the foregoing modifications may be incorporated in a single oligonucleotide, or even at a single nucleoside within an oligonucleotide.

[0258] The antisense compounds of the present disclosure include chimeric oligonucleotides. "Chimeric oligonucleotides" contain two or more chemically distinct regions, each consisting of at least one monomer unit (i.e., a nucleotide, in the case of an oligonucleotide compound). These oligonucleotides typically contain at least one region in which the oligonucleotide has been modified to confer increased resistance to nuclease degradation, increased cellular uptake, increased stability, and / or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Cleavage of RNA:RNA hybrids can be achieved in a similar manner through the action of endoribonucleases such as RNAse L, which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

[0259] The chimeric antisense compounds of the present disclosure can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, and / or oligonucleotide mimetics.Such compounds are also referred to in the art as hybrids or gapmers.Representative US patents that teach the preparation of such hybrid structures include, but are not limited to, US 5,013,830, US 5,149,797, US 5,220,007, US 5,256,775, US 5,366,878, US 5,403,711, US 5,491,133, US 5,565,350, US 5,623,065, US 5,652,355, US 5,652,356, and US 5,700,922.

[0260] Exemplary Oligonucleotides Exemplary antisense platforms known in the art include, but are not limited to, morpholino, first-generation oligo, second-generation oligo, gapmer, siRNA, LNA, BNA, or oligomimetic such as peptide nucleic acid. Oligonucleotides can be naked or formulated in liposomes. Oligonucleotides can be bound to or not bound to a delivery vehicle for cells. Oligonucleotides can be bound to or not bound to an endosomal release agent.

[0261] In one embodiment, the antisense compound is a second-generation phosphorothioate-backbone 2'-MOE-modified chimeric oligonucleotide gapmer designed to hybridize to the 3' untranslated region of VLA-4 mRNA. In one embodiment, the oligonucleotide selectively inhibits VLA-4 expression in both primary human cells and several human cell lines by hybridizing to RNA encoding VLA-4 and CD49, the α4 integrin subunit of the α4β7 integrin.

[0262] In one embodiment, the oligonucleotide is a 19-sodium salt of a 3'→5' phosphorothioate oligonucleotide (20-mer), also known as a 3-9-8 MOE gapmer, having a molecular weight of 7230 daltons, in which nucleotides 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) (2'MOE) modified ribonucleosides (2'-O-(2-methoxyethylribose)), nucleotides 4 to 12 from the 5' end are 2'-deoxyribonucleosides, all of which have 5-methylcytosines, and nucleotides 13 to 20 from the 5' end are 2'-O-(2-methoxyethyl) modified ribonucleosides.

[0263] In one embodiment, the sequence of the oligonucleotide is (SEQ ID NO:1): 5'- Me C Me UG AGT Me CTG TTT Me U Me C Me CAMe U Me U Me C Me U-3' (SEQ ID NO: 1).

[0264] The empirical formula of this oligonucleotide is: C 233 H 327 N 60 O 129 P 19 S 19 Na 19 is.

[0265] The ability of antisense oligonucleotides against the CD49d alpha chain of VLA-4 to selectively inhibit VLA-4 in immune cells prevents important safety events such as PML that are characterized by the administration of antibody and small molecule inhibitors of VLA-4, which are pan-VLA-4 inhibitors that affect all cells expressing VLA-4.

[0266] In one embodiment, all uracils are 5-methyluracil (MeU). Typically, oligonucleotides are synthesized using 2-methoxyethyl modified thymidine rather than 5-methyluracil.

[0267] In one embodiment, all pyrimidines are C5 methylated (ie, U, T, C are C5 methylated).

[0268] In one embodiment, the sequences of the oligonucleotides may each be named according to accepted oligonucleotide nomenclature, indicating the following OO-linked phosphorothioate internucleotide linkages: 2'-O-Methoxyethyl-5-methylcytidylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethylguanosilyl-(3'→5' O,O-phosphorothioyl)-2'-O-deoxyadenosilyl-(3'→5' O,O-phosphorothioyl)-2'-O-deoxyguanosilyl-(3'→5' O,O-phosphorothioyl)-thymidylyl-(3'→5' O,O-phosphorothioyl)-2'-deoxy-5-methylcytidylyl-(3'→5' O,O-phosphorothioyl)-thymidylyl-(3'→5' O,O-phosphorothioyl)-2'-deoxyguanosilyl-(3'→5' O,O-phosphorothioyl)-thymidylyl-(3'→5' O,O-phosphorothioyl)-thymidylyl-(3'→5' O,O-phosphorothioyl)-thymidylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridylyl-(3'→5' O,O-phosphorothioyl)-2'-methoxyethyl-5-methylcytidylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methylcytidylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-adenosilyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridylyl-(3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methylcytosine, (3'→5' O,O-phosphorothioyl)-2'-O-methoxyethyl-5-methyluridylyl-19 sodium salt.

[0269] RNA interference In some embodiments, nucleic acid therapeutic target inhibitors are siRNAs or miRNAs that act by RNA interference to reduce mRNA encoding a therapeutic target disclosed herein.

[0270] In some embodiments, the nucleic acid therapeutic target inhibitor is an siRNA against C5a. In some embodiments, the siRNA against C5a is semdisilane (Alnylam).

[0271] The term " RNA interference ", " RNAi " or " gene silencing " generally refers to the process that double-stranded RNA molecules reduce the expression of the nucleic acid sequence with which they share substantial or complete homology.However, it has been shown that non-RNA double-stranded molecules can also be used to achieve RNA interference (see, for example, US2007 / 0004667).

[0272] Nucleic acid molecules are typically RNA but can include chemically modified nucleotides and non-nucleotides.

[0273] The double-stranded region may be at least 19 contiguous nucleotides, e.g., about 19 to 23 nucleotides, or may be longer, e.g., 30 or 50 nucleotides, or 100 or more nucleotides. Full-length sequences corresponding to the entire gene transcript may be used. Preferably, they are about 19 to about 23 nucleotides in length.

[0274] The degree of identity of the double-stranded region of the nucleic acid molecule to the target transcript should be at least 90%, more preferably 95-100%. The nucleic acid molecule may, of course, contain unrelated sequences that may function to stabilize the molecule.

[0275] As used herein, the term "short interfering RNA" or "siRNA" refers to a nucleic acid molecule containing ribonucleotides that can inhibit or down-regulate gene expression, for example, by mediating RNAi in a sequence-specific manner, and the double-stranded portion is less than 50 nucleotides in length, preferably about 19 to about 23 nucleotides in length. For example, an siRNA can be a nucleic acid molecule containing self-complementary sense and antisense regions, where the antisense region contains a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. An siRNA can be assembled from two separate oligonucleotides, one strand being the sense strand and the other strand being the antisense strand, where the antisense and sense strands are self-complementary.

[0276] As used herein, the term siRNA is intended to be equivalent to other terms used to describe nucleic acid molecules that can mediate sequence-specific RNAi, such as microRNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotides, short interfering nucleic acids (siNA), short interfering modified oligonucleotides, chemically modified siRNA, and post-transcriptional gene silencing RNA (ptgsRNA). In addition, as used herein, the term RNAi is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, or epigenetics. For example, siRNA molecules can be used to epigenetically silence genes at both the post-transcriptional and pre-transcriptional levels. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules can be attributed to siRNA-mediated modification of chromatin structure to alter gene expression.

[0277] "shRNA" or "short hairpin RNA" refers to an RNA molecule in which less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, base pair with a complementary sequence located on the same RNA molecule, separated by an unpaired region of at least about 4 to about 15 nucleotides, such that the sequence and the complementary sequence form a single-stranded loop on the stem structure created by the two base-complementary regions.

[0278] Included shRNAs are dual or bi-finger and multi-finger hairpin dsRNAs, where the RNA molecule contains two or more of such stem-loop structures separated by a single-stranded spacer region.

[0279] Once designed, a nucleic acid molecule containing a double-stranded region can be produced by any method known in the art, for example, by in vitro transcription, recombinantly, or by synthetic means.

[0280] Nucleotide modifications or analogs can be introduced to improve the properties of nucleic acid molecules, including increased nuclease resistance and / or increased ability to permeate cell membranes. Thus, the terms "nucleic acid molecule" and "double-stranded RNA molecule" include, but are not limited to, synthetically modified bases such as inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl-, and other alkyl-adenines, 5-halouracil, 5-halocytosine, 6-azacytosine, and 6-azathymine, pseudouracil, 4-thiuracil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyladenine, 8-hydroxyladenine and other 8-substituted adenines, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine and other substituted guanines, other aza and deazaadenines, other aza and deazaguanines, 5-trifluoromethyluracil, and 5-trifluorocytosine.

[0281] Chemically modified siRNAs particularly suitable for in vivo delivery are described, for example, in WO2014 / 201306 and WO2007 / 051303. siRNAs targeting most human gene mRNAs are known in the art and / or commercially available. For example,

[0282] Aptamers In some embodiments, the nucleic acid therapeutic target modulator is an aptamer. In some preferred embodiments, the aptamer therapeutic target modulator is a therapeutic target inhibitor, whereby the aptamer therapeutic target inhibitor specifically binds to the therapeutic target and reduces the activity of the therapeutic target protein, for example, by reducing the interaction of the therapeutic target with a binding partner or receptor, or by interfering with enzymatic activity.

[0283] Aptamers, sometimes referred to as "therapeutic aptamers," are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules that bind to protein targets by folding into a three-dimensional structure similar to that of antibodies. Aptamers can be isolated by a method called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). One aptamer has been approved by the U.S. Food and Drug Administration (FDA) to treat macular degeneration in the eye, and numerous others are in preclinical or clinical trials. For a review, see Nimjee et al. (2017).

[0284] Protein-encoding nucleic acids In some embodiments, the nucleic acid therapeutic target modulator encodes a protein or peptide, whereby delivery of the nucleic acid (e.g., as an expression construct) to cells of a subject to be treated results in expression of the encoded protein or peptide therapeutic modulator after administration. In some embodiments, the encoded protein or peptide, when expressed in cells expressing the therapeutic target, functions as an inhibitor of the therapeutic target by inhibiting the interaction of the therapeutic target with one or more of its interaction partners, cognate receptors, or cognate ligands, or by functioning as a dominant-negative variant of the therapeutic target.

[0285] In some embodiments, the therapeutic target inhibitor comprises a nucleic acid in the form of an expression construct for expression of a protein or peptide that inhibits the activity of the therapeutic target.

[0286] In some embodiments, the expression construct is provided as a plasmid, optionally with a non-viral delivery vehicle or conjugate (e.g., an in vivo transfection agent). In other embodiments, the expression construct is provided for delivery in a recombinant virus for expression of the encoded protein.

[0287] In some embodiments, the therapeutic target inhibited is tumor necrosis factor receptor superfamily member 1A. In some embodiments, the nucleic acid inhibitor of tumor necrosis factor receptor superfamily member 1A is an expression construct encoding a fusion protein comprising the extracellular and transmembrane domains of human TNF receptor 1 and the intracellular domain of Fas receptor. In some embodiments, the just-described expression construct is provided in a recombinant virus. In some embodiments, the recombinant virus is VB-111 (Ofranergene obadenovec) (VBL Therapeutics, Israel), a replication-defective adenovirus 5.

[0288] In some embodiments, when the expression construct uses a modulator of therapeutic target activity, the modulator is an activator of the therapeutic target. In some preferred embodiments, the expression construct encodes a wild-type version of a therapeutic target protein to increase the expression level of the therapeutic target protein to compensate for a deficiency of the therapeutic target protein associated with NPASCs, as disclosed herein. In some preferred embodiments, the expression construct for activating a therapeutic target is directed to increasing the expression / activity of a therapeutic target selected from the following: (xiii) B cell receptor CD22, (xiv) antithrombin-III, (xv) protein farnesyltransferase / geranylgeranyltransferase type 1 subunit α, (xvi) amyloid A4 protein, (xvii) thyroid peroxidase, (xviii) IgG, (xix) palmitoyl protein thioesterase, and (xx) Growth hormone-releasing hormone (GHRH).

[0289] Those skilled in the art will appreciate that the expression vector can be delivered to the cells of the subject to be treated in vivo using any of several transfection methods known in the art, such as recombinant viral transduction, liposome-based transfection, electroporation, or nanoparticle-based transfection.

[0290] As used herein, an "expression vector" is a DNA or RNA vector capable of causing expression of one or more nucleic acids in a host cell. The vector is typically a plasmid or a recombinant virus. Any suitable expression vector can be used, including, but not limited to, a plasmid or a viral vector. In some embodiments, the viral vector is a retroviral, lentiviral, adenoviral, herpesviral, or adeno-associated viral vector.

[0291] Such vectors contain one or more promoters for expressing polynucleotides such as dsRNA for gene silencing. Suitable promoters include, but are not limited to, retroviral LTR, SV40 promoter, and human cytomegalovirus (CMV) promoter. Cellular promoters such as eukaryotic promoters, including, but not limited to, histone, RNA polymerase III (for shRNA or miRNA expression), and β-actin promoter, can also be used. The selection of a suitable promoter will be clear to those skilled in the art from the teachings contained herein.

[0292] In embodiments, some nucleic acids encode programmable nucleases that inhibit therapeutic target activity by inactivating or reducing expression of the corresponding therapeutic target gene when the nucleic acid therapeutic target inhibitor is administered. As used herein, the term "programmable nuclease" refers to a nuclease that is "targeted" ("programmed") to recognize and edit a predetermined genomic location. In some embodiments, the encoded polypeptide is a programmable nuclease that is "targeted" or "programmed" to introduce a genetic modification into the therapeutic target gene or its regulatory region. In some embodiments, the genetic modification is a deletion or substitution in the therapeutic target gene or its regulatory region.

[0293] In some embodiments, programmable nucleases can be programmed to recognize genomic locations through a combination of DNA-binding zinc finger protein (ZFP) domains. ZFPs recognize specific 3-bp in DNA sequences, and a combination of ZFPs can be used to recognize specific genomic locations. In some embodiments, programmable nucleases can be programmed to recognize genomic locations through transcription activator-like effector (TALE) DNA-binding domains. In alternative embodiments, programmable nucleases can be programmed to recognize genomic locations through one or more RNA sequences. In alternative embodiments, programmable nucleases can be programmed with one or more DNA sequences. In alternative embodiments, programmable nucleases can be programmed with one or more hybrid DNA / RNA sequences. In alternative embodiments, programmable nucleases can be programmed with one or more of an RNA sequence, a DNA sequence, and a hybrid DNA / RNA sequence.

[0294] Programmable nucleases that can be used in accordance with the present disclosure include, but are not limited to, RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-cas (CRISPR-associated) system, zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs), and Argonautes.

[0295] In some embodiments, the nuclease is an RNA-guided engineered nuclease (RGEN). In some embodiments, RGEN is derived from an archaeal genome or a recombinant version thereof. In some embodiments, RGEN is derived from a bacterial genome or a recombinant version thereof. In some embodiments, RGEN is derived from a type I (CRISPR)-cas (CRISPR-associated) system. In some embodiments, RGEN is derived from a type II (CRISPR)-cas (CRISPR-associated) system. In some embodiments, RGEN is derived from a type III (CRISPR)-cas (CRISPR-associated) system. In some embodiments, the nuclease is a class I RGEN. In some embodiments, the nuclease is a class II RGEN. In some embodiments, RGEN is a multi-component enzyme. In some embodiments, RGEN is a single-component enzyme. In some embodiments, RGEN is CAS3. In some embodiments, RGEN is CAS10. In some embodiments, RGEN is CAS9. In some embodiments, the RGEN is Cpf1 (Zetsche et al., 2015). In some embodiments, the RGEN is targeted by a single RNA or DNA. In some embodiments, the RGEN is targeted by two or more RNAs and / or DNAs. In some embodiments, the programmable nuclease can be a DNA-programmed Argonaute (WO14 / 189628).

[0296] In other embodiments, the nucleic acid therapeutic target modulator, i.e., either a therapeutic target inhibitor or a therapeutic target activator, is a synthetic, chemically modified mRNA. In the case of a nucleic acid therapeutic target inhibitor, the modified mRNA encodes a protein that inhibits the therapeutic target of interest. In other embodiments, when a nucleic acid therapeutic target activator is used, the modified mRNA encodes a therapeutic target protein and increases its expression level in the treated subject, at least in cells that normally endogenously express the therapeutic target protein. Chemically modified mRNAs and their synthesis are described in detail, for example, in WO 2011 / 130624. Typically, chemically modified mRNAs contain (i) a 5' synthetic cap to enhance translation, (ii) modified nucleotides that confer RNAse resistance and an attenuated cellular interferon response or otherwise significantly reduce translation efficiency, and (iii) a 3' polyA tail. Typically, chemically modified mRNAs are synthesized in vitro from a DNA template containing an SP6 or T7 RNA polymerase promoter operably linked to an open reading frame encoding the protein to be expressed. The synthesis reaction of chemically modified mRNA is carried out in the presence of a mixture of modified and unmodified nucleotides. In some embodiments, the modified nucleotides included in the in vitro synthesis of chemically modified mRNA are pseudouridine and 5-methylcytosine. A key step in cellular mRNA processing is the addition of a 5' cap structure, which is a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide. The cap is enzymatically methylated at the N-7 position of guanosine to form mature mCAP. When preparing chemically modified mRNA, the 5' cap is typically added before administration / in vivo transfection to stabilize the modified mRNA and significantly enhance translation. In some embodiments, a 4:1 mixture of cap analog and GTP is used in the transcription reaction to obtain 5'-capped chemically modified mRNA. In a preferred embodiment, the anti-reverse cap analog (ARCA), 3'-O-Me-m7G(5')ppp(5')G, is used to generate chemically modified mRNA that can be efficiently translated in human cells.Systems for in vitro synthesis are commercially available, as exemplified by the mRNAExpress™ mRNA Synthesis Kit (System Biosciences, Mountain View, Calif.). The synthesis and use of such modified RNAs for in vitro and in vivo transfection are described, for example, in WO2011 / 130624 and WO2012 / 138453.

[0297] Recombinant viruses A variety of recombinant virus types are suitable for expressing the therapeutic inhibitors or therapeutic target inhibitors disclosed herein. Preferably, the recombinant virus used is a replication-defective recombinant virus.

[0298] In some embodiments, the administered recombinant virus is a DNA virus. Suitable types of DNA viruses include adenoviruses, adeno-associated viruses (AAVs), herpes simplex viruses (HSVs), retroviruses, and lentiviruses. Methods for designing, producing, and using such types of recombinant DNA viruses have been established in the art, and are exemplified in Fukazawa et al. (2010) and "Gene Therapy Protocols" for noviruses, "Adeno-Associated Virus: Methods and Protocols" for AAVs, "Cody et al. (2013) and "Herpes Simplex Virus: Methods and Protocols" for HSVs, "Gene Therapy Protocols Vol. 1: Production and In Vivo Applications of Gene Transfer Vectors" and Amer (2014) for retroviruses, and Merten et al. (2016) and Emeagi et al. (2013) for lentiviruses. In some preferred embodiments, the recombinant virus used in the therapeutic methods is an adenovirus, hi other preferred embodiments, the recombinant virus is a lentivirus.

[0299] In other embodiments, the administered recombinant virus is a recombinant, replication-defective RNA virus, suitable for use in replication-defective or replication-competent RNA viruses, including alphaviruses (e.g., Sindbis or Semliki Forest viruses), flaviviruses (e.g., Kunjin virus), paramyxoviruses (e.g., Sendai virus), rhabdoviruses (e.g., vesicular stomatitis virus), and orthomyxoviruses (e.g., influenza A virus). Methods for the design, production, and use of these types of recombinant RNA viruses are established in the art and are exemplified by Lundstrom (2015) and Quetglas et al. (2010) for alphaviruses, Hoang-Le et al. (2009) and Usme-Ciro et al. (2013) for flaviviruses, Cattaneo (2010) for paramyxoviruses, Finke et al. (205) and Chang et al. (2010) for rhabdoviruses, and US 8,475,806 for orthomyxoviruses.

[0300] Examples of suitable promoters for driving expression of biotherapeutic agents from recombinant viruses in the methods described herein include, but are not limited to, constitutive promoters such as the CMV, CAG, EF-1-I, HSV1-TK, SV40, β-actin, and PGK promoters. In other embodiments, the promoter is an inducible promoter, such as one containing a TET operator element. In certain embodiments, target-selective promoters are used to drive expression of biotherapeutic agents in vivo in specific cell types of the subject being treated.

[0301] In some embodiments, when two or more proteins (e.g., two different therapeutic targets or two different isoforms of a therapeutic target) are expressed from a recombinant virus, the recombinant virus contains an expression cassette encoding a polycistronic mRNA (a "polycistronic expression cassette"), which, when translated, results in independent polypeptides containing different amino acid sequences or functions. In some embodiments, the polycistronic expression cassette encodes a "polyprotein" containing multiple polypeptide sequences separated by encoding 2A peptide sequences from picornaviruses (e.g., foot-and-mouth disease virus (FMDV)). The 2A peptide sequence acts cotranslationally by preventing the normal peptide bond formation between the conserved glycine and the final proline, causing the ribosome to skip to the next codon and cleaving the nascent peptide between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the "upstream" protein, while a proline is added to the N-terminus of the "downstream" protein. This allows the nascent polypeptide sequence to be cleaved into separate polypeptides during translation. See, e.g., Trichas et al. (2008).

[0302] In other embodiments, the polycistronic expression cassette may incorporate one or more internal ribosome entry site (IRES) sequences between the open reading frames incorporated into the polycistronic expression cassette. IRES sequences and their uses are known in the art, for example, as exemplified in Martinez-Sales (1999).

[0303] In some embodiments, the recombinant viruses used in this method have targeted tropism (e.g., tropism for a specific cell type), as outlined in Bucholz et al. (2015). Suitable targeting moieties for incorporation into the surface of recombinant viral capsids include ligands that bind to cell surface receptors overexpressed by cancer cells. For example, in AAV (Munch et al., 2013), the Her2 / neu receptor, which is frequently overexpressed in breast cancer cells, can be targeted by incorporating a designed ankyrin repeat protein (DARPin) ligand, as has been done for lentiviruses (Munch et al., 2011). In another example, recombinant lentiviruses are engineered to target P-glycoprotein, which is overexpressed on the surface of melanoma cells, by incorporating an antibody into the viral capsid surface (Morizono et al., 2005).

[0304] Administration and Dosing Regimen of NPASC Therapeutic Target Modulators In some embodiments, a method of treating a human subject identified as suffering from NPASC or a method for preventing such a condition comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of at least one inhibitor or activator of a therapeutic target disclosed herein, or a pharmaceutically acceptable salt, pharmaceutically acceptable N-oxide, pharmaceutically active metabolite, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof.

[0305] Therapeutic target inhibitors or therapeutic target activators are administered to prevent, cure, or at least partially arrest symptoms in patients already suffering from and / or diagnosed with NPASC. The therapeutically or prophylactically effective amount will depend on the severity and course of one or more NPASC symptoms, the patient's health status, COVID status, weight, and initial response to treatment. Determining such therapeutically effective amounts by routine experimentation (including, but not limited to, dose escalation clinical trials) is considered well within the skill of one of ordinary skill in the art.

[0306] In prophylactic applications, the therapeutic target modulators disclosed herein are administered to patients identified as susceptible to or otherwise at high risk of developing NPASC. Such an amount is defined as a "prophylactically effective amount or dose," i.e., a dose sufficient to prevent or reduce infection. In this use, the precise amount will also depend on the specific condition, the patient's health, weight, timing, etc. It is considered well within the skill of one of ordinary skill in the art to determine such a prophylactically effective amount by routine experimentation (e.g., a dose-escalation clinical trial).

[0307] If the subject's condition does not improve, based on reliable medical advice, administration of the NPASC therapeutic target modulator may be given continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily discontinued for a period of time (i.e., a "drug holiday"). The length of the drug holiday may vary from 2 days to 1 year, including, by way of example only, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, or 60 days. The dose reduction during the drug holiday may be 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0308] The amount of a given NPASC therapeutic target modulator (i.e., inhibitor or activator) that would be suitable as a therapeutically effective dose will vary depending on factors such as the type and potency of the therapeutic target modulator administered, the severity / stage of NPASC, the characteristics (e.g., weight) of the subject or host requiring treatment, and previous or concurrent treatments, but may nevertheless be routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, for example, the specific agent administered, the route of administration, the condition being treated, and the subject or host being treated. In general, however, doses used for adult human treatment typically range from 0.02 to 5,000 mg per day, or for small molecule therapeutics, from about 1 to 1,500 mg per day. The desired dose may be provided in a single dose or as divided doses administered simultaneously (or closely spaced), or at appropriate intervals, e.g., two, three, four, or more times per day.

[0309] In some embodiments, the administration of a therapeutic target modulator will be based on the determined level of the therapeutic target to be modulated in the subject. For example, if the therapeutic target modulator administered is a therapeutic target inhibitor, the dose of the therapeutic target inhibitor administered to a first subject to be treated will be higher than that of a second subject to be treated if the first subject expresses a higher level of the therapeutic target to be inhibited than that of a second subject to be treated, assuming all other factors to be considered (e.g., subject's body weight) are the same or similar. On the other hand, if the therapeutic target modulator administered is a therapeutic target activator, the dose of the therapeutic target activator administered to a first subject to be treated will be higher than that of a second subject to be treated if the first subject expresses a lower level of the therapeutic target to be activated than that of a second subject to be treated, assuming all other factors to be considered (e.g., subject's body weight) are the same or similar.

[0310] In some embodiments, the methods of treatment disclosed herein include determining the level of the therapeutic target level or activity level of the therapeutic target to be modulated.

[0311] The aforementioned ranges are merely suggestions, as there are many variables associated with each individual treatment regime, and significant deviations from these recommendations are not uncommon. Such dosages may vary depending on several variables, including, but not limited to, the activity of the NPASC therapeutic target modulator used, the severity of one or more NPASC symptoms being treated, the mode of administration, and the judgment of the practitioner.

[0312] The toxicity and therapeutic efficacy of such treatment regimens are determined by the LD 50 (lethal dose in 50% of the population) and ED 50 These can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determination of the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD 50 and ED 50 Therapeutic indexes can be expressed as a ratio between ED and . NPASC therapeutic target inhibitors or activators that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in human and non-human subjects. The dosage of such compounds is preferably ED 1000 or ED 1000 with minimal toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0313] Therapeutic formulation Any of the modulators of NPASC therapeutic targets disclosed herein may be formulated, either alone or in combination with pharmaceutical compositions, for administration to a human subject via any conventional means, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intrapleural), oral, intranasal, or transdermal routes of administration.

[0314] Therapeutic agents may be formulated into any suitable dosage form, including, but not limited to, injectable formulations, aqueous oral dispersions for oral ingestion by the subject to be treated, liquids, mists, gels, syrups, elixirs, slurries, suspensions and the like, solid oral dosage forms, controlled release formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0315] Pharmaceutical preparations for oral use can be prepared by mixing one or more solid excipients with one or more of the therapeutic agents described herein, optionally adding suitable excipients, and then milling the resulting mixture and processing the granular mixture to obtain tablets or dragee cores. Suitable excipients include fillers (e.g., sugars including lactose, sucrose, mannitol, or sorbitol), cellulose preparations (e.g., corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), or others (e.g., polyvinylpyrrolidone (PVP or povidone) or calcium phosphate, etc.). If necessary, disintegrants such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate) may be added.

[0316] Pharmaceutical solid dosage forms can comprise, in addition to a therapeutic agent, one or more pharmaceutically acceptable excipients such as a compatible carrier, binder, filler, suspending agent, flavoring agent, sweetener, disintegration agent, dispersing agent, surfactant, lubricant, coloring agent, diluent, solubilizing agent, humectant, plasticizer, stabilizer, penetration enhancer, wetting agent, antifoaming agent, antioxidant, preservative, or one or more combinations thereof.

[0317] Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol, and the like.

[0318] Suitable fillers for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0319] Disintegrants are often used in formulations to release the therapeutic agent from the solid dosage form matrix as efficiently as possible, especially when the dosage form is compressed with a binder. Disintegrants help break down the dosage form matrix by swelling or capillary action when moisture is absorbed into the dosage form. Suitable disintegrants for use in the solid dosage forms described herein include natural starches such as corn starch or potato starch, pregelatinized starches such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, celluloses such as wood products, methylcrystalline cellulose, e.g., Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Examples of suitable crosslinking agents include, but are not limited to, Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or crosslinked cellulose (e.g., crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®), crosslinked carboxymethylcellulose, or crosslinked croscarmellose, crosslinked starch (e.g., sodium starch glycolate), crosslinked polymers (e.g., crospovidone), crosslinked polyvinylpyrrolidone, alginates (e.g., alginic acid or alginates), clays (e.g., Veegum® HV (magnesium aluminum silicate)), gums (e.g., agar, guar, carob, karaya, pectin, or tragacanth), sodium starch glycolate, bentonite, natural sponge, surfactants, resins (e.g., cation exchange resins), citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combined starches, and the like.

[0320] Binders impart cohesiveness to solid oral dosage form formulations: in powder-filled capsule formulations, they aid in the formation of a plug that can be filled into soft or hard shell capsules, and in tablet formulations, they ensure that the tablet remains intact after compression and help to ensure blend uniformity before the compression or filling step. Materials suitable for use as binders in the solid dosage forms described herein include carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropyl methylcellulose (e.g., Hypromellose USP Pharmacoat-603), hydroxypropyl methylcellulose acetate stearate (Aqoate HS-LF and HS), hydroxyethyl cellulose, hydroxypropyl cellulose (e.g., Klucel®), ethyl cellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acid, bentonite, gelatin, polyvinylpyrrolidone / vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, sugars (e.g., sucrose (e.g., Dipac®), glucose, dextrose, Examples of suitable sucrose-containing gums include, but are not limited to, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), lactose), natural or synthetic gums (e.g., acacia, tragacanth, ghatti gum), isapol shell mucilage, starch, polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone® XL-10, and Povidone® K-12), larch arabinogalactan, Veegum®, polyethylene glycol, wax, sodium alginate, and the like.

[0321] Powder-filled gelatin capsule formulations typically use binder levels of 20-70%. Binder levels in tablet formulations can be determined by direct compression, wet granulation, roller compaction, or the use of other excipients, such as fillers, that can act as moderate binders themselves. Those skilled in the art can determine the binder level for their formulations, but binder levels of up to 70% are common in tablet formulations.

[0322] Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumarate, alkali metal and alkaline earth metal salts (e.g., aluminum, calcium, magnesium), zinc, stearic acid, sodium stearate, magnesium stearate, zinc stearate, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol or methoxypolyethylene glycol (e.g., Carbowax™, PEG4000, PEG5000, PEG6000), propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

[0323] Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrins), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins, and the like.

[0324] Suitable humectants for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodium oleate, sodium lauryl sulfate, magnesium stearate, docusate sodium, triacetin, vitamin E TPGS, and the like.

[0325] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide (e.g., Pluronic® (BASF)), and the like.

[0326] Suitable suspending agents for use in the solid dosage forms described herein include polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol (e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), vinylpyrrolidone / vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinyl ... These include, but are not limited to, resorbate-80, hydroxyethylcellulose, sodium alginate, gum acanthia, gums (e.g., tragacanth gum, acacia gum, guar gum, xanthan (including xanthan gum)), sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

[0327] It should be understood that there is considerable overlap between the additives used in the solid dosage forms described herein.Therefore, the additives listed above should be considered as merely exemplary and not limiting of the types of additives that can be included in the solid dosage forms described herein.The amount of such additives can be easily determined by those skilled in the art according to the specific properties desired.

[0328] The liquid pharmaceutical dosage form for oral administration may be an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

[0329] The aqueous suspensions and dispersions described herein can remain homogeneous for at least 4 hours, as defined in the USP Pharmacists' Pharmacopeia (2005 Edition, Chapter 905). Homogeneity should be determined by a consistent sampling method for determining the homogeneity of the entire composition. In one embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 1 minute. In another embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 30 seconds. In yet another embodiment, no agitation is required to maintain a homogeneous aqueous dispersion.

[0330] In addition to the additives listed above, the liquid formulation can also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers.Exemplary emulsifiers include ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, sodium lauryl sulfate, sodium docusate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils (e.g., cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, or mixtures of these substances.

[0331] Injectable preparations Formulations suitable for intramuscular, subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection may also contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0332] For intravenous injection, the therapeutic agents described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, suitable formulations may include aqueous or non-aqueous solutions, preferably containing physiologically compatible buffers or excipients. Such excipients are generally known in the art.

[0333] Parenteral injections may involve bolus injection or continuous infusion. Injectable formulations may be provided in unit dosage form, e.g., in ampoules or multi-dose containers, with an added preservative. The pharmaceutical compositions described herein may be in a form suitable for parenteral injection as sterile suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the therapeutic agent in water-soluble form. Additionally, suspensions of the therapeutic agent may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the therapeutic agent, allowing for the preparation of highly concentrated solutions. Alternatively, the therapeutic agent may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. The therapeutic agents described herein may be in unit dosage forms suitable for single administration of precise dosage amounts. In unit dosage forms, the formulation is divided into unit doses containing appropriate amounts of one or more therapeutic agents. The unit dosages may be in the form of packages containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose non-reclosable containers. Alternatively, multi-dose non-reclosable containers may be used, in which case the composition typically contains a preservative. By way of example only, formulations for parenteral injection may be provided in unit dosage forms, including, but not limited to, ampoules or multi-dose containers, with an added preservative. [Example]

[0334] Example 1 Proteomic analysis of 7300 plasma proteins The majority of plasma samples were initially obtained from subjects enrolled in a previous study of Neuro-COVID long-haulers at the Northwestern Medicine Neuro-COVID Clinic (Chicago, USA), as reported by Visvabharathy et al. (www.medrxiv.org / content / 10.1101 / 2021.08.08.21261763) entitled "Neuro-COVID long-haulers exhibit broad dysfunction in T cell memory generation and responses to vaccination." (The "Northwestern Study") The Northwestern Study was designed to compare humoral and cellular immune responses in NPASC subjects with those found in healthy COVID convalescent patients and healthy controls. A total of 111 prevaccinated participants comprised outpatients and non-hospitalized patients. Of the 56 NPASC subjects, 48 ​​were not hospitalized for pneumonia or hypoxemia, and all had NPASC symptoms for at least 6 weeks after infection, and for an average of approximately 4 to 6 months. Some subjects received vaccinations during the course of the reported study. The demographics and neurological symptoms of the patients used in the above reported study are tabulated and reproduced here for reference purposes in Table 3. [Table 4]

[0335] Plasma samples from the Northwestern study were kindly provided by Dr. Igor Koralnik of the Northwestern Medicine Neuro-COVID Clinic (Chicago, USA / Northwestern University). A commercially available aptamer-based proteomics assay, SomaScan®, was used herein to analyze patient heparinized plasma samples and determine the relative fluorescence units (RFU) of over 7,300 proteins for three patient groups: Group 1: 48 patients currently suffering from Neuro-PASC (NP) who had never been hospitalized with COVID (positive PCR or IgG antibody test). 42 of the 48 subjects were from the Northwestern study mentioned above, plus 6 plasma samples from eligible subjects. Group 3: 20 patients (convalescent controls) who recovered from SARS-CoV-2 infection and did not show symptoms of PASC or NPASC, but had never been hospitalized with COVID (positive PCR or IgG antibody test). Seventeen of the 24 patients were from the Northwestern study mentioned above, plus three eligible controls. Group 4: 24 healthy control (HC) subjects (patients never infected with SARS-CoV-2). Plasma samples were from 24 of the 31 HC patients from the Northwestern study described above.

[0336] Thus, the plasma samples for this study consisted of 83 samples (out of 103) from groups 1, 3, and 4 from the Visvabharathy et al. study (supra), further selected from non-hospitalized and non-vaccinated subjects. Six and three additional samples were included from groups 1 and 3, respectively, for a total of 92 samples. Group 2 samples from the study reported at Northwestern Hospital were from hospitalized subjects with NPASC and were not included. One patient sample was obtained from another site, designated CL-2, and designated study group 2, but was actually non-hospitalized. Therefore, this was added to the 47 subjects present in group 1, resulting in 48 subjects, all designated NP (hereafter referred to as group 1, but the pooled group is shown as group 1+2 in the plots).

[0337] Multiplexed proteomics assays are performed with the SomaScan assay using approximately 7,300 aptamers (called SOMAmers). Briefly, in an illustrative example, a sample is incubated with a mixture of SOMAmers, each containing biotin, a photocleavable group, and a fluorescent tag, followed by capture of all SOMAmer-protein complexes on streptavidin beads. The beads are stringently washed to remove unbound proteins, and after labeling bead-associated proteins with biotin under controlled conditions, the complexes are released from the beads into solution by UV light irradiation and diluted into a high concentration of anionic competitor, dextran sulfate. The biotin originally part of the SOMAmers remains on the beads. In combination with dilution, the anionic competitor selectively disrupts non-cognate complexes; since only the proteins now contain biotin, the complexes are recaptured on a second set of beads, and unbound SOMAmers are removed by a second stringent wash. The SOMAmers, which remain bound to the beads, are eluted under high pH denaturing conditions and hybridized to sequence-specific complementary probes printed on standard DNA microarrays.

[0338] The result is a mixture of SOMAmers that quantitatively reflects the protein concentration in the original sample. The modified nucleotides in the SOMAmers are designed to maintain canonical base pairing (in the DNA duplex, the 5-position pyrimidine addition is oriented toward the major groove of the DNA) and hybridize effectively to unmodified DNA oligonucleotides on the array. Capture of the SOMAmers on the hybridization array allows for quantitative determination of the protein present in the original sample by converting the assay signal (relative fluorescence units, RFU) to analyte concentration. Thus, the Somascan assay exploits the dual nature of SOMAmer aptamers as molecules capable of both folding into complex three-dimensional structures—the basis of their unique binding properties—and hybridizing to specific capture probes.

[0339] Example 2 Identification of proteins for statistical analysis and likelihood testing The results of the levels in the different groups, NP (also referred to as Group 1 and Group 2, or simply Group 1), CC in Group 3, and HC in Group 4, were analyzed using both parametric and nonparametric statistical analyses. ANOVA and Kruskalis-Wallis (KW) statistical analysis were used to compare NP in Group 1, CC in Group 3, and HC in Group 4, and p-values ​​were adjusted using the Benjamini-Hochberg false discovery rate (FDR) and the highly stringent Bonferroni statistical test. Proteins of interest were evaluated using Somalogic®'s Dataviz software. Plasma proteins with a Bonferroni-adjusted value of <0.05 were identified (see Table 2). Plasma proteins with an FDR of <0.02 were identified. The median percent change in NP in Group 1 relative to convalescent control (CC) and healthy control (HC) patients was calculated for proteins of interest that met statistical significance. The median ANOVA results and Bonferroni and FDR results for the biomarkers are shown in Table 2. These proteins were entered into Genetrail 3 to identify associated pathways. The identified protein biomarkers had various functions in neuronal, autoimmune, viral, vascular, adipose, and coagulation pathways, as well as in inflammation and fibrosis, integrin, and antigen presentation pathways.

[0340] Using both parametric and non-parametric statistical analysis, the results of levels in the different groups, NP (also referred to as Group 1 and Group 2, or simply Group 1), were also analyzed compared to Group 3 (CC), and NP was also analyzed compared to Group 4. T-test, median results between NP in Group 1 versus CC in Group 3 and between NP in Group 1 versus HC in Group 4 are also shown in Table 2. T-test and U-test analyses were performed with p-values ​​adjusted using Benjamini-Hochberg false discovery rate (FDR) and Bonferroni statistical tests, and proteins of interest were identified using Dataviz software from Somalogic®.

[0341] No statistical differences were found when comparing groups 3 + 4 using FDR or Bonferroni adjusted p-value tests. Therefore, T-tests and U-tests, ANOVA and KW were used to determine the median results between group 1 NPs versus group 3 and group 4 controls (Table 2).

[0342] Table 1 shows proteins found to have different circulating levels, derived from three different statistical analyses to identify key molecules associated with NPASCs. Whisker plots of the circulating levels of each of the indicated biomarkers (1–11) in the NP, CC, and HC groups are shown in Figures 1–11.

[0343] Example 3 Exemplary Biomarker Combinations In another aspect, the present invention provides a combination of two or more biomarkers identified herein to assay for NPASC potential in a subject or sample, or to monitor a subject's progression to NPASC, such as a subject with Covid or NPASC or a subject undergoing therapy.

[0344] The method comprises: The method includes measuring (i) the level of gliomedin, and (ii) the level of C5a anaphylatoxin, and / or (iii) the level of TGF beta 1, and / or (iv) the level of Gal3ST1. C5a and TGF beta 1 are two markers of inflammation and fibrosis. The combination of markers enhances the sensitivity of the method in a population and detects more subjects with NPASC. Preferably, the NPASC or NPASV sample is compared with the gliomedin level, TGF beta 1, and C5a levels of an HC reference, although the levels can also be compared with those of a CC reference. Preferably, the NPASC or NPASV sample is compared with the Gal3ST1 level of a CC reference.

[0345] In this way, the status of all subjects with one or more symptoms of NPASC could potentially be verified with a combination of markers to ensure time to recovery, progress treatment, and provide evidence to employees and insurance companies.

[0346] To maximize specificity, it is useful to measure levels of all 11 biomarkers listed in Table 1, especially in convalescent subjects who have recovered from Covid (as opposed to subjects whose Covid status has not yet been determined).

[0347] Example 4 Therapeutic target analysis Among the proteins identified as having different levels between NPASCs and control groups based on the statistically stringent criteria described above, a subset was filtered / prioritized as therapeutic targets based on: (1) the percentage difference between Group 1 and Groups 3+4, (2) the direction of the difference (higher or lower levels), (3) Genetrail3-related pathway analysis summarized in Table 5 below, and (4) the presence of at least one therapeutic agent (approved or in clinical trials) against the target or target pathway. [Table 5]

[0348] As part of the pathway analysis, a statistical test of FDR<0.02 was performed to identify pathways (the number of "hits" identified was statistically significant). This pathway statistical analysis is summarized in Table 6 below. [Table 6-1] [Table 6-2]

[0349] A set of therapeutic target proteins was identified based on this analysis, as summarized in Table 7 below. This includes proteins found to have higher levels in Group 1 (NPASC subjects) compared to Group 3 (healthy control subjects) and therapeutic agents that modulate the therapeutic target, as well as therapeutic target proteins found to have lower levels in Group 1 compared to Group 3. Figures 12 and 14 show exemplary therapeutic target proteins CD33, VEGFD, and TGF-β1 that were found to have higher levels in the plasma of NPASC subjects than in control subjects. Figure 13 shows exemplary target proteins thyroid peroxidase and amyloid A4 that were found to have lower levels in the plasma of NPASC subjects than in control subjects. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0350] Example 5 Phase 2 Clinical Trial of ATL1102 to Treat NPASC (Prophetic Example) The Phase 2 study will evaluate the potential efficacy and safety of ATL1102, administered subcutaneously at a dose of 25 mg once weekly for 24 weeks, in subjects experiencing non-hospitalized non-patient-specific respiratory syndrome (NPASC) (≥12 weeks but <6 months after COVID-19). The Phase 2 study is a randomized, double-blind, placebo-controlled study in patients with NPASC and cognitive impairment. Patients and researchers will not know whether they are receiving a placebo or 25 mg of ATL1102 per week. Treatment will be self-administered subcutaneously at home, with subjects returning to the clinic every 4 weeks. The treatment-to-placebo ratio is 1:1 for a total of 34 subjects, both male and female.

[0351] Outcome measures at baseline and at weeks 6 (day 42), 12 (day 84), 18 (day 126), and 24 (day 168).

[0352] Primary outcome measure - Change from baseline to endpoint (weeks 12 and 24) in selected cognitive tests 1. Baseline Digit Symbol Substitution Test (DSST) CogState Online Cognitive Battery 2. The Trail Making Test (TMT)-A / B is used to assess changes in cognitive function. 3. The Rey Auditory Verbal Learning Test (RAVLT) is used to assess changes in verbal memory. 4. Assess subjective changes in cognitive function using the Perceived Deficit Change Questionnaire, 20 items (PDQ-20). 5. Change from baseline in PROMIS® daily cognitive function score 6. Change from Baseline in the NIH Toolbox Test: Attention, Processing Speed, Executive Function, and Working Memory Impairments 7. Diary of long-term NPASC-related symptom severity scores during the treatment phase 8. Number of symptom-free days for NPASC-related symptoms that were present at the start of study treatment (Day 0), based on self-assessment using a daily symptom diary. Symptoms scored as mild, moderate, or severe at baseline will be scored as absent (or none). 9. Progression (or worsening) of NPASC throughout the day a. Moderate at baseline is scored as severe throughout the day b. Mild at baseline is scored as moderate or severe throughout the day c. Baseline absence is scored as mild, moderate, or worsening throughout the day

[0353] Secondary outcome measures - Change from baseline to endpoint (weeks 12 and 24) selected from fatigue and sleep studies 1. Use the Fatigue Severity Scale (FSS) to assess changes in the severity and impact of fatigue. Prevalence of fatigue in patients with depression and / or anxiety post-COVID-19 2. The mental fatigue questionnaire score is a self-reported measure. 3. Change from baseline in daily PROMIS® fatigue score 4. Change from baseline in PROMIS® daily sleep disturbance score 5. Change from baseline in PROMIS-57 patient-reported T-scores - physical function, anxiety, depression, fatigue, sleep disturbance, pain interference, and overall pain score

[0354] Other outcome measures: Exploratory 1. Changes from baseline in CD3+CD4+CD49d+ and CD3+CD8+CD49d+ T cell counts, and CD3- NK and B cell counts 2. Changes from baseline in transforming growth factor beta 1 (TGF beta 1), thrombospondin-1, and LTBP4; protein levels and / or activity levels. 3. Daily change in CRP from baseline 4. Changes from baseline in daily serum cytokine and chemokine levels 5. Plasma biomarkers that can predict and / or affect pharmacodynamic indices of pharmacological activity: drug versus placebo (SomaScan®)

[0355] Inclusion Criteria: 1. Subjects >18 years of age at the time of signing the informed consent form. 2. Male or female with NPASC who have cognitive impairment (e.g., confusion, difficulty thinking, decreased attention, impaired executive function, and memory) and potentially neurological symptoms of one or more of the following: headache, paresthesia (tingling and numbness), taste disorder, smell disorder, muscle pain, post-traumatic stress disorder (PTSD), sleep disorder, anxiety, and depression, and fatigue. 3. Non-hospitalized post-COVID-19 patients with proven SARS-CoV-2 positivity and RT-PCR confirmed. 4. Subjects experiencing prolonged neurological symptoms (e.g., cognitive impairment, fatigue) for 12 weeks or more after a negative COVID-19 RT-PCR test. 5. Subjects who have not fully recovered from NPASC for at least 12 weeks despite a negative SARS-COV-02 test. 6. Subjects who have been experiencing symptoms that interfere with normal daily activities for at least 12 weeks. Symptoms must be new, i.e., the subject was not receiving medical treatment for the symptoms prior to COVID-19: Extreme fatigue - low energy, not getting enough sleep despite a strong need for sleep, and feeling overworked. Cognitive impairment

[0356] Exclusion criteria: 1. Test positive for SARS-CoV-2 infection (acute infection) at screening, including by nasal swab specimen or another FDA-approved test. 2. Subjects who have fully recovered from COVID-19 and have new onset of extreme symptoms not attributable to COVID-19. 3. Subjects with serious comorbidities will be excluded. For example: o Liver enzymes are >2x ULN. eGFR <60 ml / min according to the CKD-EPI formula Hb is <11 mg / dL. o Platelet count is <100K. ○ Lower than normal blood count 4. History of neurological disorders before COVID-19 infection. 5. PASC affecting musculoskeletal, gastrointestinal, pulmonary, and neurological PASC (NPASC).

[0357] Those skilled in the art will appreciate that numerous variations and / or modifications can be made to the present invention, as illustrated in the specific embodiments, without departing from the spirit or scope of the invention as broadly described. For example, in a study, subjects with elevated TGF-beta1 levels above a threshold, who have been diagnosed as possibly suffering from NPASC based on the disclosed methods, are treated with ATL1102. For example, in a crossover study, after the first six months of treating patients with a placebo, the patients are treated with ATL1102 for six months, and the patients treated with ATL1102 for the first six months are treated with a placebo for the next six months. Therefore, the present embodiments should be considered in all respects to be illustrative and not restrictive.

[0358] It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the present invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0359] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0360] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of these matters form part of the prior art or were common general knowledge in the art relevant to the present invention as existing before the priority date of each claim of this application.

[0361] References Visvabharathy et al.: / / www.medrxiv.org / content / 10.1101 / 2021.08.08.21261763. Graham E.L and Koralnik I.J et al.“Persistent neurological symptoms and cognitive dysfunction in non-hospitalized COVID-19 “Long Haulers” Ann. Clinical and Translational Neurol 2021, 8(5):1073-1085 Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999 Rose et al., A Laboratory Course Manual, Cold Spring Harbor Laboratory, Cold Spring Hampshire et al (EClinical Medicine 2021 Sept; 39:10144 Pinzon et al.J.Infection and Public Health 2022; 15: 856 -860 Gold L, Ayers D, Bertino J et al.Aptamer based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5(12), e15004 (2010) Gold L, Walker JJ, Wilcox SK, Williams S. Advances in human proteomics at high scale with the SOMAscan proteomics platform. N. Biotechnol. 29(5), 543-549 (2012). Eaton BE. The joys of in vitro selection: chemically dressing oligonucleotides to satiate protein targets. Curr. Opin. Chem.Biol.1(1), 10-16 (1997) Vaught JD, Bock C, Carter J et al.Expanding the chemistry of DNA for in vitro selection. J.Am. Chem.Soc. 132(12), 4141-4151 (2010). Wild D. “The Immunoassay Handbook” Nature Publishing Group, 4 th Edition, 2013 Sousa-Pereira et al., (2019) Antibodies 8(4): 57 Remington’s Pharmaceutical Sciences, 1990 The Proteomics Protocols Handbook Ed John M. Walker Humana Press Inc, 2005 Proteomic and Metabolomic Approaches to Biomarker Discovery 2 nd Edition, Eds. Haleem Issaq, Timothy Veenstra, 2019 Academic Press ISBN:9780128186077 Ausubel (Ed) Current Protocols in Molecular Biology, 5th Edition, John Wiley & Sons, Inc, NY, 2002. Rundstrom, G et al.Whole Blood. Clinical Chemistry 53, 342-348 (2007) Glorio-Paulet et al J Agric Food Chem 48 (5):1678-1682, 2000 S. Geisse, B. Voedisch Methods Mol.Biol., 899 (2012), pp.203-219 Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; European Journal of Immunology 6: 511-519, 1976 Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Edition, CSHLP, CSH, NY, 2001

Claims

1. A method for determining the likelihood of neurological acute post-acute sequelae (NPASC) of COVID-19 in a subject, comprising measuring or having measured the level of at least one of the biomarkers: C5a anaphylatoxin (C5a) or gliomedin in a biological sample from the subject.

2. The method according to claim 1, comprising measuring or having measured the level of C5a.

3. The method according to claim 1, comprising measuring or having measured the level of gliomedin.

4. The method according to any one of claims 1 to 3, comprising measuring or having measured the level of C5a and the level of gliomedin.

5. The method according to any one of claims 1 to 4, further comprising determining, or having determined, that the subject is more likely to have NPASC if the measured levels of C5a, gliomedin, or both exceed threshold levels of C5a, gliomedin, or both.

6. The method according to any one of claims 1 to 4, further comprising measuring or having measured a level of at least one biomarker selected from the list consisting of transforming growth factor β1 (TGFβ1), galactosylceramide sulfotransferase (Gal3ST1), interferon (IFN) lambda-1, growth hormone-releasing hormone (GHRH), lymphocyte function-associated antigen 3 (LFA-3), Fas ligand (FASLG), transgerin, immunoglobulin heavy chain constant gamma 1 (IgG1), and glycoprotein NMB (GPNMB).

7. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, and TGFβ1.

8. The method according to claim 7, further comprising determining, or having determined, that the subject is more likely to be NPASC if one or more measured levels of C5a, gliomedin, or TGFβ1 exceed a threshold level for C5a, gliomedin, TGFβ1, or any combination thereof.

9. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, TGFβ1, and Gal3ST1.

10. The method according to claim 9, further comprising determining, or having determined, that the likelihood of NPASC in the subject is higher if the measured levels of one or more of C5a, gliomedin, or TGFβ1 are above the threshold level of C5a, gliomedin, TGFβ1, or any combination thereof, and the level of Gal3ST1 is below the threshold level of Gal3ST1.

11. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, Gal3ST1, IFN-Lambda-1, and GHRH.

12. The method according to claim 11, further comprising determining, or having determined, that the likelihood of NPASC in the subject is higher if (i) one or more measured levels of C5a, gliomedin, or GHRH are above a threshold level for C5a, gliomedin, GHRH, or any combination thereof, and (ii) one or more measured levels of Gal3ST1 or IFN-Lambda-1 are below a threshold level for Gal3ST1, IFN-Lambda-1, or any combination thereof.

13. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFN-Lambda-1, and LFA-3.

14. The method according to claim 13, further comprising determining, or having determined, that the likelihood of NPASC in the subject is higher if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, or LFA-3 is above the threshold level of C5a, gliomedin, TGFβ1, LFA-3, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1 or IFN-Lambda-1 is below the threshold level of Gal3ST1, IFN-Lambda-1, or any combination thereof.

15. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFN-Lambda-1, GHRH, and LFA-3.

16. The method of claim 15, further comprising determining, or having determined, that the likelihood of NPASC in the subject is higher if (i) the measured level of one or more of C5a, gliomedin, GHRH, or LFA-3 is above the threshold level of C5a, gliomedin, GHRH, LFA-3, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1 or IFN-Lambda-1 is below the threshold level of Gal3ST1, IFN-Lambda-1, or any combination thereof.

17. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFN-Lambda-1, FASLG, and transgerin.

18. The method according to claim 17, further comprising determining, or having determined, that the likelihood of NPASC in the subject is higher if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, FASLG, or transgerin is above the threshold level of C5a, gliomedin, TGFβ1, FASLG, transgerin, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1 or IFN-Lambda-1 is below the threshold level of Gal3ST1, IFN-Lambda-1, or any combination thereof.

19. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, TGFβ1, Gal3ST1, IFN-Lambda-1, GHRH, GPNB, and IgG1.

20. The method according to claim 19, further comprising determining, or having determined, that the subject is more likely to have NPASC if (i) the measured level of one or more of C5a, gliomedin, TGFβ1, transgerin, or GPNB is above the threshold level of C5a, gliomedin, TGFβ1, transgerin, GPNB, or any combination thereof, and (ii) the measured level of one or more of Gal3ST1, IFN-lambda-1, or IgG1 is below the threshold level of Gal3ST1, IFN-lambda-1, IgG1, or any combination thereof.

21. The method according to claim 6, comprising measuring or having measured the levels of C5a, gliomedin, TGFβ1, IFN-lambda-1, LFA-3, IgG1, and GPNMB.

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