BIOMARKERS AND USES THEREOF IN DIAGNOSIS AND TREATMENT OF NEUROLOGICAL POST ACUTE SEQUELAE OF COVID 19 (NPASC)

The present application claims priority from U.S. Provisional Patent Application Serial No. 63/398,363 filed on 16 August 2022 and AU 2023900376 filed on 16 February 2023, the entire contents of each of which are incorporated herein by reference.

TECHNICAL FIELD

The application relates broadly to the field of diagnostics, and in particular to methods and kits for diagnosis and/or treatment of Neural Post Acute Sequelae of COVID (“NPASC”).

BACKGROUND

Bibliographic details of references in the subject specification are also listed at the end of the specification.

Reference to any prior art in this specification is not, and should not be taken as, acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

It is understood that sub-optimal levels or functioning of biological molecules in the human system can dysregulate homeostasis and profoundly affect essential biological functions such as gene expression regulation, cell division and differentiation, development and repair. Ultimately, such structure-function disorder can lead to various diseases and pathogenesis, including nervous system diseases.

A pandemic of coronavirus disease caused by SARS-CoV-2 infection has dramatically affected more than 500 hundred million people worldwide since 2019. SARS-CoV-2 causes COVID-19 defined often as the acute symptoms lasting until 4 weeks from onset of the initial symptoms. A significant subset of infected subjects develop a range of symptoms that appear to persist post such acute CO VID- 19 symptoms or complication some long after viral infection and the acute symptoms of COVID-19 have passed. This "long COVID" has been termed Post Acute Sequelae of COVID-19 (PASC) and symptoms can persist and/or last for 6 weeks, up to 12 weeks and/or beyond to 26 weeks, and/or 1 and 2 years. PASC can affects the musculoskeletal, digestive, pulmonary, neurological and other systems. Neurological PASC (NPASC) is a subset of PASC in subjects having neurological symptoms and, including includes cognitive deficits (e.g., reported as brain fog, difficulty thinking, poor attention, executive function and memory impairment etc)), headaches, parasthesia (tingling numbness), dysgeusia, anosmia, myalgia, Post Traumatic Stress Disorder (PTSD), sleep disturbances, anxiety and depression, with fatigue considered a neurological symptom being the most prevalent (Pinzon et al. J. Infection and Public Health 2022; 15: 856 -860 ).

A study by Hampshire et al. (EClinical Medicine 2021 Sept; 39: 10144) supports the hypothesis that individuals who have been infected with SARS-CoV-2 may have persistent, objectively measurable cognitive deficits after carefully controlling for pre- morbid IQ, pre-existing medical conditions, socio-demographic factors and mental health symptoms. People who had recovered from COVID-19, including those no longer reporting symptoms, exhibited significant cognitive deficits versus controls when controlling for age, gender, education level, income, racial-ethnic group, pre-existing medical disorders, tiredness, depression and anxiety. The deficits were of substantial size for both people who had been hospitalised, who comprised a small proportion of the COVID-19 subjects, and also for non-hospitalised cases, who comprised the majority of subjects infected with SARS-CoV-2 who had biological confirmation of COVID- 19. NPASC subjects are observed to have symptoms as assessed with numerous methods.

These methods may have been established whilst looking for symptoms of neurological disorders frequently seen in subjects with other nervous system diseases such as neurodegenerative, neuropsychiatric, neurodevelopmental or neuropathological conditions. Diagnosis and monitoring of NPASC includes standardised cognitive functions tests that aim to measure aspects of processing speed (e.g., pattern comparisons processing speed test), attention and executive memory, (e.g,. inhibitory control and attention test), executive function (e.g., dimensional change card sort test), and working memory (e.g., list sorting working memory tests) and the like. Aspects of such conditions may be assessed through in depth interviews and questionnaires such as the NIH Toolbox v2.1 instrument run by researchers and clinicians expressed as T-scores adjusted for age, education, gender and race, ethnicity, with a score of 50 representing the normative mean/median of the US population and a standard deviation of 10. Diagnosis may also be via PROMIS-57 a questionnaire for self-reporting different aspects of physical, mental, and social health the like. Diagnosis of NPASC is currently limited by clinical assessment such as those using the above criteria. In some conditions scans or biopsies can also inform diagnosis and treatments. NPASC treatment or therapy may include counselling, memory and olfactory training.

In terms of the causal mechanisms, immunology and virology studies, metabolomics, genomics, proteomics, and pathway analysis have yet to be sufficiently characterized to identify the myriad processes involved at various levels, from molecular biological, cellular immune responses, to tissues and organs involved in PASC or NPASC. The key endogenous molecules affecting nervous system pathology are poorly understood. Multimodal neuroimaging method, including magnetic resonance imaging techniques are being used to investigate nervous system diseases at the structural- functional level but research is at a relatively early stage and the key molecular mechanism and pathways underlying the observed pathology remains generally elusive.

Given the large number of people affected with NPASC globally, and how long people remain affected by NPASC, the absence of established treatments for this condition, there is an urgent need to identify biomarkers, preferably non-invasive biomarkers, of NPASC that will facilitate diagnosis and the development of treatments, treatment testing and monitoring. This will be useful for the PASC subjects post proof of a viral infection with SARS-CoV-2 and the many NPASV subjects that are without tests to confirm COVID-19 but nevertheless show neurological symptoms as assessed with current methods available, and for NPASV unrelated to SARS-CoV-2. The tests may be useful for subjects that have tested positive with SARS-CoV and MERS-CoV and such subjects without confirmation of infection.

SUMMARY

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 taken to provide explicit support for both meanings or for either meaning. As used herein, the term "about", unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, even more preferably +/- 1%, of the designated value.

Throughout this specification the word "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.

As used herein, the singular form "a", "an" and "the" include singular and plural references unless the context indicates otherwise.

By "about" is meant a measurement, quantity, level, activity, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference measurement, quantity, level, activity, value, number, frequency, percentage, dimension, size, amount, weight or length.

Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. The present invention has been described and exemplified with respect to the long term neurological sequelae observed with viral infection with SARS-CoV-2 however the invention extends to neurological post-acute sequelae of viral infection (NPASV) with pathogenic viruses other than SARS-Cov-2, such as other coronavirus infections or others known in the art to be accompanied by long term neurological sequelae.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l (SEQ ID NO: 1), <400>2 (SEQ ID NO:2), etc. Sequence identifiers of biomarkers are described in the Tables. A sequence listing may be provided after the claims for any sequences described in the specification.

In accordance with the present invention, the inventor considered that while gene expression studies provide an indication of canonical proteins expressed in a subject, protein studies such as 2D-elecrophoresis and increasingly, proteomics offers a more forensic investigation into the amount and form of a given protein in a given biological niche in a subject, and, coupled with further analytic techniques, may offer the skilled person insights into diagnostic and therapeutic targets.

The present invention is based, at least in part, upon the finding of a number of endogenous circulatory proteins whose levels are surprisingly altered in NPASC and are able, individually or in combination, to serve as (i) non-invasive biomarkers for subject and sample assessment or, in some cases, (ii) therapeutic targets for treatment of NPASC as disclosed herein.

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

In some embodiments the method also includes measuring or having measured a level of at least one of the biomarkers selected from the list consisting of Transforming Growth Factor pi (TGFpi), Galactosylceramide sulfotransferase (Gal3STl), interferon (IFN) lambda- 1, Growth Hormone Releasing Hormone (GHRH), Lymphocyte Function Associated Antigen 3 (LFA-3), Fas Ligand (FASLG), Transgelin, Immunoglobulin Heavy Constant Gamma 1 (IgGHl), Glycoprotein NMB (GPNMB), and Antithrombin- III.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, and TGFpi. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of one or more of C5a, Gliomedin, or TGFpi is above a threshold level for C5a, Gliomedin, TGFpi, or any combination thereof.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, TGFpi, and Gal3STl. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of one or more of C5a, Gliomedin, or TGFpi is above a threshold level for C5a, Gliomedin, TGFpi, or any combination thereof; and the level of Gal3STl is below a threshold level for Gal3STl.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, Gal3STl, IFN lambda-1, and GHRH. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of (i) one or more of C5a, Gliomedin, or GHRH is above a threshold level for C5a, Gliomedin, GHRH, or any combination thereof; and (ii) the level of Gal3STl or IFN lambda-1 is below a threshold level for Gal3STl, IFN lambda- 1, or any combination thereof.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, and LFA-3. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of: (i) one or more of C5a, Gliomedin, TGFpi, or LFA-3 is above a threshold level for C5a, Gliomedin, TGFpi, LFA-3, or any combination thereof; and (ii) the measured level of level of one or more of Gal3STl or IFN lambda-1 is below a threshold level for Gal3STl, IFN lambda-1, or any combination thereof.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, GHRH, and LFA-3. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of: (i) one or more of (i) one or more of C5a, Gliomedin, GHRH, or LFA-3 is above a threshold level for C5a, Gliomedin, GHRH, LFA-3, or any combination thereof; and (ii) the measured level of level of one or more of Gal3 ST 1 or IFN lambda- 1 is below a threshold level for Gal3 STI, IFN lambda- 1, or any combination thereof.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, FASLG, and Transgelin. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of: (i) one or more of C5a, Gliomedin, TGFpi, FASLG, or Transgelin is above a threshold level for C5a, Gliomedin, TGFpi, FASLG, Transgelin, or any combination thereof; and (ii) the measured level of level of one or more of Gal3 ST 1 or IFN lambda- 1 is below a threshold level for Gal3 STI, IFN lambda- 1, or any combination thereof.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, TGFpi, GaBSTl, IFN lambda-1, GHRH, GPNB and IgGHl.

In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of: (i) one or more of C5a, Gliomedin, TGFpi, GHRH, or GPNB is above a threshold level for C5a, Gliomedin, TGFpi, GHRH, GPNB, or any combination thereof; and (ii) the measured level of level of one or more of GaBSTl, IFN lambda-1, or IgGHl is below a threshold level for GaBSTl, IFN lambda-1, IgGHl, or any combination thereof.

In some embodiments the method includes measuring or having measured a level of C5a, Gliomedin, TGFpi, GaBSTl, IFN lambda-1, GHRH, LFA-3, IgGHl, and GPNMB. In some embodiments the method also includes determining or having determined a higher likelihood of NPASC in the subject when the measured level of: (i) one or more of C5a, Gliomedin, TGFpi, GHRH, LFA-3, or GPNB is above a threshold level for C5a, Gliomedin, TGFpi, LFA-3, GPNB, or any combination thereof; and (ii) the measured level of level of one or more of GaBSTl, IFN lambda-1, or IgGHl is below a threshold level for IFN lambda- 1, IgGHl, or any combination thereof.

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.

In some preferred embodiments the at least one biomarker is measured by one or more immunoassays. In some embodiments the one or more immunoassays include a multiplexed immunoassay. In some embodiments the one or more immunoassay or the multiplexed immunoassay includes an ELISA immunosassay. In other embodiments the at least one biomarker level is measured by use of a slow off-rate modified aptamer that binds specifically to the protein the level of which is being measured.

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

In other embodiments the level of the at least one biomarker is determined by mass spectroscopy.

In some embodiments the subject has COVID-19 or has had COVID-19.

In a related aspect provided herein is a method for treating NPASC in a subject that included identifying the subject as having a high likelihood of suffering from NPASC based on any of the foregoing methods, and providing or having provided to the subject an NPASC therapy to the subject.

In some embodiments, where the subject diagnosed as having a high likelihood of suffering from NPASC based on the methods disclosed herein, and where the level of TGFpi in the subject is above the threshold, NPASC therapy includes administering or having administered to the subject a therapeutically effective amount of an antisense oligonucleotide to a4 integrin. In some preferred embodiments the subject to which the antisense oligonucleotide is to be administered is not a subject identified as suffering from multiple sclerosis or Duchenne muscular dystrophy (DMD). In some embodiments the antisense oligonucleotide is ATL-1102, the nucleotide sequence of which consists of SEQ ID NO:1:

5' - ^Me c ^Me UG AGT ^Me CTG TTT ^Me U ^Me CMeC A ^Me U ^Me U ^Me C ^Me U - 3' (SEQ ID

NO: 1) wherein, a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at the positions 1 to 3 from the 5' end are 2'-O-(2- methoxy ethyl) modified ribonucleosides; c) the nucleotides at the positions 4 to 12 from the 5' end are 2'- deoxy rib onucl eosi des ; d) the nucleotides at the positions 13 to 20 from the 5' end are 2'-O- (2- methoxyethyl) modified ribonucleosides; and e) all cytosines are 5-methylcytosines ( ^Me C), or a pharmaceutically acceptable salt thereof or stereoisomer thereof. In a related aspect is the use of antisense oligonucleotide to integrin a4 integrin in the manufacture of a medicament for treatment of NPASC in a subject identified as having a high likelihood of suffering from from NPASC based on the methods disclosed herein, and where the level of TGFpi in the subject is above the threshold.

In a further aspect provided herein is a method for treating a human subject identified as suffering from or at risk of suffering from neurological post-acute sequelae of COVID-19 (NPASC), the method comprising administering or having administered to the subject a therapeutically effective amount of a modulator of a therapeutic target selected from the group consisting of:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta-1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll);

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin;

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

In some embodiments the modulator of the therapeutic target is an inhibitor of the therapeutic target. In some embodiments, where the modulator to be administered is an inhibitor, the inhibitor is to a therapeutic target selected from the list consisting of:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58 (iii) Transforming growth factor-beta-1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll); and

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin.

In other embodiments the modulator of the therapeutic target is an activator of the therapeutic target. In some embodiments, where the modulator to be administered is an activator, the activator is to a therapeutic target selected from the list consisting of:

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xiii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

In some embodiments the activator of the therapeutic target comprises a nucleic acid encoding the therapeutic target.

In another aspect provided herein is a method for treating a human subject identified as suffering from or at risk of suffering from neurological post-acute sequelae of COVID-19 (NPASC), the method comprising administering or having administered to the subject a therapeutically effective amount of an inhibitor of a therapeutic target selected from the list consisting of:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta-1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33; (vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll); and

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin.

In some embodiments, the method includes administering a therapeutically effective amount of a TGFpi inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Complement C5b-C6 complex inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Complement Lymphocyte function-associated antigen 3 CD58 inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Vascular endothelial growth factor D (VEGF-D) inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Myeloid cell surface antigen CD33 inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of an Alcohol dehydrogenase IB inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Tumor necrosis factor receptor superfamily member 10B inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Tumor necrosis factor receptor superfamily member 1 A inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a Cathepsin S inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a TLR4: Lymphocyte antigen 96 complex inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a BcL2 like 1 (Bcl2Ll) inhibitor.

In some embodiments, the method includes administering a therapeutically effective amount of a C5a inhibitor. In some embodiments the therapeutic target inhibitor to be administered is a small molecule inhibitor. In some embodiments the method includes administering the small molecule TGF-pi inhibitor Pirfenidone (CAS 53179-13-8). In other embodiments the method includes administering the small molecule Alcohol dehydrogenase IB inhibitor Fonepizole (CAS 7554-65-6). In some embodiments the method includes administering the small molecule Cathepsin S inhibitor Petesicatib (CAS 1252637-35-6). In some embodiments the method includes administering the small molecule TLR4: Lymphocyte antigen 96 complex inhibitor Resatorvid (CAS 243984-11-4) or Eritoran (CAS 185954- 98-7). In some embodiments the Bcl2Ll inhibitor is Navitoclax/ ABT-263 (CAS 923564-51-6) or Obatoclax (CAS 803712-67-6).

In other embodiments the therapeutic target inhibitor to be administered is an antibody inhibitor. In some embodiments the method includes administering the antibody Complement C5b-C6 complex inhibitor Ravulizumab. In some embodiments the method includes administering the antibody Myeloid cell surface antigen CD33 inhibitor Gemtuzumab. In some embodiments the method includes administering the antibody Tumor necrosis factor receptor superfamily member 10B inhibitor Conatumumab or Lexatumumab. In some embodiments the method includes administering the antibody Tumor necrosis factor receptor superfamily member 1A inhibitor Atrosimab or TNF Receptor-One Silencer (TROS).

In other embodiments the therapeutic target inhibitor to be administered is a fusion protein inhibitor. In some embodiments the method includes administering the protein fusion Lymphocyte function-associated antigen 3 CD58 inhibitor Alefacept. In some embodiments the method includes administering the protein fusion VEGF-D inhibitor OPT-302.

In other embodiments the therapeutic target inhibitor to be administered includes a nucleic acid inhibitor. In some embodiments the nucleic acid includes: an antisense oligonucleotide (ASO), an siRNA, a miRNA, an aptamer, or a sgRNA. In some embodiments the treatment method includes administering the antisense oligonucleotide TGF pi inhibitor ATL1102 (comprising the nucleotide sequence corresponding to SEQ ID NO: 1). In some embodiments the Growth Hormone Releasing Hormone (GHRH) inhibitor or the C5a inhibitor is an siRNA. In some embodiments the siRNA C5a inhibitor is Cemdisiran.

In some embodiments the therapeutic target inhibitor to be administered includes an expression construct. In some embodiments the expression construct is provided in a recombinant virus. In some embodiments any of the foregoing treatment methods include administering in combination therapeutically effective amounts of inhibitors of at least two of the therapeutic targets disclosed herein.

In some embodiments of any of the foregoing methods, the therapeutically effective amount of the inhibitor to be administered is based on a level of the therapeutic target or therapeutic target activity in the subject or an activity level of the therapeutic target in the subject.

In some embodiments any of the foregoing methods include determining a level of the therapeutic target or an activity level of the therapeutic target in the human subject following the administration.

In some embodiments any of the foregoing methods also include the step of diagnosing a human subject or having the human subject diagnosed as suffering from or at high risk of suffering from NPASC prior to the administration step.

In a further aspect provided herein is the use of a modulator of a therapeutic target in the manufacture of a medicament for treatment of neurological post-acute sequelae of COVID-19 (NPASC), wherein the therapeutic target is selected from the group consisting of:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta-1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll);

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin;

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid idase;

(xviii) IgG (xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

In some embodiments the modulator to be included in the medicament is an inhibitor of the therapeutic target. In some embodiments, where the modulator to be included in the medicament is an inhibitor, the therapeutic target is selected from the group consisting of:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta- 1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll); and

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin.

In other embodiments the modulator to be included in the medicament is an activator of the therapeutic target. In some embodiments, where the modulator to be included is an activator, the therapeutic target is selected from the group consisting of:

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein farnesyltransferase/geranylgeranyl transferase type- 1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

In a further aspect provided herein is an inhibitor of a therapeutic target selected from the list consisting of:

(i) Complement C5b-C6 complex; (ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta-1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll); and

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin for use in treatment of neurological post-acute sequelae of CO VID-19 (NPASC).

In another aspect provided herein is an activator of a therapeutic target selected from the list consisting of:

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH) for use in treatment of neurological post-acute sequelae of COVID-19 (NPASC).

In some embodiments of any of the foregoing treatment methods, the subject subject was identified as suffering from NPASC, based on the level in a biological sample obtained from the subject of at least one, two, or more NPASC-associated circulatory biomarkers selected from the group consisting of:

Prostaglandin-H2 D-isomerase,

TNF receptor superfamily member 1 A,

SLIT and NTRK-like protein 2,

Carbohydrate sulfotransferase 15,

Ribonuclease T2,

Alpha- 1,6-mannosylgly coprotein 6-beta-N-acetylglucosaminyltransferase A,

Ephrin type-B receptor 2, Thrombospondin-4,

Alanyl-tRNA editing protein A,

Galactosylceramide sulfotransferase,

C5a anaphylatoxin (complement component),

Protein phosphatase IF (PPM1F), and

Neutrophil cytosol Factor 2 (NCF2).

In some embodiments a treatment method includes the step of measuring in a biological sample from the subject the level of: (i) protein phosphatase IF (PPM1F) and the level of (ii) C5a anaphylatoxin, and/or Neutrophil cytosol Factor 2 (NCF2). In other embodiments a treatment method includes the step of measuring in a biological sample from the subject the level of: (i) protein phosphatase IF (PPM1F) and the level of (ii) C5a anaphylatoxin, and/or Neutrophil cytosol Factor 2 (NCF2), and (iii) the level of Galactosylceramide Sulfotransferase and/or the level of Prostaglandin D Synthase (PDGS).

In a further aspect provided herein is a kit or panel for determining the likelihood of neurological post-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.

In some embodiments the kit or panel comprises specific binding agents for at least each of the biomarkers C5a anaphylatoxin (C5a) and Gliomedin and Transforming Growth F actor P 1 (TGFP 1 ) .

In some embodiments the kit or panel also includes one or more specific binding agents for at least one protein selected from the group consisting of: Transforming Growth Factor pi (TGFpi), Galactosylceramide sulfotransferase (Gal3STl), interferon (IFN) lambda- 1, Growth Hormone Releasing Hormone (GHRH), Lymphocyte Function Associated Antigen 3 (LFA-3), Fas Ligand (FASLG), Transgelin, Immunoglobulin Heavy Constant Gamma 1 (IgGHl), Glycoprotein NMB (GPNMB), and Antithrombin III.

Symptoms associated with NPASC are known and developing in the art and include brain related symptoms including Anxiety/Depression, Sleep Disturbances, PTSD, Cognitive disturbances, and Headaches. In some embodiments, subjects may have olfactory dysfunction. For example, to identify subjects suffering from significant neurological disturbances subjects may complete a cognitive function evaluation with the National Institutes of Health (NIH) Toolbox v2.1 instrument, which includes 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). Accordingly in some embodiments clinical or self-assessment for NPASC includes an assessment of one or more than one of processing speed, attention and executive memory, executive function working memory, "brain fog", numbness/tingling, headaches, dizziness, fatigue, sleep disturbances, depression, and anxiety. Self assessment may, for example, employ a suitable symptom checker App.

The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows two box and whisker plots illustrating the differences between C5a anaphylatoxin levels within and between groups, comparing (i) Group 1+2 NPASC subjects (NP) and Group 4 healthy controls (HC), with a 100% increase in the median of NP vs HC and (ii) Group 1+2 NP and Group 3 COVID convalescent subjects (CC) showing an 88% increase in the median in NP vs CC.

FIGURE 2 shows box and whisker plots illustrating the difference between levels of Gliomedin comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between Gliomedin comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 31% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subjects in Group 3 CC with a 41% increase in the median in NP vs CC.

FIGURE 3 shows box and whisker plots illustrating the difference between levels of TGFbetal comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between TGFbetal comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 27% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subject in Group 3 CC with a 17% increase in the median in NP vs CC.

FIGURE 4 shows box and whisker plots illustrating the difference between levels of Galactose-3-O-Sulfotransf erase (Gal3STl) (AKA “Galactosylceramide sulfotransferase”) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between Gal3STl comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 23% decrease in the median of NP vs HC, and (ii) Group 1+2NP and 20 subject in Group 3 CC with a 22% decrease in the median in NP vs CC.

FIGURE 5 shows box and whisker plots illustrating the difference between levels of Interferon lambda- 1 (IFN lambda- 1) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between IFN lambda- 1 comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 29% decrease in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subject in Group 3 CC with a 14% decrease in the median in NP vs CC.

FIGURE 6 shows box and whisker plots illustrating the difference between levels of Growth Hormone Releasing Hormone (GHRH) (AKA “Somatoliberin”) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between GHRH comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 34% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subject in Group 3 CC with a 16% increase in the median in NP vs CC.

FIGURE 7 shows box and whisker plots illustrating the difference between levels of Lymphocyte Function-Associated Antigen 3 (LFA-3) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between LFA-3 comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 17% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subjects in Group 3 CC with a 5% increase in the median in NP vs CC.

FIGURE 8 shows box and whisker plots illustrating the difference between levels of Fas Ligand (FASLG) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between FASLG comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 46% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subjects in Group 3 CC with a 16% increase in the median in NP vs CC.

FIGURE 9 shows box and whisker plots illustrating the difference between levels of Transgelin comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between Transgelin comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 39% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subjects in Group 3 CC with a 53% increase in the median in NP vs CC.

FIGURE 10 shows box and whisker plots illustrating the difference between levels of Glycoprotein NMB (GPNMB) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between GPMB comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 16% increase in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subjects in Group 3 CC with a 25% increase in the median in NP vs CC.

FIGURE 11 shows box and whisker plots illustrating the difference between levels of Immunoglobulin Heavy Constant Gamma 1 (IgGHl) comparing NP Group 1+2 and Group 3 CC and Group 4 HC. The Figure illustrates the difference between IgGHl comparing (i) Group 1+2 NP and 24 subjects in Group 4 healthy controls (HC) with a 18% decrease in the median of NP vs HC, and (ii) Group 1+2 NP and 20 subject in Group 3 CC with a 23% decrease in the median in NP vs CC.

FIGURE 12 Exemplary identified therapeutic targets CD33 and VEGFD are higher in subjects suffering from NPASC. Scatter plots showing plasma levels of CD33 (top panel) and VEGFD (bottom panel) NPASC subjects (Groups 1 + 2), CO VID convalescent subjects not having NPASC (Group 3), and healthy control subjects that never had COVID (Group 4). Protein levels were measured by SomaScan® proteomic analysis (Y axis represents relative fluorescence units). The levels of CD33 and VEGFD are 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 suffering from NPASC. Scatter plots showing plasma levels of Thyroid peroxidase (top panel) and Amyloid A4 (bottom panel) NPASC subjects (Groups 1 + 2), COVID convalescent subjects not having NPASC (Group 3), and healthy control subjects that never had CO VID (Group 4). Protein levels were measured by SomaScan® proteomic analysis (Y axis represents relative fluorescence units). The levels of Thyroid peroxidase and Amyloid A4 are significantly lower in NPASC subjects (false discovery rate - FDR = 0.014 and 0.014, respectively).

FIGURE 14 Exemplary identified therapeutic target Transforming growth factor-01 is higher in subjects suffering from NPASC. Scatter plots showing plasma level TGF- 1 in NPASC subjects (Groups 1 + 2), COVID convalescent subjects not having NPASC (Group 3), and healthy control subjects that never had COVID (Group 4). Protein level was measured by SomaScan® proteomic analysis (Y axis represents relative fluorescence units). The level of TGF-01 is significantly higher in NPASC subjects (false discovery rate - FDR = 0.011).

TABLE 1 List of biomarkers used in the diagnostic methods described herein. TABLE 2 Statistical tests and values indicating biomarkers used in the methods described herein.

TABLE 3 Exemplary biomarker combinations and diagnostic sensitivity and specificity characteristics for use in the methods described herein.

TABLE 4 provides subject information from the study of Visvabharathy et al. ://www.medrxiv.org/content/10.1101/2021.08.08.21261763 (2021). The subject sample for the present disclosure comprises in Group 1+2, 48NP subjects who are nonhospitalized, 42 of whom were from the non hospitalized group of 48 subjects in Visvabharathy et al. The subject sample herein comprises in Group 3, 20 CC subjects who are non hospitalized consisting 17 of the 24 in Visvabharathy, and 24 HC subjects of the 31 HC subjects from Visvabharathy et al.

TABLE 5 Summary of pathway analysis undertaken/pathways identified to assist in identification of NPASC therapeutic targets.

TABLE 6 Summary of pathways of interest identified by statistical analysis of hit frequency (FDR<0.02).

TABLE 7 Therapeutic Targets Identified by ANOVA FDR < 0.02 (Group 1 vs Group 3).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meanings 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 are particularly directed to Wild D. "The Immunoassay Handbook" Nature Publishing Group, 4 ^th 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 2 ^nd Edition, Eds. Haleem Issaq, Timothy Veenstra, 2019 Academic Press ISBN:9780128186077, Chapter 17. Top-down mass spectrometry for protein molecular diagnostics, structure analysis, and 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 for definitions and terms of the art and other methods known to the person skilled in the art. The present methods can be done in any convenient format known in the art.

The term "coronavirus" or "Coronaviridae" , refers to viruses known by the common name of "Coronavirus" or "CoV" which are enveloped, positive sense, singlestranded RNA viruses. There are two subfamilies of Coronaviridae, Letovirinae and Orthocoronavirinae. In some embodiments, the CoV is selected from the genera Alphacoronavirus (alphaCoV), Betacoronavirus (betaCoV), Gammacoronavirus (gammaCoV) and 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 betaCoV is selected from human coronavirus HKU1 (HCoV- HKU1), Human coronavirus OC43 (HCoV-OC43), Severe acute respiratory syndrome- related coronavirus (SARS-CoV), Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), Middle-East respiratory syndrome-related coronavirus (MERS-CoV), murine hepatitis virus (MHV) and/or bovine coronavirus (BCoV). In some embodiments, the CoV is capable of infecting a human. In some embodiments, the CoV capable of infecting a human is selected from: SARS-CoV-2, HCoV-OC43, HCoV- HKU1, HCoV-229E, HCoV-NL63, SARS-CoV, and MERS-CoV or a subtype of variant 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 an embodiment, SARS- CoV-2 is SARS-CoV-2 hCoV-19/ Australia/VICO 1/2020. In some embodiments, SARS- COV-2 comprises the sequences as described in NCBI Reference Sequence: NC_045512.2 (ancestral Hu-1). In some embodiments, SARS-CoV-2 comprises the sequence as described in GenBank: MN908947.3 or a pathogenic variant thereof. World Health Organization (WHO) labels include Alpha, Beta, Gamma, Delta, and Omicron with lineages and clades as outlined and updated from time to time on Wikipedia https://en.wikipedia.org/wiki/Variants_of_SARS-CoV-2

The present invention is predicated in part upon the determination by the inventor/s that the level of a number of specific proteins is significantly modulated in the circulating blood of subjects with neural PASC. Accordingly, the measured level of one or more of these NPASC associated biomarkers can be used to identifying the likelihood of NPASC in a subject, the risk of developing NPASC in a pathogenic coronavirus infected (e.g., COVID-19) subject or a healthy subject Further, the measured biomarker level may be used to determine those who are likely to be responsive to treatment with one or more modulators to these specific biological molecules, or in combination with other NPASC therapies. For one or more of these specific biological molecules, their level is significantly depressed in the circulatory system of subjects with NPASC. Accordingly, treatments are contemplated that elevate the level of a specific biological molecule identified herein.

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

Proteomic plasma profiles were identified for three subject groups. Group 1 was subjects with NPASC, Group 3 was subjects recovered from CO VID without PASC or NPASC, Group 4 was health control subjects as described in the examples.

Level data may be measured or expressed with any convenient parameter, including mass concentration and light emission, relative fluorescence units (RFU). With certain assays and samples, level data may be compared to healthy adult control normalized RFU eg SomaScan® EDTA plasma level data of NPASC subject may be generated and compared to the healthy adult control nRFU for each Somamer, with healthy control values. The healthy control values are a robust point estimate generated during assay validation of the aptamers, and values are the median of 1000 individuals from an adult US population, both males and females, ages varying between 18-80; there is no healthy dataset matching the CC subjects with EDTA plasma.

Comparing two or more levels includes determining or obtaining a value for the difference or similarity between levels, e.g., visual or machine/computer based comparisons.

Level data from subjects may conveniently be summarised or represented graphically to facilitate data comparison and interpretation. As the skilled person will appreciate graphical representation of data may take many different forms and represent or concentrate on different aspects of the data depending upon the requirements of the analysis and the user, and this may be modified. For example the user may be a computer program or a person. Graphical representations of data include tables, line plots, charts (e.g., pie chart, heat maps, bubble charts, zoom charts, chord diagrams, graphs, signatures, bar codes, box plots, and combinations of these and others.

Box and whisker plots conveniently summarise comparative data derived from a subject or population, or from a database and represent levels by highlighting factors such as the distribution, spread and skewness in dataset and any outliers/unusual observations/extreme values through summary data such as median, lower and upper quartile, minimum and maximum values, first quartile and third quartile, the bottom and third quartile, the whisker (thin vertical line connecting the minimum and maximum (non-outlier) values to the box), and bee swarm data shows individual levels, median, mode, average. The median (or inter quartile range) is considered a particularly useful measurement of the central tendency(especially in the case of a skewed distribution), and it the point at which there are an equal number of data points whose values lie above and below the median value. In addition, to showing median, first and third quartile and maximum and minimum values, the box and whisker chart is also used to depict determine mean, standard deviation, mean deviation and quartile deviation. In the Examples, DataViz is employed wherein the upper whisker is also known as the upper fence, and the lower whisker is also known as the minimum (or lower fence).

As described further herein state of the art proteomics assays were used to identify, from about 7300 plasma proteins, plasma proteins differentially present between groups of plasma samples representing disease (NPASC). COVID convalescent control and healthy controls, from different groups. Of the tens of plasma proteins identified by Bonferroni analysis by the different parametric and non-parametric tests, and few hundred of plasma proteins identified as differentially present by a False Discovery Rate analysis with the parametric and non-parametric tests, the inventor/s undertook a range of studies including pathway analysis for those that were statistical significant to identify proteins that would serve as biomarkers for assessing the likelihood of NPASC in the sample or subject.

In some embodiments, NPASC associated circulatory biomarkers were identified as shown in table selected from Table 1. It has been determined that the level of each of these proteins is substantially either elevated or depressed in NPASC subjects relative to reference values from non-NPASC subjects as described and illustrated herein in Table 1 and Table 2.

TABLE 1 - Biomarker List

** GHRH statistically significant with the Bonferroni KW non-parametric test = 0.047 TABLE 2 Biomarkers and Statistical Tests

As used herein, the term “sensitivity” in reference to a diagnostic or prognostic method disclosed herein, refers to the ability of the method to correctly identify a subject as suffering from NPASC, /.< ., the total proportion of individuals within a group of tested subjects that is correctly identified as suffering from NPASC.

As used herein, the term “specificity” in reference to a diagnostic or prognostic method disclosed herein, refers to the ability of the method to identify a subject who is not suffering from NPASC, /.< ., the total proportion of individuals within a group of tested subjects that is correctly identified as not suffering from NPASC.

Taken together, the sensitivity and specificity define the accuracy of a diagnostic test, where 100% sensitivity would detect all subjects suffering from NPASC in a given group of subjects (/.< ., no false negatives for NPASC), and 100% specificity would detect all subjects who are not suffering from NPASC (/.< ., no false positives for NPASC). A test with 100% sensitivity and 100% specificity provides 100% accuracy. In some embodiments of the methods the test identifies subjects with at least 70% accuracy, e.g., 75%, 80%, 85%, 90%, 95%, or another level of accuracy from at least 70% accuracy to 100% accuracy depending on the subject subpopulation and/or assay. The skilled person will appreciate that diagnostic assays having the same level of accuracy can achieve this based differing combinations of underlying sensitivity sensitivity. For example, where a diagnostic assay having 90% accuracy may, comprise 95% sensitivity and 85% specificity, or 85% sensitivity and 95% specificity or a different combination of sensitivity and specificity levels as would be understood by those in the art. A 90% accuracy in a group of subjects, may end up with a 70%, 75%, 80%, 85%, 90% or greater accuracy in a different independent group of subjects, and/or or a smaller or larger study, or depending on the assay.

In some embodiments of the methods disclosed herein measuring a level of C5a and/or gliomedin either or both of which are higher than a threshold level identifies a subject suffering from NPASC with about 88% sensitivity. In other embodiments where a third marker, TGFpi (higher than threshold) is included, the test provides about 98% sensitivity. Alternatively if Gal3STl is included among the three biomarker panel rather than TGFpi, the sensitivity is about 92%. If both TGF pi and Gal3STl are measured, in addition to C5a and Gliomedin, the sensitivity is about 98% as outlined in Table 3.

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

In other embodiments where a third marker, TGFpi (normal) or an alternative third marker, Gal3STl (normal), are included, the test still provides about an average 84% specificity for the two controls. Alternatively if included among the biomarker panel is interferon lambda- 1 (normal level) together with GHRH (normal level) the specificity increases to about 100% for healthy controls (HC). If additionally interferon lambda-1 (normal level), together with LFA-3 (normal level) is included the specificity increases to about 85% for the Convalescent controls (CC). When using these targets for these two control subject groups if measuring also a level of C5a and/or gliomedin that is potentially about 92.5% average specificity as outlined in Table 3. An additional up to 4 markers FASLG (normal) and Transgelin (normal), GPNMB (normal) and IgGHl (normal level) each in a combination with at least Interferon lambda- 1 (normal level) on top of C5a and/or gliomedin provide potentially up to 100% specificity as outlined in Table 3.

In other embodiments where a third marker, TGFpi (normal) or an alternative third marker, Gal3STl (normal), are included, the test still provides about an average 84% specificity for the two controls. Alternatively if included among the biomarker panel is interferon lambda- 1 (normal level) together with GHRH (normal level) the specificity increases to about 100% for healthy controls (HC). If additionally interferon lambda-1 (normal level), together with LFA-3 (normal level) is included the specificity increases to about 85% for the Convalescent controls (CC). When using these targets for these two control subject groups if measuring also a level of C5a and/or gliomedin that is potentially about 92.5% average specificity as outlined in Table 3. An additional up to 4 markers FASLG (normal) and Transgelin (normal), GPNMB (normal) and IgGHl (normal level) each in a combination with at least Interferon lambda- 1 (normal level) on top of C5a and/or gliomedin provide potentially up to 100% specificity as outlined in Table 3.

Assessed T-test, U-test, ANOVA & KW Bonferroni < 0.05 targets - identified markers for diagnosis

In some embodiments, the level of a biomarker is detected using specific binding agents such as without limitation antibody or antigen-binding fragments of antibodies that bind to the biomarker and directly or indirectly provide a detectable signal that can be quantified visually or by instrument. The term "level" or "levels" also encompasses ratios of level/s of biomarkers determined using the binding agent detection protocol and quantification.

As used herein, "immunoassay" refers to immune assays, typically, but by no means exclusively sandwich assays, capable of detecting and quantifying a desired biomarker. The immunoassay may be one of a range of immune assay formats known to the skilled addressee. In some preferred embodiments an immunoassay to be used is an ELISA format assay.

While microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).

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

In operation, a protein sample (see, e.g., U.S. Pat. App. Pub. 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. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to 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 known to be invariant.

In an illustrative example, fluorescence labelling can be used for detecting protein bound to the array. The same instrumentation as used for reading 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 are labeled with different fluorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g, available from PerkinElmer Lifesciences). Planar waveguide technology (e.g., available from Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot). Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on 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), resonance light scattering (e.g., available from Genicon Sciences) and atomic force microscopy (e.g., available from BioForce Laboratories). A microfluidics system for automated sample incubation with arrays on glass slides and washing has been co-developed by NextGen and Perkin Elmer Life Sciences.

Electrochemiluminescence (ELICA), enzyme-linked immunosorbet type assay (ELISA) and Luminex eg, LabMAP, magnetic Luminex, immunoassays are examples of suitable assays to detect levels of the biomarkers.

In the bead-type immunoassays, the Luminex LabMAP system can be utilized. The LabMAP system incorporates polystyrene microspheres that are dyed internally with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, an array is created consisting of different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere 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 fluorochrome coupled to a reporter molecule quantifies the biomolecular interaction that has occurred at the microsphere surface. Microspheres are interrogated individually in a rapidly flowing fluid stream as they pass by two separate lasers in the Luminex analyzer. High-speed digital signal processing classifies the microsphere based on its spectral address and quantifies the reaction on the surface in a few seconds per sample.

Lateral flow assays and more recently non-lateral flow and microfluidics provide a useful set up for biomarker screening. Such assays can be qualitative, quantitative or semi quantitative. In microfluidic devices, small volumes of liquid are moved through microchannels generated in, for example, a chip or cartridge. A wide range of detection reagents are available including metal nanoparticles, coloured or luminescent materials. Resonance enhanced adsorption (REA) of bioconjugated metal nanoparticles offers rapid processing times and other advantages. These devices have been combined with barcode technologies to identify the patient and the analyte being tested. Computer software and hardware for assessing input data are encompassed by the present disclosure.

A wide range of methods for the detection of antibody to specific antigens are also known. For example, the enzyme-linked immunosorbent assay (ELISA), Western and dot blot assays, and radio-immunoassay (RIA) are routinely used in laboratories. Arrays and high throughput screening methods are also employed. General formats and protocols for the conduct of various formats of ELISA are disclosed in the art and are known to those of skill in the field of diagnostics. For example, reference may be made to Chapter 11 of Ausubel (Ed) Current Protocols in Molecular Biology, 5th Edition, John Wiley & Sons, Inc, NY, 2002. Rundstrbm, G et al. describe lateral Flow immunoassay using Europium (III) Chelate Microparticles and Time-Resolved A wide Fluorescence for eosinophils and neutrophils in Whole Blood. Clinical Chemistry 53, 342-348 (2007), incorporated herein.

Qualitative assays providing an intermediate or definitive diagnosis require integrated cutoffs, gates or windows that permit scoring of samples as likely or not to have a condition. Instrument readers and software are often employed to collate data and process it through a diagnostic algorithm or decision tree.

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

These biomarker assays may be been combined with barcode or ledger technologies to identify the patient and the biomarker being tested. Computer software and hardware for assessing input data are encompassed by the present disclosure. Point- of-care devices and arrays and high throughput screening methods are also contemplated. In some embodiments the capture portion is a test line.

Qualitative assays providing an intermediate or definitive diagnosis require integrated thresholds, gates or windows that permit scoring of samples as likely or not to have a condition. Instrument readers and software are often employed to collate data and process it through a diagnostic algorithm or decision tree.

The presence of complexes may be evaluated using ELISA-type procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two- site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. The skilled person will appreciate that the selection and implementation of a known label system involves only routine experimentation.

Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an binding agent is immobilized on a solid or semi-solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an binding agent-antigen complex, a second binding agent specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of binding agent-antigen-labelled binding agent. 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). The results may be qualitative or quantitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of marker. Variations on the forward assay include a simultaneous assay, in which both sample and labelled binding agent are added simultaneously to the bound binding agent. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is generally a biological sample comprising biological fluid, and is most conveniently a whole blood sample such as capillary or venous blood that may be treated with an anticoagulant.

A "solid substrate" is suitable for some assays and refers to, for example, a material having a rigid or semi-rigid surface or surfaces, which may be regular or irregular, and may take the form of beads, resins, gels, spheres, microspheres, particles, fibres or other geometric configurations or physical forms. A solid substrate typically comprises a material that is applicable in medical, biochemical or biological assays, for example, substrate used in apheresis, column chromatography for purification or separation of biological molecules or organic molecules and ELISA assays. Solid substrates may be porous or non-porous.

Solid substrates or surfaces for immobilizing biomarkers or binding agents. Nonlimiting examples of solid substrates include for example, polymers such as polysaccharides, in particular polysaccharides having a molecular weight of 100 kDa or more, such as agarose. Agarose may be in particulate form, which optionally can be cross-linked. A particular example of agarose is Sepharose, or cellulose, which can be cross-linked. Other polymers appropriate as a substrate include, for example, carboxylated polystyrene. Solid substrates may be provided in the form of magnetic beads. Glass is also an appropriate substrate material. Any suitable blood or plasma filtration column or system can be adapted for the present process. Non-limiting examples include 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. of Japan.

In a typical forward sandwich assay, a first binding agent having specificity for a marker is either covalently or passively bound to a solid or semi-solid support. The support is typically glass or a polymer, the most commonly used polymers being nitrocellulose, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene or mixture or derivatives of these. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the polymer-binding agent complex to the 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 of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37° C. including 25° C.) to allow binding of any subunit present in the binding agent. Following the incubation period, the binding agent subunit solid phase is washed and incubated with a second binding agent specific for a portion of the antigen. The second binding agent is linked to a detectable marker which is used to indicate the binding of the second binding agent to the antigen.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific binding agent which may or may not be labelled with a detectable marker. Depending on the amount of target and the strength of the signal from the detectable marker, a bound target may be detectable by direct labelling with the binding agent. Alternatively, a second labelled binding agent, specific to the first binding agent is exposed to the target-first binding agent complex to form 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 of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide or electrophoretic tag, so as to facilitate identification of the amino acid sequence of each library member. These improved beadbased methods are described in International Publication No. WO 93/06121.

In other embodiments, the method is a liquid phase method. In one example of a liquid phase immunoassay (see for example U.S. Pat. No. 6,632,603) the sample is contacted with an agent capable of binding a marker and a detector agent comprising a visually detectable agent such as colloidal gold or silver labelled. The test sample is applied by flowing onto a defined zone of an insoluble porous support film having a pore size impassable to a complex formed between the marker and its target, and if present, with a binding substance and a detector substance, but passable to the binding substance and detector substance while remaining uncomplexed in the absence of the desired target. If the target is present in the test specimen, the detector substance binds with the target and the binding substance to form a visually inspectable complex on the surface of the porous support film. After application of the test sample to the porous support, the surface of the porous support is visually inspected for colour to determine the presence and quantity or the absence of the marker being assayed.

In another assay, magnetic antibodies that bind to markers are used to tag markers and a high Tc superconducting quantum interference device is used to measure the amount of target protein. A liposome immunomigration, liquid-phase competition strip immunoassay is, for example, described in Glorio-Paulet et al J Agric Food Chem 48 (5): 1678-1682, 2000.

Antibody-based methods are more sensitive than 2-D gels or MS and can detect analytes in the sub-nM range due to the high affinity of antibodies for their targets (typically nM to pM). These are widely used in single analyte tests and are proposed herein for multiple assay types. However for multiplex or miniplex assays such as bead based assays, cross reactivities caused by antibodies reduces sensitivity and specificity. 2D electrophoresis provides forensic analysis of proteins in a sample and mass spectrometry based approaches are used by themselves or in conjunction 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. Proteomics assays based on affinity molecules that include additional proofreading steps have the best combination of sensitivity and specificity, and are preferred.

Proximity ligation assay (PLA) is a highly sensitive technique for multiplex detection of biomarkers in plasma with little or no interfering background signal. Antibodies with proximity ligation and SomAmers are providing very sensitive and specific multiplexed assays for proteomics. PLA was originally demonstrated with aptamers and this is easier to implement than with antibodies and has much higher proofreading potential. DNA aptamers belong to a group of molecules that are less expensive alternatives to antibodies. These alternatives include peptide aptamers isolated by phage and ribosomal display technologies, but DNA aptamers stand out as the simplest and most economic to produce. One reason why Gold et al. (Gold L, Ayers D, Bertino J et al. Aptamer based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5(12), el5004 (2010). have succeeded in developing highly multiplexed assays is that their slow off-rate aptamers (SOMAmers) have amino acidlike 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 was developed 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 J et al. Expanding the chemistry of DNA for in vitro selection. J. Am. Chem. Soc. 132(12), 4141-4151 (2010). It forms part of an approach that is advancing in the direction of hybrid molecules that resemble peptides that are compatible with enzymatic e.g., PCR amplification.

One proteomics method for biomarker assessment is 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, small volumes of plasma are incubated overnight and allowed to bind with oligonucleotide- labeled antibody pairs to form specific DNA duplexes. The templates are then extended and pre-amplified, and the individual protein markers were measured using high- throughput microfluidic real-time PCR. The resulting Ct values are normalized against an extension control, an inter-plate control, and adjusted with a correction factor according to the manufacturer’s instructions to calculate a normalized protein expression value (NPX) in log2 scale. Raw expression values are then batch corrected by normalizing to the overlapping reference samples within each plate.

In one example of a proteomics affinity capture assay (Gold et al PLos ONE 5 (12), el5004 (2010)) SOMAmers are mixed with the target sample (purified protein or plasma) and incubated to bind to equilibrium. In Catch-1 bound SOMAmer(S)- protein(P) complexes are captured onto streptavidin beads (SA) and the proteins are tagged with biotin (NHS- biotin) and fluorescent label (F) (NHS Alexa 647). Unbound proteins are washed away. Bound complexes are released from the beads by cleaving the photo-cleavable linker with ultraviolet light. In Catch-2 SOMAmer-protein complexes are captured onto monomeric avidin beads, washed, and eluted from the beads with 2 mM biotin. At this stage, SOMAmer-protein complexes are subjected to a kinetic challenge. Specific complexes survive the challenge and non-specific complexes dissociate. In the final step, Catch-3, bound complexes are captured onto primer beads by DNA primer that is complementary to a portion of the SOMAmer and any remaining unbound protein resulting from the kinetic challenge is washed away. Finally, the captured complexes are dissociated with 20 mM NaOH and the target protein is eluted for analysis by PAGE.

A suitable "biological sample" for the methods and kits as described herein includes any sample containing or suspected of containing biomarkers for detection including, but not limited to, biological fluids e.g. whole blood or a fraction thereof, a blood product, plasma, a mucosal surface sample such as serum, saliva, nasal swab, throat swab, respiratory swab, nasal scrapings, nasal washings, respiratory tract washing, lung washings, gut samples, faeces or gingivo creviscular fluid. In one particularly advantageous embodiment, the sample is whole blood or a part or derivative thereof comprising circulating plasma proteins. In some embodiments, the sample is purified or partially purified. In an embodiment the sample is plasma. In an embodiment, the sample is serum. The blood, plasma or serum sample is, in some embodiments, treated with an anticoagulant, such as citrate, EDTA and heparin. In an embodiment, biological samples are collected from a subject at two more time points. In an embodiment, the sample is stored for a period of time at about 4°C, at about 15°C or about 24°C before use. In an embodiment, the sample is dried, freeze dried or snap frozen. Non-limiting examples of a blood product include whole blood, serum, plasma, the like, and a combination thereof. A blood product may be devoid or depleted of cells, or may include cells (e.g., red blood cells, platelets and/or lymphocytes). Reference to a "blood sample" includes a plasma sample or a serum sample or other blood part comprising proteins derived from a blood sample. Whole blood refers to an essentially unprocessed sample of blood from a subject, generally anticoagulant has been added when the blood is obtained by venous collection, or it may or may not be omitted if the blood is obtained by fingerprick or fingerstick collection and used without delay at point of care. In some embodiments, EDTA plasma is employed to analyse subject samples. In some embodiments, different subject groups may one type of anticoagulant.

The "subject" contemplated herein is generally a human subject and may also be referred to as a patient, individual or recipient. The human subject may be neonatal or an infant, child, adolescent, teenager, young adult, adult or elderly adult of male or female gender. Reference subjects are often selected groups of subjects referred to as a population of subjects/patients. In some embodiments, biological samples for testing are from reference subjects such as those who have recovered from the "infection" phase of a viral infection, for example the convalescent controls (CC) in the present study or healthy control subjects (HC). In some embodiments, biological samples for testing are from subjects who have recovered from the "infection" phase of a viral infection, however samples may also be collected from subjects in individual or general population screening to exclude a likelihood or risk of NPASC. Reference samples may reflect different subgroups and populations as known in the art. Any subject group can be usefully employed as a reference population including "health" or "unhealthy" subjects such as the immunocompromised, or age, ethnicity or gender-specific populations.

In some embodiments, the subject is a human and includes different populations of humans. The present invention extends, however, to primates, livestock animals, laboratory test animals including fish models, companion animals and avian species as well as non-mammalian animals such as fish and reptiles. The assay has applications, therefore in human, livestock, veterinary and wild-life therapy and diagnosis.

The term therefore includes humans and a wide range of mammalian, non-mammalian, avian or other animals including wild and domesticated animals, horses, camelids, rabbits, rodents, guinea pigs, dogs, pets, pests and potential vehicles for emerging infectious diseases. In some embodiments, the subject is a mammalian or avian animal species. In an embodiment, the mammal is a human. In relation to subjects, the subjects may be at risk of, suspected of or diagnosed with an infectious disease or pathogen, a condition, C0VID/C0VID-19 due to SARs-CoV-2 infection including any emerging coronavirus variant or disease, and NPASC.

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

In some embodiments, the aptamer comprises a biotin or other member of a binding pair, a photocleavable group and a fluorescent or luminescent tag. Modified apatamers with protein like binding sites are advantageously used in some embodiments.

In some embodiments, the binding agent is conjugated to a detectable marker or microparticles comprising a detectable marker, that may provide a detectable signal. Biomarker-binding agent complexes may directly or indirectly provide a detectable signal that can be quantified visually or photometrically including fluorometrically (by instrument reader).

In some embodiments, the binding agent may be conjugated to a detectable marker or microparticles comprising a detectable marker, that provide a detectable signal. Illustrative particles and methods are described in Rundstrbm, G et al.

The term "binding agent" and like terms, refers to any compound, composition or molecule capable of specific or substantially specific (that is with limited crossreactivity) binding to the biomarker. The "binding agent" generally has a single specificity. Notwithstanding, binding agents having multiple specificities for two or more biomarkers are also contemplated herein. Alternatively, ‘combibodies’ comprising non-covalent associations of VH and VL domains, can be produced in a matrix format created from combinations 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 region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.

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.

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

Polyclonal antibodies can be generated by immunization of an animal. Monoclonal antibodies can be prepared according to standard (hybridoma) methodology. Antibody derivatives and analogs, including humanized antibodies can be prepared recombinantly by isolating a DNA fragment from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. Phage display and aptamer technology is described in the literature and permit in vitro clonal amplification of antigen-specific binding reagents with very affinity low cross-reactivity. Phage display reagents and systems are available commercially, and include the Recombinant Phage Antibody System (RPAS), commercially available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey and the pSKAN Phagemid Display System, commercially available from MoBiTec, LLC of Marco Island, Florida. Aptamer technology is described for example and without limitation in US Patent Nos. 5,270,163; 5,475,096; 5,840,867 and 6,544,776.

Antibodies are often used in immunoassays because of the ability to produce them in large quantities and the homogeneity of the product . The preparation of hybridoma cell lines for monoclonal antibody production is derived by fusing an immortal cell line and lymphocytes sensitized against the antigen of interest or can be done by techniques which are well known to those who are skilled in the art. (See, for example, 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 or more recent references Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Edition, CSHLP, CSH, NY, 2001). The DNA that encodes antibodies can be manipulated in vitro and introduced back into lymphoid cell lines, thus allowing the production of genetically-engineered antibodies. In the last decade, the use of transient mammalian expression systems for the production of native complex proteins has increased (see S. Geisse, B. Voedisch Methods Mol. Biol., 899 (2012), pp. 203-219) boosted by the publication of efficient transfection protocols and the availability of suspension cell lines growing at high density. For transient expression, mainly the HEK-293 and CHO-K1 cell lines have been applied. Both cell lines can be adapted to suspension culture, and sub clones are available which grow in chemically defined medium at high cell densities. With its ease of transfection, high expression yield and native human glycosylation, the human embryonic kidney 293 cell line may be used.

Antibodies (and their smaller formats such as scFv and Fab fragments can be produced in any cell type known in the art. Cell-free protein synthesis, also termed in vitro translation, facilitates the production of a given target protein by utilizing the translational machinery without using the living cell. Cell-free systems have been successfully used for the high-throughput production of protein libraries as well as for 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, since no timeconsuming cloning steps are required prior to protein production. Antibodies of a given target protein takes approximately one to two days, whereas cell-based expression, including the cloning procedure and cell-transformation, may take up to two weeks. Phage display technology is the most commonly used technique for the in vitro selection and evolution of antibody fragments.

Alternatives to antibodies as specific binding agent are described in the literature and are recognised in the field for their potential to improve inter alia, assay reproducibility and stability. A review of antibody alternatives is provided by McLeod et al The Scientist February 2016 incorporated herein, and includes aptamers and affimers. Any such binding agent may be employed in the present assays and kits without undue experimentation.

Illustrative binding agents include antigen-binding constructs comprising a protein scaffold which is linked to one or more epitope-binding domains wherein the antigen-binding construct has at least two antigen binding sites at least one of which is from an epitope binding domain and at least one of which is from a paired VH/VL domain.

In one broad embodiment, the method comprises (i) measuring or having measured the level of at least one of the biomarkers C5a or Gliomedin, preferably measuring or having measured the level of both of these biomarkers. In other embodiments, the method includes measuring the level of at least one more biomarker level selected from:

1. Gliomedin (UniProt Q6ZMI3)

2. C5a anaphylatoxin (UniProt P01031)

3. Transforming Growth Factor pi (UniProt P01137)

4. Galactosylceramide sulfotransferase (UniProt C9JIS3)

5. Inteferon lambda-1 (UniProt G9C945)

6. Growth Hormone Releasing Hormone (AKA “Somatoliberin”) (UniProt P01286)

7. Lymphocyte Function Associated Antigen 3 (UniProt Pl 9256)

8. FASLG (UniProt P48023)

9. Transgelin (UniProt Q7Z517)

10. Immunoglobulin Heavy Constant Gamma 1 (UniProt P01857)

11. Glycoprotein NMB (UniProt Q14956)

In some embodiments the method includes measuring the level of Gliomedin, C5a, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of biomarkers 3-11 listed above in any combination.

In some preferred embodiments the method includes measuring or having measured a level of each of: C5a, Gliomedin, and TGFpi. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, and Gal3STl. In some embodiments the biomarkers to be included are: C5a, Gliomedin, Gal3STl, IFN lambda- 1, and GHRH. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, and LFA-3. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, GHRH, and LFA-3. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, GHRH, FASLG, and Transgelin. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda- 1, GHRH, GPNB and IgGHl. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, IFN lambda-1, LFA-3, IgGHl, and GPNMB. In some embodiments the biomarkers to be included are: C5a, Gliomedin, TGFpi, Gal3STl, IFN lambda-1, GHRH, LFA-3, FASLG, Transgelin, IgGHl, and GPNMB.

In some embodiments, a further step is (iii) analysing levels from (i) and/or (ii) relative to preselected thresholds or gates derived from levels determined from reference subjects/populations to derive a score indicating whether a subject is likely or not to have NPASC. The skilled person will be able to design various gating strategies to optimise testing and reporting The level of linear or reproducible non-linear correlation needed for the marker to serve as an accurate indication risk will vary depending upon the marker and the assay employed. Generally a correlation of not less than about 70% will be effective. 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 analysis can be conducted within the NP patients with and without the various neurological symptoms such as those in Table 1 below, and other neurological and other symptoms such as diabetes, obesity, and gender.

The term "differentially present" and the like, is used herein to describe the level of biomarker, and refers to an increase or decrease in the amount of subject sample detected biomarker relative to the amount detected in a reference subject or population. In some embodiments the amount is expressed as a mean, median, mode amount from a reference population or sub population. The term encompasses a higher or lower level of biomarker in a test sample relative to a reference value. In certain embodiments, a biomarker is differentially present if its level 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 no more 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 a corresponding biomarker in a reference sample obtained from a reference subject or population. In certain embodiments, biomarkers are differentially present as determined through application of algorithms and optionally the allocation of a score.

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 determined to have a known disease status. In some embodiments, the reference sample is a healthy control sample or healthy control samples from a selected reference population of healthy subjects. In some embodiments, the reference sample is a sample from a COVID-19 patient who has recovered without signs of COVID-19 during the acute phase of COVID-19 or soon Post the acute phase of COVID-19 like the Convalescent controls or a subject that has recovered from NPASC or a population thereof. Ideally with like severity of COVID-19, in the present case non hospitalized patients, but if NPASC was studies in hospitalized patients, then the reference sample of subjects that have recovered COVID-19 should be similar. In some embodiments, the reference sample is a population or subpopulation of health subjects. In some embodiments, the reference value is determined or pre-determined from an earlier sample of the same subject undergoing testing. In some embodiments, the subject has NPASC as determined from an established diagnostic tool/protocol such as a questionnaire, neurology assessment etc.

The reference value may also be determined in the practise of the method or it may be a pre-determined value. In either case, the value may be a level, combination of levels, score, threshold, range, against which the level in the sample is compared, wherein the method comprises assigning a score for the sample based on the level of the at least one biomarker.

Reference to a protein biomarker includes a modified form thereof such as a mutant, isoform, post-transcriptional form, post-translationally modified form etc. A modified form includes a derivative, polymorphic variant, truncated form (truncate) and aggregated or multimeric forms or forms having expansion elements (e.g. amino acid expansion elements).

An "altered" level means an increase or elevation or a decrease or reduction in the level or ratio of one or more biomarker. The determination of the levels of the biomarkers enables establishment of a diagnostic rule based on the levels or ratios and may be relative to a reference level. Alternatively, the diagnostic rule is based on the application of a statistical, analysis of variance and/or a machine learning procedures. Such an algorithm uses relationships between biomarkers and disease or healthy status observed in reference subjects to infer relationships which are then used to predict the status of subject with unknown status. An algorithm may be employed which provides a visually detectable score or index of probability that a patient is not indicated for NPASC or is indicated for NPASC. In some embodiments, an algorithm performs a multivariate or univariate analysis function.

A positive score for NPASC likelihood or risk of same in accordance with the present disclosure permits prompt administration of NPASC therapy. Accordingly, the disclosure extends to methods of treatment and prophylaxis involving screening subjects according to herein disclosed methods and depending upon the outcome of the assay, administering an NPASC therapy to the subject. Accordingly, in another expression, the present disclosure teaches the use of the herein disclosed assays and kits and algorithms in diagnosis and treatment and/or prophylaxis of NPASC.

The present methods and kits and diagnostics are for use in the treatment and/or prophylaxis of conditions associated with NPASC. Included herein is the monitoring of subjects before and/or after treatment. Accordingly, in some embodiments the present description provides a method for evaluating in a biological sample from a subject with neurological post- viral symptoms, NPASV, NPASC or at risk of same, long COVID, COVID-19 or at risk of same the method comprising the steps of:

(i) contacting the sample with a binding agent that binds specifically to one of Biomarkers 1 to 11 in the sample and forms a biomarker-binding agent complex a;

(ii) simultaneously with (i) and/or (ii) or sequentially, contacting the sample with a second binding agent that binds specifically to a in the sample and forms to one of Biomarkers 1 to 11 complex b;

(iii) employ the amount of complex a and complex b to determine a likelihood that the subject has a likelihood of NPASC or NPASCoV.

In some embodiments, the method comprises directly or indirectly scoring the sample as comprising a healthy control level of, a convalescent COVID level or other reference level of one or two or more of biomarkers 1 to 11.

In some embodiments method includes determining a ratio of multiple biomarkers selected from biomarkers 1 to 11 from samples from the subject.

In some embodiments method includes scoring a likelihood of NPASC when biomarker levels in the subject sample are altered compared to the predetermined mean plus 2 or more standard deviations of a healthy reference population.

In some embodiments method includes scoring a likelihood of NPASC when biomarker levels in the subject sample are differentially present when compared to the predetermined mean plus 2 or more standard deviations of a healthy reference population.

In some embodiments a method includes scoring a likelihood of NPASC when biomarker levels in the subject sample are above the predetermined mean plus 2 or more standard deviations of a healthy control population or a convalescent control population. In some embodiments, the biomarker is one or more of: C5a or Gliomedin. In other embodiments the biomarkers to be measured are:

(i) C5a and/or Gliomedin and

(ii) one or more of: Transforming Growth Factor pi (TGFpi), Galactosyl ceramide sulfotransferase (Gal3STl), interferon (IFN) lambda- 1, Growth Hormone Releasing Hormone (GHRH), Lymphocyte Function Associated Antigen 3 (LFA-3), Fas Ligand (FASLG), Transgelin, Immunoglobulin Heavy Constant Gamma 1 (IgGHl), and Glycoprotein NMB (GPNMB). In some embodiments the method includes scoring a likelihood of NPASC when biomarker levels are below or above the predetermined mean plus 2 or more standard deviations from healthy control or covid convalescent control population values.

In some embodiments the required level is plus 3 or more standard deviations of reference population values, e.g., median values.

The assay described herein permits integration into existing or newly developed pathology architecture or platform systems. For example, the method described herein allows a user to determine the status of a subject with respect to a neuropathophysiological condition associated with, the assay including:

(a) receiving data in the form of levels of any one or more of Biomarkers 1 to 11 or one or more of those set forth in Tablel in a test sample from a subject via a communications network;

(b) processing the subject data via an algorithm which provides a score or disease index value by comparing levels and/or ratios of corresponding biomarkers to those from predetermined reference levels.

In some embodiments, an indication of the status of the subject to the user is transferred via a communications network. It will also be appreciated that in one example, the end stations can be hand-held devices, such as PDAs, mobile phones, or the like, which are capable of transferring the subject data to the base station via a communications network such as the Internet, and receiving the reports. When a server is used, it is generally a client server or more particularly a simple object application protocol (SOAP). This analysis may employ a principal component analysis.

Aspects of the present disclosure provide numerical values in various ranges. Slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, these ranges are intended as a continuous range including every value between the minimum and maximum values. In addition, the present disclosure extends to ratios of biomarker levels providing a numerical value associated with a state of NPASC, development or monitoring thereof. Methods for Treatment of NPASC

As disclosed herein the level of a number of proteins in subjects identified as suffering from NPASC are significantly altered from those who have recovered from COVID without NPASC and/or from subjects who were never infected with COVID. Without being bound by theory, it is believed that such proteins may underlie an aberrant immune response to CO VID infection that persists into the post-acute phase. By data mining and pathway analysis a subset of such proteins have been identified as therapeutic targets NPASC in the treatment methods disclosed herein.

Accordingly, the methods described herein include treating a subject identified as suffering from or at risk of suffering from neurological post-acute sequelae of COVID- 19 (NPASC), the method comprising administering or having administered to the subject a therapeutically effective amount of a modulator of a therapeutic target selected from the list consisting of:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta- 1 (TGF-pi);

(iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll);

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin;

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein farnesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

In some embodiments, where an activity or expression level of a therapeutic target is to be reduced, the therapeutic modulator is an inhibitor of a therapeutic target selected from among:

(i) Complement C5b-C6 complex;

(ii) Lymphocyte function-associated antigen 3 CD58

(iii) Transforming growth factor-beta-1 (TGF-pi); (iv) Vascular endothelial growth factor D;

(v) Myeloid cell surface antigen CD33;

(vi) Alcohol dehydrogenase IB;

(vii) Tumor necrosis factor receptor superfamily member 1 A;

(viii) Tumour Necrosis Factor Receptor Superfamily (TNFRSF)lOb;

(ix) Cathepsin S;

(x) TLR4: Lymphocyte antigen 96 complex

(xi) BCL2 like 1 (Bcl2Ll); and

(xii) Complement Component 5a (C5a)/Complement 5 anaphylotoxin.

In other embodiments, where an activity or expression level of a therapeutic target is to be increased, the therapeutic modulator is an activator of a therapeutic target selected from among:

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

In some embodiments a therapeutic target from among (i)-(xii) listed above is to be activated. In some embodiments a therapeutic target from among (xiii)-(xx) above is to be inhibited.

Symptoms and diagnostic tests for identifying subjects suffering from NPASC are known in the art. See, e.g., Pinzon et al., (2022). For example, to identify subjects suffering from significant neurological disturbances subjects may complete a cognitive function evaluation with the National Institutes of Health (NIH) Toolbox v2.1 instrument, which includes 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, a diagnostic test for NPASC includes a method of identifying a subject as suffering from NPASC based on the level of one or more NPASC biomarkers or NPASC biomarker combinations as disclosed herein.

NPASC Therapeutic Target Modulation

The NPASC treatment and/or prevention methods disclosed herein comprise administering to a subject identified as in need thereof a modulator of an NPASC therapeutic target as disclosed herein. In some embodiments the modulator is a therapeutic target inhibitor. In other embodiments the modulator to be administered is a therapeutic target activator.

Therapeutic target inhibition, as used herein, refers to target-specific or selective reduction of one or more of net gene expression, net protein levels, or a function/activity (e.g., a protein-protein interaction or an enzymatic activity) of a therapeutic target as disclosed herein. In some preferred embodiments a therapeutic target to be inhibited is selected from among the following:

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-pi) (GenBankNo. KAI4042861);

(iv) Vascular endothelial growth factor D (GenBank No. CAA03942);

(v) Myeloid cell surface antigen CD33 (GenBank No. AAK83654);

(vi) Alcohol dehydrogenase IB (GenBank No. AAH33009);

(vii) Tumor necrosis factor receptor superfamily member lA(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 (Bcl2Ll) (GenBank No. KAI4005058); and

(xii) Complement component 5a (C5a) (GenBank No. NP_001726.2) Therapeutic target activation, as used herein, refers to target-specific or selective increase of one or more of net gene expression, net protein levels, or a function/activity (e.g., a protein-protein interaction or an enzymatic activity) of a therapeutic target as disclosed herein. In some preferred embodiments a therapeutic target to be activated is selected from among the following:

Exemplary NPASC Therapeutic Targets to be Activated

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III (GenBank No. NP 000479.1);

(xv) Protein farnesyltransferase/geranylgeranyl transferase 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)

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

Activation of a therapeutic target may include at least about a 10% to a 200% increase in therapeutic target activity level or expression in the presence of, or resulting from, a given dose of the therapeutic target activator relative to therapeutic target activity level 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 in therapeutic target activity level or expression from about 10% to about 200%.

Examples of types of NPASC therapeutic target modulators useful for the invention include, but are not limited to, small molecule modulators, antibody modulators, polypeptide/protein modulators, nucleic acid modulators, peptide modulators, and peptidomimetic modulators. Small Molecules

In some embodiments, a therapeutic target modulator to be administered is a small molecule therapeutic target modulator. In some preferred embodiments the small molecule therapeutic target modulator is a therapeutic target inhibitor.

In some embodiments, the small molecule therapeutic target inhibitor binds specifically to the therapeutic target and reduces an activity of the therapeutic target protein, e.g., by reducing the interaction of the therapeutic target with binding partners or a receptor, or by interfering with an enzymatic activity.

In some embodiments the therapeutic target to be inhibited with a small molecule inhibitor is TGF-pi. In some embodiments the small molecule TGF-pi inhibitor to be administered is Pirfenidone (CAS 53179-13-8).

In other embodiments the therapeutic target to be inhibited is Alcohol dehydrogenase IB. In some embodiments the small molecule Alcohol dehydrogenase IB inhibitor is Fonepizole (CAS 7554-65-6).

In other embodiments the therapeutic target to be inhibited is Cathepsin S. In some embodiments the small molecule Cathepsin S inhibitor to be administered is Petesicatib (CAS 1252637-35-6).

In other embodiments the therapeutic target to be inhibited is the TLR4: Lymphocyte antigen 96 complex. In some embodiments the small molecule TLR4: Lymphocyte antigen 96 complex inhibitor to be administered is Resatorvid (CAS 243984-11-4) or Eritoran (CAS 185954-98-7).

In further embodiments the therapeutic target to be inhibited is Bcl2Ll . In some embodiments the small molecule Bcl2Ll inhibitor to be administered is Navitoclax (CAS 923564-51-6) or Obatoclax (CAS 803712-67-6).

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

In some embodiments, a small molecule therapeutic target inhibitor or activator to be administered is be a precursor compound, commonly referred to as a “prodrug” which is inactive or comparatively poorly active, but which, following administration, is converted (i.e., metabolised) to a an active therapeutic target inhibitor or activator. In those embodiments, the small molecule compound that is administered may be referred to as a prodrug. Alternatively or in addition, the a modulator compound that is administered may be metabolized to produce active metabolites which inhibit or activate a therapeutic target in a population of cells in the subject to be treated as compared the population of cells in the absence of administration of the small modulator compound. The use of such active metabolites is also within the scope of the present disclosure.

Depending on the substituents present in a modulator compound, the compound may optionally be present in the form of a salt. Salts of compounds which are suitable for use in the described methods are those in which a counter-ion is pharmaceutically acceptable. Suitable salts include those 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, such as alkane carboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, such as saturated or unsaturated di carboxylic acids, such as hydroxy carboxylic acids, such as amino acids, or with organic sulfonic acids, such as (Ci-4)-alkyl- or aryl- sulfonic acids which are substituted or unsubstituted, for example by halogen. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidien, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tbutyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine. Corresponding internal salts may also be formed. Those skilled in the art of organic and/or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallised. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates, such as hydrates, exist when the drug substance incorporates solvent, such as water, in the crystal lattice in either stoichiometric or non-stoichiometric amounts. Drug substances are routinely screened for the existence of solvates such as hydrates since these may be encountered at any stage. Accordingly it will be understood that the compounds useful for the present invention may be present in the form of solvates, such as hydrates. Solvated forms of the compounds which are suitable for use in the invention are those wherein the associated solvent is pharmaceutically acceptable. For example, a hydrate is an example of a pharmaceutically acceptable solvate.

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

Antibodies

In some embodiments, a therapeutic target modulator to be administered 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 binds specifically to the therapeutic target and reduces an activity of the therapeutic target protein, e.g., by reducing the interaction of the therapeutic target with binding partners or a receptor, or by interfering with an enzymatic activity.

In some embodiments the therapeutic target to be inhibited with an antibody inhibitor is the Complement C5b-C6 complex. In some embodiments the antibody Complement C5b-C6 complex inhibitor to be administered is Ravulizumab or a monoclonal antibody that competes for binding to the same epitope as Ravulizumab.

In other embodiments the therapeutic target to be inhibited with an antibody inhibitor is the Myeloid cell surface antigen CD33. In some embodiments the antibody Myeloid cell surface antigen CD33 inhibitor to be administered is Gemtuzumab or a monoclonal antibody that competes for binding to the same epitope as Gemtuzumab.

In other embodiments the therapeutic target to be inhibited with an antibody inhibitor is the Tumor necrosis factor receptor superfamily member 10B. In some embodiments the antibody Tumor necrosis factor receptor superfamily member 10B inhibitor to be administered is Conatumumab, Lexatumumab or a monoclonal antibody that competes for binding to the same epitope as either of Conatumumab or Lexatumumab. In other embodiments the therapeutic target to be inhibited with an antibody inhibitor is the the Tumor necrosis factor receptor superfamily member 1 A. In some embodiments the antibody Tumor necrosis factor receptor superfamily member 1 A inhibitor to be administered is Atrosimab, TNF Receptor-One Silencer (TROS) (see Fischer et al., 2020), or a monoclonal antibody that competes for binding to the same epitope as either of these.

In some embodiments an inhibitor of an NPASC therapeutic target disclosed herein is an antibody or a binding fragment thereof that binds to the therapeutic target and inhibits its interaction with binding partners, target proteins, and/or receptor(s).

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 modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light (VL) and heavy chain (VH) variable regions which may be joined directly or through a linker, or Fd fragments containing the heavy chain variable region and the CHI domain.

A scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody". Also encompassed are fragments of antibodies such as Fab, (Fab')2 and FabFc2 fragments which contain the variable regions and parts of the constant regions. Complementarity determining region (CDR)-grafted antibody fragments and oligomers of antibody fragments are also encompassed. The heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region. The antibody may be of animal (for example mouse, rabbit or rat) or human origin or may be chimeric or humanize.

As used herein the term "antibody" includes these various forms. Using the guidelines provided herein and those methods well known to those skilled in the art which are described in the references cited above and in such publications as Harlow & Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, (1988) the antibodies for use in the methods of the present invention can be readily made.

The antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain in which the light and heavy chains may be joined directly or through a linker. As used herein a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed. Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.

In another embodiment, recombinantly produced single chain scFv antibody, preferably a humanized scFv, is used in the methods of the invention.

In one embodiment, the antibodies have the capacity for intracellular transmission. Antibodies which have the capacity for intracellular transmission include antibodies such as camelids and llama antibodies, shark antibodies (IgNARs), scFv antibodies, intrabodies or nanobodies, for example, scFv intrabodies and VHH intrabodies. Yeast SPLINT antibody libraries are available for testing for intrabodies which are able to disrupt protein-protein interactions. Such agents may comprise a cellpenetrating peptide sequence or nuclear-localizing peptide sequence such as those disclosed in Constantini et al. (2008). Also useful for in vivo delivery are Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al. (2005).

In addition, the antibodies may be fused to a cell penetrating agent, for example a cell-penetrating peptide. Cell penetrating peptides include Tat peptides, Penetratin, short amphipathic peptides such as those from the Pep-and MPG-families, oligoarginine and oligolysine. In one example, the cell penetrating peptide is also conjugated to a lipid (C6-C18 fatty acid) domain to improve intracellular delivery (Koppelhus et al., 2008). Examples of cell penetrating peptides can be found in Howl et al. (2021) and Deshayes et al. (2008). Thus, the invention also provides the therapeutic use of antibodies fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to a cell-penetrating peptide sequence.

Proteins

In some embodiments, a therapeutic target modulator to be administered 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 binds specifically to the therapeutic target and reduces an activity of the therapeutic target protein, e.g., by reducing the interaction of the therapeutic target with binding partners or a receptor, or by interfering with an enzymatic activity.

In some preferred embodiments the protein therapeutic target inhibitor is a fusion protein. In some embodiments the therapeutic target to be inhibited with a fusion protein inhibitor is the Lymphocyte function-associated antigen 3 (LFA3) CD58. In some embodiments the fusion protein Lymphocyte function-associated antigen 3 CD58 inhibitor to be administered is Alefacept, a fusion protein comprising the first extracellular domain of human LFA3 fused to the hinge segment and constant regions of human IgGl.

Peptides

In some embodiments a therapeutic target inhibitor or a therapeutic activator to be used in the methods of treatment disclosed herein is a peptide. In some embodiments a peptide inhibits one or more activities of a NPASC therapeutic target as disclosed herein. In other embodiments a peptide activates or has similar activity on its own as a NPASC therapeutic target disclosed herein. In some embodiments, where the therapeutic target to be activated is, Growth Hormone Releasing Hormone, the peptide is synthetic growth hormone analogue modified peptide MR 409 (CAS 1445155-39-4).

Peptides suitable for use in the methods of the invention may be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods. Preferably, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430 A peptide synthesizer (Applied Biosystems, Foster City, Calif.), according to manufacturer's instructions. Other methods of synthesizing peptides, either by solid phase methodologies or in liquid phase, 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 detachable bond by reacting with a carboxyl group in a C-terminal amino acid. For example, in some cases an insoluble resin support used is p-hydroxymethylphenoxymethyl polystyrene (HMP) resin. Other useful resins include, but are not limited to: phenylacetamidomethyl (PAM) resins for synthesis of some N-methyl-containing peptides (this resin is used with the Boc method of solid phase synthesis; and MBHA (p-methylbenzhydrylamine) resins for producing peptides having C-terminal amide groups. During the course of peptide synthesis, branched chain amino and carboxyl groups may 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 groups). Side-chain functional groups consistent with Fmoc synthesis may be protected with the indicated protecting groups as follows: arginine (2,2,5,7,8-pentamethylchroman-6-sulfonyl); asparagine (O-t-butyl ester); cysteine glutamine and histidine (trityl); lysine (t-butyloxycarbonyl); serine and tyrosine (t-butyl). Modification utilizing alternative protecting groups for peptides and peptide derivatives will be apparent to those of skill in the art.

Peptidomimetics

In some embodiments a therapeutic target inhibitor is a peptidomimetic. All peptides are susceptible to enzymatic degradation in vivo. Therefore, peptidomimetics which retain or even enhance the biological activity of the basic peptide but have a greater circulating half life are particularly advantageous for use in the treatment methods of the invention.

While a peptidic backbone is characterised by one or more internal peptide bonds, a peptide will have peptide bonds linking each amino acid residue. Thus, a compound wherein one or more amide bond has been replaced by an alternative linker but wherein at least one amide bond remains is considered a peptidomimetic.

Peptidomimetic backbones will generally be linear or linear strings of fused cyclic groups which mimic the peptide backbone.

A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein the peptide bonds have been replaced, often by more stable linkages. By ' stable' is meant more resistant to enzymatic degradation by hydrolytic enzymes. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc. Chapter 14 of "Drug Design and Development", Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pub provides a general discussion of prior art techniques for the design and synthesis of peptidomimetics. Suitable amide bond surrogates include the following groups: N-alkylation, retro-inverse amide, thioamide, thioester, phosphonate, ketomethylene, hydroxymethylene, fluorovinyl, vinyl, methyleneamino, methylenethio, alkane and sulfonamido. Peptides and peptidomimetics will generally have a backbone of 4 to 20, preferably 7 to 16 atoms in length. Molecules having backbones at the upper end of these ranges will generally comprise beta and/or gamma amino acids or their equivalents.

Nucleic Acids

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

In some embodiments, where the therapeutic target inhibitor to be administered 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, a miRNA, or a sgRNA as disclosed herein.

Antisense Oligonucleotides

In some embodiments, where the therapeutic target inhibitor to be administered comprises a nucleic acid therapeutic target inhibitor, the nucleic acid therapeutic target inhibitor is an antisense oligonucleotide (ASO) targeting a mRNA encoding a NPASC therapeutic target as disclosed herein. In other embodiments the ASO targets the mRNA encoding a non-therapeutic target protein, but the level of which can indirectly affect the level of the therapeutic target protein.

In some embodiments, where a subject is diagnosed as having a likelihood of suffering from NPASC based on the methods disclosed, and the subject was identified as having level of TGFpi that is above the threshold level as disclosed herein, treatment for NPASC includes administering to the subject a therapeutically effective amount of an antisense oligonucleotide to a4 integrin, as targeting a4 integrin has previously been shown to inhibit TGFpi activity or signalling by reducing thrombospondin- 1 (TSP-1), increasing Latent-transforming growth factor beta-binding protein 4 (LTBP-4), and/or increasing CXC motif chemokine ligand 16 (CXCL16) as disclosed in WO 2023/039643. In some preferred embodiments the subject to be treated is not a subject that has been identified as suffering from multiple sclerosis (MSC) or Duchenne muscular dystrophy (DMD). In some preferred embodiments the antisense oligonucleotide consists of:

5' - ^Me c ^Me UG AGT ^Me CTG TTT ^Me U ^Me CMeC A ^Me U ^Me U ^Me C ^Me U - 3' (SEQ ID NO: 1) wherein, a) each of the 19 internucleotide linkages of the oligonucleotide is an O,O-linked phosphorothioate diester; b) the nucleotides at the positions 1 to 3 from the 5' end are 2'-O-(2- methoxy ethyl) modified ribonucleosides; c) the nucleotides at the positions 4 to 12 from the 5' end are 2'- deoxy rib onucl eosi des ; d) the nucleotides at the positions 13 to 20 from the 5' end are 2'-O- (2- methoxyethyl) modified ribonucleosides; and e) all cytosines are 5-methylcytosines ( ^Me C), or a pharmaceutically acceptable salt thereof or stereoisomer thereof.

Antisense oligonucleotides to a4 integrin

The present disclosure provides antisense oligonucleotides for inhibiting expression of a4 integrin. Such antisense oligonucleotides are targeted to nucleic acids encoding the a4 integrin chain of VLA-4 or a4p7 integrin. The term "inhibits" as used herein means any measurable decrease (e.g., 10%, 20%, 50%, 90%, or 100%) in a4 integrin expression.

In some embodiments an ASO is administered as an activator of a therapeutic target as disclosed herein. The skilled person in the art is aware that in many cases, if not all, “background” missplicing events of wild type gene mRNA transcripts results in a portion of untranslatable (e.g. frame-shifted and/or truncated) mRNAs that are eliminated by nonsense-mediated decay (NMD) that results in a lower net level of the corresponding protein isoform derived from the corresponding gene. In order to reduce the background of such missplicing events and to increase the level of full length, wild type protein, one or more ASOs are directed to hybridize with sequences on a pre-mRNA transcript that comprise cryptic splice sites having a propensity to induce missplicing. While not wishing to be bound by theory, it is believed that an ASO once hybridized with the pre- mRNA sterically hinders access of a splicing complex to the cryptic splice site thereby avoiding missplicing and NMD of a misspliced transcript. See Lim et al., (2020) for a review. Thus, in some embodiments an ASO activator of a NPASC therapeutic target is administered, where the therapeutic target is selected from among:

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH)

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 native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for the target nucleic acid and increased stability in the presence of nucleases.

In forming oligonucleotides, phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner so as to produce a fully or partially double-stranded compound. With regard to oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Antisense oligonucleotides useful in the methods of the present disclosure include, for example, ribozymes, siRNA, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligonucleotides which hybridize to at least a portion of the target nucleic acid.

Antisense oligonucleotides may be administered in the form of single-stranded, double-stranded, circular or hairpin and may contain structural elements such as internal or terminal bulges or loops. Once administered, the antisense oligonucleotides may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases, such as those in the RNase III and ribonuclease L family of enzymes.

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 native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for the target nucleic acid and increased stability in the presence of nucleases.

The oligonucleotides may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure. Reference may be made to Wan et al. Nucleic Acids Research 42 (22: 13456-13468, 2014 for a disclosure of antisense oligonucleotides containing chiral phosphorothioate linkages.

In forming oligonucleotides, phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner so as to produce a fully or partially double-stranded compound. With regard to oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. A person having ordinary skill in the art could, without undue experimentation, identify antisense oligonucleotides useful in the methods of the present disclosure.

Modified internucleoside linkages (backbones)

Antisense compounds of the present disclosure include oligonucleotides having modified backbones or non-natural intemucleoside 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.

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, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'- 5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, that is, a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, US 3,687,808, US 4,469,863, US 4,476,301, US 5,023,243, US 5,177,196, US 5,188,897, US 5,264,423, US 5,276,019, US 5,278,302, US 5,286,717, US 5,321,131, US 5,399,676, US 5,405,939, US 5,453,496, US 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. Modified oligonucleotide backbones that do not include a phosphorus atom therein include, for example, backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, US 5,034,506, US 5,166,315, US 5,185,444, US 5,214,134, US 5,216,141, US 5,235,033, US 5,264,562, US 5,264,564, US 5,405,938, US 5,434,257, US 5,466,677, US 5,470,967, US 5,489,677, US 5,541,307, US 5,561,225, US 5,596,086, US 5,602,240, US 5,610,289, US 5,602,240, US 5,608,046, US 5,610,289, US 5,618,704, US 5,623,070, US 5,663,312, US 5,633,360, US 5,677,437, US 5,792,608, US 5,646,269 and US 5,677,439.

Modified sugar and internucleoside linkages

Antisense compounds of the present disclosure include oligonucleotide mimetics where both the sugar and the intemucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with the target nucleic acid.

An oligonucleotide mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, in particular, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States 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.

The antisense compounds of the present disclosure also include oligonucleotides with phosphorothioate backbones and oligonucleotides with heteroatom backbones, for example, -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene (methylimino) or MMI backbone], -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)- CH2- and -O-N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as -0-P-0-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 having morpholino backbone structures of US 5,034,506, which is incorporated by reference herein for the disclosure of the just-mentioned oligonucleotides.

Modified sugars

Antisense compounds of the present disclosure include oligonucleotides having one or more substituted sugar moieties.

Examples include oligonucleotides comprising one of the following at the 2' position: OH; F; O-, S-, orN-alkyl; O-, S-, orN-alkenyl; O-, S- orN-alkynyl; or O-alkyl- O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl.

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, where n and m are from 1 to about 10. Further examples include of modified oligonucleotides include oligonucleotides comprising one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkenyl, alkynyl, 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 having similar properties.

In one embodiment, the modification includes 2'-methoxyethoxy (2'-O- CH2CH2OCH3 (also known as 2'-O-(2 -methoxy ethyl) or 2'-M0E) (Martin et al., 1995), that is, an alkoxyalkoxy group. In a further embodiment, the modification includes 2'- dimethylaminooxyethoxy, that is, a O(CH2)2ON(CH3)2 group (also known as 2'- DMAOE), or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl- amino-ethoxy-ethyl or 2'-DMAEOE), that is, 2'-O-CH2-O-CH2-N(CH3)2. Other modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. In one embodiment a 2'-arabino modification is 2'-F.

Similar modifications may 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 the 5' terminal nucleotide.

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

Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, US 4,981,957, US 5,118,800, US 5,319,080, US 5,359,044, US 5,393,878, US 5,446,137, US 5,466,786, US 5,514,785, US 5,519,134, US 5,567,811, 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.

A further modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 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 bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

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).

Modified nucleobases include other synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, 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 cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 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.

Further modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as, for example, a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4- b][l,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).

Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in US 3,687,808, those disclosed in J. I. Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), those disclosed by Englisch et al. (1991), and those disclosed by Y.S. Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, S.T. Crooke, B. Lebleu (editors), CRC Press, 1993.

Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotide. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. In one embodiment, these nucleobase substitutions are combined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, US 3,687,808, US 4,845,205, US 5,130,302, US 5,134,066, US 5,175,273, US 5,367,066, US 5,432,272, US 5,457,187, US 5,459,255, US 5,484,908, US 5,502,177, 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. Conjugates

Antisense compounds of the present disclosure may be conjugated to one or more moieties or groups which enhance the activity, cellular distribution or cellular uptake of the antisense compound. These moieties or groups may be covalently bound to functional groups such as primary or secondary hydroxyl groups.

Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes.

Moieties or groups that enhance the pharmacodynamic properties include those that improve uptake, enhance resistance to degradation, and/or strengthen sequencespecific hybridization with the target nucleic acid.

Moieties or groups that enhance the pharmacokinetic properties include those 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 lipid moieties such as a cholesterol moiety, cholic acid, a thioether, for example, hexyl- S-tritylthiol, a thiocholesterol, an aliphatic chain, for example, dodecandiol or undecyl residues, a phospholipid, for example, di-hexadecyl-rac-glycerol or tri ethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety.

Chimeric compounds

As would be appreciated by those skilled in the art, it is not necessary for all positions in a given compound to be uniformly modified and in fact, more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at a single nucleoside within an oligonucleotide.

Antisense compounds of the disclosure include chimeric oligonucleotides. "Chimeric oligonucleotides" contain two or more chemically distinct regions, each made up of at least one monomer unit, that is, a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide 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 which 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. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric antisense compounds of the disclosure may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, and/or oligonucleotide mimetics. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States 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.

Exemplary oligonucleotides

Illustrative antisense platforms known in the art include without limitation, morpholino, 1 ^st gen oligos, 2 ^nd gen oligo’ s, gapmer, siRNA, LNA, BNA, or oligo mimetics like Peptide Nucleic acids. Oligonucleotides may be naked or formulated in liposomes. Oligonucleotides may be linked to a delivery means to cells or not. Oligonucleotides may use an endosome release agent or not.

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 in several human cell lines by hybridizing to RNA encoding CD49, which is the a4 integrin subunit of VLA-4 and a4p7 integrin. In one embodiment, the oligonucleotide is the 19-sodium salt of a 3'— 5' phosphorothioate oligonucleotide 20mer also referred as a 3-9-8 MOE gapmer having a molecular weight of 7230 Daltons, in which the nucleotides at positions 1 to 3 from the 5' end are 2'-O-(2-methoxyethyl) (2'MOE) modified ribonucleosides (2'-O-(2- methoxyethyl ribose); the nucleotides at positions 4 to 12 from the 5' end are 2'- deoxyribonucleosides of which all cytosines are 5-methylcytosines; the nucleotides at positions 13 to 20 from the 5' end are 2'-O-(2-methoxy ethyl) modified ribonucleosides.

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 C A ^Me U ^Me U ^Me C ^Me U - 3'.

The empirical formula of the oligonucleotide is:

C233H327N60O129P19S19Nai9.

The ability of antisense oligonucleotide to CD49d alpha chain of VLA-4 to selectively inhibit VLA-4 in immune cells prevents significant safety events such as PML which have characterised administration of antibodies and small molecule inhibitors of VLA-4 which are pan VLA-4 inhibitors affecting all cells which express VLA-4.

In one embodiment, all uracils are 5 -methyluracils (MeU). Typically, the oligonucleotide is synthesized using 2-methoxyethyl modified thymidines not 5- methyluracils.

Tn one embodiment, all pyrimidines are C5 methylated (i.e., U, T, C are C5 methylated).

In one embodiment, the sequence of the oligonucleotide may be named by accepted oligonucleotide nomenclature, showing each 0-0 linked phosphorothioate internucleotide linkage:

2'-O-methoxyethyl-5-methylcytidylyl-(3'^-5' O, O-phosphorothioyl)-2'-O- methoxyethyl-5-methyluridylyl-(3'^5' O, O-phosphorothioyl)-2'-O- methoxyethylguanosylyl-(3'^-5' O, O-phosphorothioyl)-2'-O-deoxyadenosylyl-(3'^5' O, O-phosphorothioyl)-2'-O-deoxyguanosylyl-(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'-deoxyguanosylyl- (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'-methoxyethyl-5-methylcytidylyl- (3'— >5' O, O-phosphorothioyl)-2'-O-methoxyethyl-5-adenosylyl-(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.

RNA Interference

In some embodiments a nucleic acid therapeutic target inhibitor is an siRNA or a miRNA, which act to reduce mRNA encoding a therapeutic target disclosed herein by RNA interference.

In some embodiments the nucleic acid therapeutic target inhibitor is an siRNA against C5a. In some embodiments the siRNA against C5a is Cemdisiran (Alnylam).

The terms "RNA interference", "RNAi" or "gene silencing" refer generally to a process in which a double-stranded RNA molecule reduces the expression of a nucleic acid sequence with which the double-stranded RNA molecule shares substantial or total homology. However, it has been shown that RNA interference can also be achieved using non-RNA double stranded molecules (see, for example, US 20070004667).

The nucleic acid molecules are typically RNA but may comprise chemically- modified nucleotides and non-nucleotides.

The double-stranded regions should be at least 19 contiguous nucleotides, for example about 19 to 23 nucleotides, or may be longer, for example 30 or 50 nucleotides, or 100 nucleotides or more. The full-length sequence corresponding to the entire gene transcript may be used. Preferably, they are about 19 to about 23 nucleotides in length.

The degree of identity of a double-stranded region of a nucleic acid molecule to the targeted transcript should be at least 90% and more preferably 95-100%. The nucleic acid molecule may of course comprise unrelated sequences which may function to stabilize the molecule.

The term "short interfering RNA" or "siRNA" as used herein refers to a nucleic acid molecule which comprises ribonucleotides capable of inhibiting or down regulating gene expression, for example by mediating RNAi in a sequence-specific manner, wherein the double stranded portion is less than 50 nucleotides in length, preferably about 19 to about 23 nucleotides in length. For example the siRNA can be a nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary.

As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid (siNA), short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant 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 level or the pre-transcri phonal level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules can result from siRNA mediated modification of chromatin structure to alter gene expression.

By "shRNA" or "short-hairpin RNA" is meant an RNA molecule where less than about 50 nucleotides, preferably about 19 to about 23 nucleotides, is base paired with a complementary sequence located on the same RNA molecule, and where said sequence and complementary sequence are separated by an unpaired region of at least about 4 to about 15 nucleotides which forms a single-stranded loop above the stem structure created by the two regions of base complementarity.

Included shRNAs are dual or bi-finger and multi -finger hairpin dsRNAs, in which the RNA molecule comprises two or more of such stem-loop structures separated by single-stranded spacer regions.

Once designed, the nucleic acid molecules comprising a double-stranded region can be generated by any method known in the art, for example, by in vitro transcription, recombinantly, or by synthetic means.

Modifications or analogs of nucleotides can be introduced to improve the properties of the nucleic acid molecules. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes. Accordingly, the terms “nucleic acid molecule” and “double-stranded RNA molecule” includes synthetically modified bases such as, but not limited to, inosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8-halo adenine, 8- aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8- substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5 -trifluoromethyl uracil and 5-trifluoro cytosine.

Chemically modified siRNAs particularly suited for in vivo delivery are described in the art in, e.g. , WO2014201306, W02007051303. siRNAs targeting most human gene mRNAs are known in the art and/or available commercially, e.g.,

Aptamers

In some embodiments a 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 binds specifically to the therapeutic target and reduces an activity of the therapeutic target protein, e.g., by reducing the interaction of the therapeutic target with binding partners or a receptor, or by interfering with an enzymatic activity.

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 conformation, similar to antibodies. Aptamers can be isolated by a method called systematic evolution of ligands by exponential enrichment (SELEX). One aptamer has received approval from the US Food and Drug Administration (FDA) to treat macular degeneration of the eye, and numerous others are in preclinical or clinical trials. See Nimjee et al., (2017) for a review.

Nucleic acids encoding proteins

In some embodiments a nucleic acid therapeutic target modulator encodes a protein or peptide, whereby delivery of the nucleic acid (e.g., as an expression construct) to cells in the subject to be treated following administration results in expression of an encoded protein or peptide therapeutic modulator. In some embodiments the encoded protein or peptide, once expressed in cells expressing the therapeutic target, acts as an inhibitor of the therapeutic target by inhibiting interaction of the therapeutic target with one or more of its interaction partners, a cognate receptor, or a cognate ligand; or by acting as a dominant negative variant of the therapeutic target. In some embodiments a therapeutic target inhibitor comprises a nucleic acid in the form of an expression construct for expression of a protein or peptide that inhibits an activity of the therapeutic target.

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

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

In some embodiments, where an expression construct is used 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 the therapeutic target protein so as to increase the expression level of the therapeutic target protein so as to compensate for a deficit in the therapeutic target protein associated with NPASC as disclosed herein. In some preferred embodiments, an expression construct for therapeutic target activation is directed to expression/increased activity of a therapeutic target selected from among:

(xiii) B-cell receptor CD22;

(xiv) Antithrombin-III;

(xv) Protein famesyltransferase/geranylgeranyl transferase type-1 subunit alpha;

(xvi) Amyloid A4 protein;

(xvii) Thyroid peroxidase;

(xviii) IgG

(xix) Palmitoyl protein thioesterase; and

(xx) Growth Hormone Releasing Hormone (GHRH).

The skilled person will appreciate that expression vectors can be delivered in vivo to cells in a subject to be treated using any of a number of transfection methods known in the art, e.g., recombinant virus transduction, liposome-based transfection, electroporation, or nano-particle based transfection.

As used herein, an "expression vector" is a DNA or RNA vector that is capable of effecting expression of one or more nucleic acids in a host cell. The vector is typically a plasmid or recombinant virus. Any suitable expression vector can be used, examples of which include, but are not limited to, a plasmid or viral vector. In some embodiments, the viral vector is a retrovirus, a lentivirus, an adenovirus, a herpes virus, or an adeno- associated viral vector.

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

In some embodiments, where a nucleic acid therapeutic target inhibitor is to be administered, the nucleic acid encodes a programmable nuclease which inhibits therapeutic target activity by inactivating or reducing expression of the corresponding therapeutic target gene. As used herein, the term “programmable nuclease” relates to nucleases that are “targeted” (“programmed”) to recognize and edit a pre-determined genomic location. In some embodiments the encoded polypeptide is a programmable nuclease “targeted” or “programmed” to introduce a genetic modification into the therapeutic target gene or regulatory region thereof. In some embodiments, the genetic modification is a deletion or substitution in the therapeutic target gene or in a regulatory region thereof.

In some embodiments, the programmable nuclease may be programmed to recognize a genomic location by a combination of DNA-binding zinc-finger protein (ZFP) domains. ZFPs recognize a specific 3-bp in a DNA sequence, a combination of ZFPs can be used to recognize a specific a specific genomic location. In some embodiments, the programmable nuclease may be programmed to recognize a genomic location by transcription activator-like effectors (TALEs) DNA binding domains. In an alternate embodiment, the programmable nuclease may be programmed to recognize a genomic location by one or more RNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more DNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more hybrid DNA/RNA sequences. In an alternate embodiment, the programmable nuclease may be programmed by one or more of an RNA sequence, a DNA sequences and a hybrid DNA/RNA sequence.

Programmable nucleases that can be used in accordance with the present disclosure include, but are not limited to, RNA-guided engineered nuclease (RGEN) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-cas (CRISPR-associated) system, zinc-finger nuclease (ZFN), transcription activator-like nuclease (TALEN), and argonautes.

In some embodiments, the nuclease is a RNA-guided engineered nuclease (RGEN). In some embodiments the RGEN is from an archaeal genome or is a recombinant version thereof. In some embodiments the RGEN is from a bacterial genome or is a recombinant version thereof. In some embodiments the RGEN is from a Type I (CRISPR)-cas (CRISPR-associated) system. In some embodiments the RGEN is from a Type II (CRISPR)-cas (CRISPR-associated) system. In some embodiments the RGEN is 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 the RGEN is a multi-component enzyme. In some embodiments the RGEN is a single component enzyme. In some embodiments the RGEN is CAS3. In some embodiments the RGEN is CASIO. In some embodiments the RGEN is CAS9. In some embodiments the RGEN is Cpfl (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 more than one RNA and/or DNA. In some embodiments the programmable nuclease may be a DNA programmed argonaute (WO 14/189628).

In other embodiments a nucleic acid therapeutic target modulator, /.< ., 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 initerest. In other embodiments, where a nucleic acid therapeutic target activator is used, the modified mRNA encodes the therapeutic target protein to increase its expression level in the subject to be treated in at least the cells that normally express the therapeutic target protein endogenously. Chemically modified mRNAs and their synthesis is described in detail in, e.g., WO 2011/130624. Typically, chemically modified mRNAs comprise (i) a 5' synthetic cap for enhanced translation; (ii) modified nucleotides that confer RNAse resistance and an attenuated cellular interferon response, which would otherwise greatly reduce translational efficiency; and (iii) a 3' poly-A tail. Typically, chemically modified mRNAs are synthesized in vitro from a DNA template comprising an SP6 or T7 RNA polymerase promoter-operably linked to an open reading frame encoding the protein to be expressed. The chemically modified mRNA synthesis reaction is carried in the presence of a mixture of modified and unmodified nucleotides. In some embodiments modified nucleotides included in the in vitro synthesis of chemically modified mRNAs are pseudo-uridine and 5-methyl-cytosine. 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 methylated enzymatically at the N-7 position of the guanosine to form mature mCAP. When preparing chemically modified mRNAs, a 5' cap is typically added prior to administration//// vivo transfection in order to stabilize the modified mRNA and significantly enhance translation. In some embodiments a 4: 1 mixture of a cap analog to GTP is used in transcription reactions to obtained 5'-capped chemically modified mRNAs. In preferred embodiments, the Anti Reverse Cap Analog (ARCA), 3'-O-Me-m7G(5')ppp(5')G is used to generate a 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 in, e.g., WO 2011/130624, and WO/2012/138453.

Recombinant Viruses

A variety of recombinant virus types are suitable for expression of a therapeutic inhibitor or therapeutic target inhibitor as disclosed herein. Preferably, a recombinant virus to be used is a replication-deficient recombinant virus.

In some embodiments, the recombinant virus to be administered is a DNA virus. Suitable types of DNA viruses include adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus, and lentivirus. Methods for design, production, and use of such types of recombinant DNA viruses are established in the art, as exemplified in Fukazawa et al. (2010) and in “Gene Therapy Protocols” for adenovirus; “Adeno- Associated Virus: Methods and Protocols” for AAV; Cody et al. (2013) and “Herpes Simplex Virus: Methods and Protocols” for HSV; “Gene Therapy Protocols Vol. 1 : Production and In Vivo Applications of Gene Transfer Vectors” and Amer (2014) for retrovirus; and Merten et al. (2016) and Emeagi et al. (2013) for lentivirus. In some preferred embodiments, the recombinant virus to be used in the treatment method is an adenovirus. In other preferred embodiments the recombinant virus is a lentivirus.

In other embodiments, the recombinant virus to be administered is a recombinant, replication-deficient RNA virus. Suitable types of replication deficient or replication- competent RNA viruses Alphavirus (e.g., Sindbis or Semliki Forest Virus), Flavivirus (e.g., Kunjin virus), Paramyxovirus (e.g., Sendai virus), Rhabdovirus (e.g., vesicular stomatitis virus), and Orthomyxovirus (e.g., influenza A virus). Methods for design, production, and use of such types of recombinant RNA viruses are established in the art, as exemplified in Lundstrom (2015) and Quetglas et al. (2010) for Alphavirus; Hoang- Le et al. (2009) and Usme-Ciro et al. (2013) for Flavivirus; Cattaneo (2010) for Paramyxovirus; Finke et al. (2005) and Chang et al. (2010) for Rhabdovirus; and U.S. 8,475,806 for Orthomyxovirus.

Examples of suitable promoters for driving expression of biotherapeutic agents from a recombinant virus in a method described herein include, but are not limited to, constitutive promoters such as, CMV, CAG, EF-l-I, HSV1-TK, SV40, P-actin, and PGK promoters. In other embodiments, a promoter is an inducible promoters, such as those containing TET-operator elements. In certain embodiments, target-selective promoters are used to drive expression of biotherapeutic agents in specific cell types in vivo in the subject to be treated.

In some embodiments, where two or more proteins (e.g., two different therapeutic targets or two different isoforms of a therapeutic target) are to be expressed from a recombinant virus, the recombinant virus contains an expression cassette encoding a polycistronic mRNA (a "polycistronic expression cassette"), which, upon translation gives rise to independent polypeptides comprising different amino acid sequences or functionalities. In some embodiments, a polycistronic expression cassette encodes a "polyprotein" comprising multiple polypeptide sequences that are separated by encoded by a picomavirus, e.g., a foot-and-mouth disease virus (FMDV) viral 2A peptide sequence. The 2A peptide sequence acts co-translationally, by preventing the formation of a normal peptide bond between the conserved glycine and last proline, resulting in ribosome skipping to the next codon, and the nascent peptide cleaving between the Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the 'upstream' protein, while the proline is added to the N-terminus of the 'downstream' protein, which during translation allow cleavage of the nascent polypeptide sequence into separate polypeptides. See, e.g., Trichas et al. (2008). In other embodiments, a polycistronic expression cassette may incorporate one or more internal ribosomal entry site (IRES) sequences between open reading frames incorporated into the polycistronic expression cassette. IRES sequences and their use are known in the art as exemplified in, e.g., Martinez-Sales (1999).

In some embodiments, a recombinant virus used in the method has targeted tropism, e.g., tropism for a particular cell type as reviewed in Bucholz et al. (2015). Suitable targeting moieities, to be incorporated into a recombinant viral capsid surface, include ligands that bind to cell surface receptors that are overexpressed by cancer cells. For example, the Her2/neu receptor, frequently overexpressed in breast cancer cells, can be targeted by incorporating a designed ankryrin repeat protein (DARPin) ligand, as has been done for lentivirus (Munch et al., 2011) in AAV (Munch et al., 2013). In another example a recombinant lentivirus is designed to target P-glycoprotein, overexpressed on the surface of melanoma cells, by incorporating an antibody into the viral capsid surface (Morizono et al., 2005).

Administration and Dosing Regimes for NPASC Therapeutic Target Modulators

In some embodiments, a method for treating a human subject identified as suffering from NPASC or preventing such a condition, includes administration of a pharmaceutical composition containing at least one inhibitor or activator of a therapeutic target as disclosed herein, or a pharmaceutically acceptable salt, pharmaceutically acceptable N-oxide, pharmaceutically active metabolite, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof, in therapeutically effective amounts to said subject.

A therapeutic target inhibitor or therapeutic target activator, is administered to prevent, cure or at least partially arrest the symptoms of a patient already suffering from and/or diagnosed as suffering from NPASC. Amounts effective for treatment or prevention 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. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial).

In prophylactic applications, a modulator of a therapeutic target disclosed herein is administered to a patient identified as susceptible to or otherwise at high risk of developing NPASC. Such an amount is defined to be a "prophylactically effective amount or dose” /.< ., a dose sufficient to prevent or reduce the onset of infection. In this use, the precise amounts also depend on the particular condition, the patient's state of health, weight, timing, etc. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).

In a case where a subject's status does improve, upon reliable medical advice, the administration of a NPASC therapeutic target modulator may be given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (z.e., a "drug holiday"). The length of the drug holiday can vary between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, or 60 days. The dose reduction during a drug holiday may be from 10%-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%.

The amount of a given NPASC therapeutic target modulator (z.e., inhibitor or activator) that will be suitable as a therapeutically effective dose will vary depending upon factors such as the type and potency of the therapeutic target modulator to be administered, the severity/stage of NPASC, the characteristics (e.g., weight) of the subject or host in need of treatment, and prior or concurrent treatments, but can nevertheless be routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated. In general, however, doses employed for adult human treatment will typically be in the range of 0.02-5,000 mg per day, or from about 1-1,500 mg per day for small molecule therapeutic agents. The desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

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

In some embodiments the treatment methods disclosed herein include determining a level of the therapeutic target level or an activity level of the therapeutic target being modulated.

The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages may be altered depending on a number of variables, not limited to the activity of the NPASC therapeutic target modulator to be used, the severity of one or more NPASC symptoms to be treated, the mode of administration, and the judgment of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. NPASC therapeutic target inhibitors or activators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human and non-human subjects. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

Formulation of Therapeutic Agents

Modulators of any of the NPASC therapeutic targets disclosed herein can be formulated either alone or in combined 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 administration routes.

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

Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the therapeutic agents described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical solid dosage forms can include, in addition to the therapeutic agents, one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof.

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, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

Suitable filling agents 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, starches, pregelatinized starch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

In order to release the therapeutic agents from a solid dosage form matrix as efficiently as possible, disintegrants are often used in the formulation, especially when the dosage forms are compressed with binder. Disintegrants help rupturing 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, but are not limited to, natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel® PH101, Avicel®PH102, Avicel®PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®.), crosslinked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a crosslinked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USP Pharmacoat-603, hydroxypropylmethylcellulose acetate stearate (Aqoate HS-LF and HS), hydroxy ethylcellulose, hydroxypropylcellulose (e.g, Klucel®), ethylcellulose (e.g, Ethocel®), and microcrystalline cellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonites, gelatin, polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone®K-12), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fillers which itself can act as moderate binder. Formulators skilled in art can determine the binder level for the formulations, but binder usage level of up to 70% in tablet formulations is common.

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 fumerate, alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

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 maltodextrin), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins and the like.

Suitable wetting agents 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, sodium docusate, triacetin, vitamin E TPGS and the like.

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.

Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, polyvinylpyrrolidone, e.g., polyvinylpyrrolidone KI 2, 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, vinyl pyrrolidone/vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, polysorbate-80, hydroxy ethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

It should be appreciated that there is considerable overlap between additives used in the solid dosage forms described herein. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in solid dosage forms described herein. The amounts of such additives can be readily determined by one skilled in the art, according to the particular properties desired.

Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

The aqueous suspensions and dispersions described herein can remain in a homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The homogeneity should be determined by a sampling method consistent with regard to determining homogeneity of the entire composition. In one embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

In addition to the additives listed above, the liquid formulations can also include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.

Injectable Formulations

Formulations suitable for intramuscular, subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), 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 the maintenance of 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 preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, 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, appropriate formulations may include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally known in the art.

Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a 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 agents in water-soluble form. Additionally, suspensions of the therapeutic agents 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 which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of a therapeutic agent to allow 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 dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more therapeutic agents. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multidose containers, with an added preservative.

EXAMPLES

EXAMPLE 1

Proteomics Analysis of 7300 plasma proteins

The majority of plasma samples were originally obtained from subjects enrolled for the conduct of a previous study on Neuro Covid long-haulers at Northwestern Medicine Neuro-COVID clinic in Chicago, USA as reported by Visvabharathy et al. www.medrxiv.org/content/10.1101/2021.08.08.21261763. ("Northwestern Study") entitled: "Neuro-COVID long-haulers exhibit broad dysfunction in T cell memory generation and responses to vaccination." The Northwestern study was designed to compare humoral and cellular immune responses in NPASC subjects with responses found in healthy COVID convalescents and healthy controls. A total of 111 prevaccination participants comprised hospital 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 six weeks post infection, and an average of approximately 4 to 6 months. Some subjects were vaccinated during the course of the reported study. Demographic and neurological symptoms of the patients used in the above reported study were tabulated and reproduced here for reference purposes in Table 3. TABLE 4

Plasma samples from the Northwestern Study were kindly provided by Dr Igor Koralnik, at the Northwestern Medicine Neuro-COVID clinic in Chicago, USA/Northwestern University. A commercial aptamer-based proteomics assay, SomaScan® was used herein to analyse patient heparinised plasma samples and the relative fluorescence units (RFU) of over 7300 proteins was determined for three patient groups: • Group 1 : 48 patients currently affected by Neuro-PASC (NP) never hospitalised for CO VID (with positive PCR or IgG antibody test). 42 of the 48 subjects were from the above Northwestern study plus 6 plasma samples from suitable subjects

• Group 3: 20 patients who have recovered from SARS-CoV-2 infection and show no symptoms of PASC or NPASC (Convalescent Controls CC) never hospitalized for CO VID (with positive PCR or IgG antibody test). 17 of 24 patients were from the above Northwestern study, plus 3 suitable subjects

• Group 4: 24 healthy controls (HC) subjects (patients have never had SARS-CoV- 2 infections). The plasma samples were from 24 patients of the 31 HC patients from the above Northwestern study.

Accordingly, plasma samples for the instant study were made up from 83 (of 103) samples in group 1, 3, and 4 from Visvabharathy et al study (above) and were further selected from the non-hospitalized and non-vaccinated subjects. An extra 6 and 3 samples were included in group 1 and 3 subjects respectively making a total of 92 samples. Group 2 samples in the Northwestern Hospital reported study were hospitalised subjects with NPASC and these were not included. One patient sample from another site called CL-2, was obtained and called study group 2 but was actually a non-hospitalised subject and so was added to the present 47 subjects in Group 1 and together the 48 subjects were called all NP (and herein after Group 1, though plots will show the pooled group as Group 1+2).

Multiplexed proteomics assays are carried out using approx.. 7300 aptamers called SOMAmers in the SomaScan Assay. Briefly, in an illustrative example, the sample is incubated with a mixture of SOMAmers each containing a biotin, a photocleavable group, and a fluorescent tag followed by capture of all SOMAmer-protein complexes on streptavidin beads. After stringent washing of the beads to remove unbound proteins and labelling of bead-associated proteins with biotin under controlled conditions, the complexes are released from the beads back into solution by UV light irradiation and diluted into a high concentration of dextran sulphate, an anionic competitor. The biotin that is originally part of the SOMAmer remains on beads. The anionic competitor coupled with dilution selectively disrupts non-cognate complexes, and because only the proteins now contain biotin, the complexes are re-captured on a second set of beads from which unbound SOMAmers are removed by a second stringent washing. The SOMAmers that remain attached to beads are eluted under high pH- denaturing conditions and hybridized to sequence-specific complementary probes printed on a standard DNA microarray' The result is a mixture of SOMAmers that quantitatively reflects protein concentrations in the original sample. The modified nucleotides in SOMAmers are designed to maintain canonical base-pairing (in a DNA duplex, adducts at the 5-position of pyrimidines are directed toward the major groove of DNA) and hybridize effectively to unmodified DNA oligonucleotides on the array The capture of SOMAmers on a hybridization array permits quantitative determination of the protein present in the original sample by converting the assay signal (relative fluorescence units, RFUs) to analyte concentration. Thus, the Somascan assay takes advantage of the dual nature of SOMAmer aptamers as molecules capable of both folding into complex three- dimensional structures, which is the basis of their unique binding properties, and hybridization to specific capture probes.

EXAMPLE 2

Statistical Analysis and proteins of interest to likelihood testing identified

The level results in the different groups, NP (also referred to as Group 1 and Group 2, or simply Group 1), Group 3 (CC) and Group 4 (HC) were analysed using both parametric and non-parametric statistical analyses. ANOVA and Kruskalis Wallis (KW) statistical analyses were used to compare Group 1 NP, Group 3 CC and Group 4 HC, with p-values adjusted using Benjamini -Hochberg false discovery rate (FDR) and very stringent Bonferroni statistical tests. Proteins of interest were assessed using Somalogic’s ® Dataviz software. Plasma proteins with Bonferroni adjusted values of <0.05 were identified (See Table 2). Plasma proteins with an FDR of (<0.02) were identified. The median % change in Group 1 NP versus convalescent control (CC) and healthy control (HC) patients was computed for the proteins of interest which met the statistical significance. The ANOVA median results and Bonferroni and FDR results are in Table 2 for the biomarkers. These proteins were inserted into Genetrail3 to identify linked pathways. The protein biomarkers identified had various functions in neural, autoimmune, viral, vascular, adipose, and clotting pathways. Also in inflammation, and fibrosis, integrin, and antigen presentation pathways.

The level results in the different groups, NP (also referred to as Group 1 and Group 2 or simply Group 1) were also analysed vs Group 3 (CC), and NP were also analyzed vs Group 4, using both parametric and non-parametric statistical analyses. T-Test, median results between Group 1 NP vs Group 3 CC and Group 1 NP vs Group 4 HC are also in Table 2. The T-test and U-test analyses was conducted with p-values adjusted using Benjamini -Hochberg false discovery rate (FDR) and Bonferroni statistical tests, and using Somalogic’s ® Dataviz software to identify proteins of interest.

Groups 3+4 comparison using FDR or Bonferroni adjusted p-values tests, found no statistical differences. T and U Tests, ANOVA and KW were thus used to determined the median results between Group 1 NP vs Group 3 and Group 4 controls Table 2.

Table 1 describes the proteins found to have differentiated circulating levels drawn based on three different statistical analyses to identify important molecules relevant to NPASC. Whisker plots of circulating levels of each of the indicated biomarkers (1-11) in the NP group, the CC group, and the HC group are shown in Figures 1-11.

EXAMPLE 3

Exemplary Biomarker Combinations

In another aspect, the present invention provides combinations of two or more biomarkers identified herein to assay for the likelihood of NPASC in a subject or sample or for monitor the progress of a subject relative to NPASC, such as in a subject with Covid or NPASC, or a subject receiving therapy.

The method comprises measuring in a biological sample from the subject the level of:

(i) Gliomedin and the level of (ii) C5a anaphylatoxin, and/or (iii) TGF beta 1 and/or (iv) Gal3STl. C5a and TGFbetal are two markers of inflammation and fibrosis. The combination of markers enhances the sensitivity of the method in the population, detecting more subjects with NPASC. Preferably the NPASC or NPASV sample is compared to Gliomedin levels and TGFbetal and C5a levels of HC reference but the levels may also be compared to levels in the CC reference. Preferably the NPASC or NPASV sample is compared to the Gal3STl of the CC reference.

In this way potentially all subjects with one or more symptoms of NPASC can have their status verified with a combination of markers, to allow for time to recover, treatment to progress, and for certificates to be provided to employees, and insurers. For maximum specificity, especially for Covid convalescent subjects that have recovered from Covid (as opposed to subjects for which Covid status had never been determined), it is useful to measure the level of all 11 biomarkers listed in Table 1.

5 EXAMPLE 4

Therapeutic target analysis

Among the proteins identified as having levels that differed between NPASC and control group(s) based on statistically stringent criteria as described above, a subset were 10 filtered/prioritised as therapeutic targets based on: (1) percentage difference between Group 1 vs Groups 3 + 4; (2) direction of the difference (higher or lower level); (3) Genetrail3 linked pathways analysis, as summarised in Table 5 below; and (4) the existence of at least one therapeutic agent for the target or target pathway (approved or in clinical trials).

15

As part of the pathway analysis, statistical tests with a FDR < 0.02 were carried out to identify pathways for which the number of identified “hits” had statistical significance. This pathway statistical analysis is summarised in Table 6 below:

20 Table 6 - Summary of pathways of interest identified by statistical analysis of hit frequency (FDR<0.02)

A series of therapeutic target proteins were identified based on this analysis as summarised in Table 7 below, which includes proteins found to have a higher level in Group 1 (NPASC subjects) vs Group 3 (healthy control subjects) and having a therapeutic agent that modulates the therapeutic targets, as well as therapeutic target proteins found to have a lower level in Group 1 vs Group 3. Figs. 12 and 14, illustrate exemplary therapeutic target proteins CD33, VEGFD, and TGF-pi found to have higher levels in plasma of NPASC subjects than in that of control subjects. Fig. 13 illustrates exemplary target proteins Thyroid peroxidase and Amyloid A4 found to have lower levels in plasma of NPASC subjects than in that of control subjects

Table 7

Therapeutic Targets Identified by ANOVA FDR < 0.02 (Group 1 vs Group 3)

EXAMPLE 5

Phase 2 Clinical Study of ATL1102 to Treat NPASC (prophetic example)

A phase 2 study is undertaken to assess the potential efficacy and safety of a 25 mg/week ATL1102 dose administered once weekly s.c for 24 weeks in subjects experiencing NPASC (> 12 weeks and < 6 months post COVID-19) without hospitalization. The phase II study is a randomized, double blinded, placebo control study in NPASC patients with cognitive function deficit. Patients and researchers are not to know if they are getting a placebo or the ATL110225mg/week. Treatment is self- administered s.c at home with subjects returning every 4 weeks to the hospital. The ratio between treated and placebo groups is 1 : 1 for a total of 34 subjects, of both genders. Outcome measures at baseline and weeks 6 (Day 42), 12 (Day 84), 18 (Day 126), and 24 (Day 168).

Primary outcome measures- Change from baseline to endpoint week 12 and 24 selected from cognitive tests

1. Baseline Digit Symbol Substitution Test (DSST) CogState Online Cognitive Battery

2. Trails Making Test (TMT)-A/B will be used to assess change in cognitive function.

3. Rey's auditory verbal learning test (RAVLT) will be used to assess change in verbal memory. 4. Change in Perceived Deficits Questionnaire, 20-item (PDQ-20) will be used to assess change in subjective cognitive functioning.

5. Change from baseline in PROMIS® Cognitive Function Score at Days

6. Change from baseline in NIH Toolbox tests: attention, processing speed, executive function and working memory impairment

7. Daily Diary of Long NPASC Related Symptom Severity Score during the treatment phase

8. Number of symptom-free days of NPASC associated symptoms that were present at the start of study treatment (Day 0) based on self-assessment using daily symptom diary, any symptoms scored as mild, moderate or severe at baseline are scored as absent (or none)

9. Progression (or worsening) of NPASC through Day a. moderate at baseline are scored as severe through Day b.mild at baseline are scored as moderate or severe through Day c. absent at baseline are scored as mild, moderate or worse through Day

Secondary outcome measures- Change from baseline to endpoint week 12 and 24 selected from fatigue tests, and sleep tests

1. Fatigue Severity Scale (FSS) will be used to assess change in severity and impact of fatigue. Prevalence of fatigue in patients with post-COVID-19 depression and/or anxiety

2. Mental Fatigue Questionnaire Score is a self-reported scale

3. Change from baseline in PROMIS® Fatigue Score at Days

4. Change from baseline in PROMIS® Sleep Disturbance Score at Days

5. Change from baseline in PROMIS-57 patient reported T scores - physical function, anxiety, depression, fatigue, sleep disturbance, pain interference and global Pain score

Other Outcome Measures: Exploratory

1. Change from baseline in CD3+ CD4+CD49d+ and CD3+CD8+CD49d+ T cell count & CD3-NK and B cells

2. Change from baseline in Transforming growth factor beta 1 (TGF betal), thrombospondin- 1 and LTBP4; protein levels and/or activity levels. 3. Change from baseline in CRP on Days

4. Change from baseline in serum cytokine and chemokine levels on Days

5. Plasma biomarkers that may predict and/or act as pharmacodynamic indicators of pharmacologic activity Drug vs placebo (SomaScan®)

Inclusion Criteria:

1. Subjects age > 18 years at the time of signing the informed consent form.

2. Male or female with NPASC -having neurological symptoms from cognitive deficits (e.g., reported as brain fog, difficulty thinking, poor attention, executive function and memory impairment etc), and potentially one or more of headaches, parasthesia (tingling numbness), dysgeusia, anosmia, myalgia, Post Traumatic Stress Disorder (PTSD), sleep disturbances, anxiety and depression, with fatigue

3. Non-hospitalized post COVID-19 patients with a documented SARS- CoV-2 positive approved RT-PCR).

4. Subjects who are experiencing prolonged neurological symptoms e.g., cognitive deficit fatigue for over 12 weeks after their COVID-19 RT-PCR negative test.

5. Subject has not fully recovered from NPASC for at least over 12 weeks despite a negative SARS-COV-02 test.

6. Subject is experiencing the symptoms for at least over 12 weeks that interferes with normal daily activities. Symptoms must be new symptoms i.e., subject had not sought medical treatment for the symptoms prior to COVID- 19: oExtreme fatigue - feeling overtired with low energy and a strong desire to sleep but unable to have good sleep. o Cognition deficits

Exclusion Criteria:

1. Tested positive for SARS-CoV-2 infection at the time of screening (acute infection) which will involve a nasal swab sample or another FDA-approved test.

2. Subjects who had recovered fully from COVID-19 and have a new onset of extreme symptoms not due to COVID-19

3. Subjects with serious co-morbidities are excluded. For example: oLiver enzymes are >2X ULN; oeGFR is <60 ml/min by the CKD- EPI equation; oHb is <11 mg/dL; oPlatelet count is <100K; oBelow Normal blood counts

4. History of neuropathy prior to COVID-19 infection.

5. PASC affecting the musculoskeletal, digestive, pulmonary, and no. Neurological PASC (NPASC).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. For instance a study where subjects diagnosed as having a likelihood of suffering from NPASC based on the methods disclosed, have high levels of TGF-betal above the threshold are treated with ATL1102. For instance a cross over study where after the first six months of treatment of patients on placebo, they are treated for six months with ATL1102 and patients on ATL1102 in the first six months are treated with placebo in the second six months. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown 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.

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

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. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. 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), el5004 (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 ID, 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.

Rundstrbm, 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