CN117222424A - Alpha-1-antitrypsin (AAT) for the treatment and/or prophylaxis of neurological disorders - Google Patents

Abstract

The present invention relates to a composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof or a vector or genetically modified cell comprising a sequence encoding AAT for use in the treatment and/or prevention of a disease or disorder of the nervous system or symptoms thereof.

Description

Alpha-1-antitrypsin (AAT) for the treatment and/or prophylaxis of neurological disorders

The present invention relates to a composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof or a vector or genetically modified cell comprising a sequence encoding AAT for use in the treatment and/or prevention of a disease or disorder of the nervous system or symptoms thereof.

A disease or condition of the nervous system is one that can significantly affect the peripheral nervous system and/or the central nervous system (PNS/CNS). In the last decade, neuroinflammation has become increasingly important in our understanding of neurological disorders. Inflammation itself may cause the disease directly or indirectly, but it does, of course, contribute to the pathogenesis of the entire Peripheral Nervous System (PNS) and Central Nervous System (CNS) diseases. Peripheral diseases such as green-barre syndrome (GBS) (Chang et al 2012), fibular muscular dystrophy (Hoyle et al 2015), neuropathic pain, fibromyalgia and other neuropathies (PN) (Martin-Aguilar, pascual-Goni and Qumol 2019), as well as central diseases including Parkinson's Disease (PD) and motor neuron disease (Marogennni et al 2020), alzheimer's Disease (AD) (Hampel et al 2020) and other dementias, multiple Sclerosis (MS) (Baecher-Allan, kaskow and Weiner2018; matthews 2019), amyotrophic Lateral Sclerosis (ALS), ischemia and traumatic brain injury, depression and autism spectrum disorders have all been associated with mechanisms of activated microglial cell driving (Skapper et al 2018).

Hereditary peripheral neuropathy constitutes a group of highly diverse disorders, the most common form of which is collectively referred to as fibular muscular dystrophy (CMT), with a global prevalence of 1:2,500 and a high degree of genetic heterogeneity involving >100 different genes (Bird, T.D.,1993,GeneReviews (R)). Among this broad genetic pattern possibilities, CMT1A is the most common form and accounts for 80% of type 1 CMTs. Characterized by an intrachromosomal repetition of the PMP22 gene (Stavrou, sargiannidou et al 2021). The major component of peripheral neuropathy is damage to myelin, either following abnormal development (myelination disorder) in its genetic form (CMT 1A-F and-X), or directly in the acquired form (acute/chronic inflammatory demyelinating polyneuropathy; AIDP/CIDP).

Myelin is produced by Schwann Cells (SCs) in PNS and is critical for proper transmission of electrical impulses in nerves. In complex neuronal/glial cross-communications (Rao and Pearse 2016) required for proper myelin modulation, a number of different signaling pathways are involved, including growth factors, integrins and cell adhesion molecules, but more importantly, critical neuregulin type 1III (NRG 1-III) signals pass through ERBB2/3 receptors (Taveggia, C.et al 2005, neuron 47 (5): 681-694.), and their proteolytic abscisse modulators, tumor necrosis factor-alpha converting enzyme TACE (also known as ADAM 17) (Fleck, D.et al 2016,J Biol Chem 291 (1): 318-333). Although a wide variety of leading edge treatment strategies (CRISPR/Cas 9 editing, viral-based gene delivery, siRNA nanoparticles) are currently being explored, none successfully completed clinical trial phase III and did not bring actual treatment to CMT (Fridman, v. And m.a. sapora, 2021,Neurotherapeutics 18 (4): 2236-2268). Furthermore, while inhibition of TACE has been shown to promote myelination, few of these strategies target the NRG1/EBRB2/3/TACE pathway. TACE/ADAM17 is a transmembrane protein comprising an extracellular zinc-dependent protease domain. In the context of CMT1A, ADAM17 is known to inhibit SC-mediated myelination by cleaving NRG1-III in the epidermal growth factor domain in a ligand-independent manner (La Marca, R.,2011,Nat Neurosci 14 (7): 857-865.). In terms of the action of the human protease α -1-antitrypsin (AAT), conflicting evidence was reported in the literature, in particular, AAT was demonstrated not to interact with TACE in 2013 (van't Wout e.f. et al 2014,Hum Mol Genet.;23 (4): 929-4), as opposed to an early report in 2010 stating that AAT does interact with TACE and inhibit its activity in a dose-dependent manner (Bergin, d.a. et al 2010,J Clin Invest 120 (12): 4236-4250.).

Various injuries caused by neurological disorders have been increasingly viewed as global public health challenges, and the burden thereof is expected to increase in the next few decades.

Diseases or disorders of the nervous system may be caused by viruses. Viruses (such as, for example, coronaviruses) cause diseases, including diseases or disorders of the nervous system, in animals and humans worldwide. Coronaviruses are RNA viruses.

Human coronavirus (HCoV) is known to cause mainly upper and lower respiratory tract infections. Examples of human coronaviruses are: the beta coronavirus causing middle east respiratory syndrome (named MERS-CoV), the beta coronavirus causing severe acute respiratory syndrome (named SARS-CoV or SARS-CoV-1), the novel coronavirus causing coronavirus disease 2019 or covd-19 (named SARS-CoV-2), the alpha coronavirus 299E, the alpha coronavirus NL63, the beta coronavirus OC43 and the beta coronavirus HKU1.

A disease or syndrome caused by SARS-CoV-2 infection is also known as COVID-19.SARS-CoV-2 infection can be either asymptomatic or result in a disease or syndrome associated with mild or severe symptoms. The most common symptoms of diseases or syndromes associated with SARS-CoV-2 (also known as SARS-CoV-19) infection are fever and cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, and reduced or altered smell or taste. Other symptoms include loss of appetite, weight loss, stomach ache, conjunctivitis, rash, lymphoma, apathy, and somnolence.

Patients with severe symptoms may develop pneumonia. A significant number of patients with pneumonia require passive oxygen therapy. Noninvasive ventilation and nasal high-throughput oxygen therapy can be applied to cases of mild to moderate non-hypercarbonated blood pneumonia. For patients with severe acute respiratory syndrome or acute respiratory distress syndrome (SARS/ARDS) and mechanical ventilation, a lung protective ventilation strategy must be implemented.

Although the major complication of coronavirus disease 2019 (COVID-19) is respiratory failure, a significant number of patients have been reported to have neurological symptoms affecting the peripheral and central nervous systems (Niazkar, zibaee et al 2020Neurol Sci 41 (7): 1667-1671; nordvig, fong et al 2021,Neurol Clin Pract 11 (2): e135-e 146). The hematopoietic pathway, retrograde/anterograde transport along the peripheral nerve, and rare direct invasion are considered possible neuro-invasion mechanisms of neurotropic viruses including SARS-CoV-2 (barres 2021Brain Behav Immun Health 14:100251;Tavcar,Potokar et al 2021,Front Cell Neurosci 15:662578). Severe cases of SARS-CoV-2 often exhibit disproportionate and aberrant inflammatory responses, including systemic upregulation of cytokines, chemokines, and pro-inflammatory signals (Najjar et al 2020,J Neuroinflammation 17 (1): 231). This systemic excessive inflammation can impair neurovascular endothelial function, disrupt the blood brain barrier, ultimately activate the CNS immune system, and lead to CNS complications (Amruta, chastain et al, 2021,Cytokine Growth Factor Rev 58:1-15).

Although recent pandemic of covd-19 has attracted attention, several other viruses have been associated with major brain diseases such as alzheimer's disease, parkinson's disease and multiple sclerosis. Diseases or syndromes associated with viral infections further include various diseases or syndromes such as inflammatory diseases and are a major burden to society.

The biological common feature of many CNS and PNS neurodegenerative diseases is the sustained and acute inflammatory response due to a carefully planned cytokine release (also known as a "cytokine storm") in the feed forward circulation. Thus, inhibition of inflammatory responses is a central goal of therapeutic strategies. However, the subtle nature of the inflammatory mechanisms behind the various mediators is not fully understood.

Thus, there is a need to improve therapies for diseases or conditions of the nervous system.

The above technical problem is solved by the embodiments disclosed herein and defined in the claims.

Thus, the invention relates in particular to the following examples:

1. a composition for use in the treatment and/or prevention of a disease or condition of the nervous system, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof.

2. A vector for treating and/or preventing a disease or condition of the nervous system comprising a nucleic acid sequence encoding an AAT protein.

3. A genetically modified cell comprising a nucleic acid sequence encoding an AAT protein for use in the treatment and/or prevention of a disease or condition of the nervous system.

4. The composition for use according to example 1, the vector for use according to example 2 or the genetically modified cell for use according to example 3, wherein the disease or disorder of the nervous system is an inflammatory disease or disorder of the nervous system.

5. The composition for use according to example 4, the vector for use according to example 4 or the genetically modified cell for use according to example 4, wherein the inflammatory disease or disorder of the nervous system is a disease or disorder mediated by bone marrow cells of the nervous system.

6. The composition for use according to any one of embodiments 1, 4 or 5, the vector for use according to any one of embodiments 2, 4 or 5 or the genetically modified cell for use according to any one of embodiments 3 to 5, wherein the disease or syndrome of the nervous system is a disease or syndrome selected from the group consisting of dementia, multiple sclerosis, amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease and huntington's disease.

7. The composition for use according to any one of embodiments 1, 4 to 6, the vector for use according to any one of embodiments 2, 4 to 6 or the genetically modified cell for use according to any one of embodiments 3 to 6, wherein the disease or disorder of the nervous system is at least one symptom of a disease or disorder of the nervous system selected from the group consisting of: tremor, memory loss, slurred speech, dizziness, vision changes, and headache.

8. The composition for use according to example 1, the vector for use according to example 2 or the genetically modified cell for use according to example 3, wherein the disease or disorder of the nervous system is a disease or disorder of the peripheral nervous system.

9. The composition for use according to example 8, the vector for use according to example 8 or the genetically modified cell for use according to example 8, wherein the disease or disorder of the peripheral nervous system is a motor and sensory neuropathy of the peripheral nervous system.

10. The composition for use according to example 9, the vector for use according to example 9 or the genetically modified cell for use according to example 9, wherein the sensory neuropathy of the peripheral nervous system is hereditary motor and sensory neuropathy of the peripheral nervous system.

11. The composition for use according to example 10, the vector for use according to example 10 or the genetically modified cell for use according to example 10, wherein the hereditary motor and sensory neuropathy of the peripheral nervous system is fibula muscular dystrophy or a symptom thereof, preferably at least one symptom selected from the group consisting of: weakness of the legs, ankle and/or foot, reduced muscle mass of the legs and/or foot, high arch, bending of the toes, reduced running ability, difficulty in lifting the foot at the ankle, abnormal gait, frequent trips or falls, hypoesthesia or unconsciousness of the legs and/or foot.

12. The composition for use according to any one of embodiments 1, 4 to 11, wherein the AAT protein, variant, isoform and/or fragment thereof is human plasma extracted.

13. The composition for use of any one of embodiments 1, 4 to 11, wherein the alpha 1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof is recombinant alpha 1-antitrypsin (rhAAT), variant, isoform and/or fragment thereof.

14. The composition for use according to any one of embodiments 1, 4 to 13, wherein the composition comprises at least one pharmaceutical carrier.

15. The composition for use according to embodiment 14, wherein the pharmaceutical carrier is a blood brain barrier permeability enhancer.

16. The composition for use according to any one of embodiments 1, 4 to 11, the vector for use according to any one of embodiments 2, 4 to 6 or the genetically modified cell for use according to any one of embodiments 3 to 7, wherein the composition, the vector or the genetically modified cell is formulated for intra-cerebral administration, intravenous injection, intravenous infusion, quantitative pump infusion, inhalable nasal spray, eye drops, skin patches, sustained release formulations, ex vivo gene therapy or ex vivo cell therapy.

Accordingly, in one embodiment, the present invention relates to a composition for treating and/or preventing a disease or condition of the nervous system, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof.

As used herein, the term "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to a clinical intervention that attempts to alter the natural course of the treated individual, and may be used to prevent or progress during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, reducing the rate of disease progression, improving or ameliorating a disease state, and alleviating or ameliorating prognosis. In some embodiments, the antibodies of the invention are used to delay the progression of a disease or to slow the progression of a disease.

As used herein, the term "disease or condition of the nervous system" refers to a group of diseases or conditions in which pathology involves the nervous system. In some embodiments, the disease or disorder of the nervous system described herein is a disease or disorder selected from the group consisting of: 12q14 microdeletion syndrome, 15q13.3 microdeletion syndrome, 15q24 microdeletion syndrome, 22q11.2 microdeletion syndrome, 22q13.3 microdeletion syndrome, 2-methylbutyryl-CoA dehydrogenase deficiency, 2q23.1 microdeletion syndrome, 2q37 deficiency syndrome, 3-alpha hydroxy acyl-CoA dehydrogenase deficiency, 3MC syndrome, XXXY syndrome, XYY syndrome, XXXXY syndrome, 5q14.3 microdeletion syndrome, 6-pyruvoyl tetrahydropterin synthetase deficiency syndrome, alscoger syndrome, beta-free lipoproteinemia, ABri amyloidosis, clear compartment deficiency, copper-free hemoglobinula, acromiosis syndrome, cartania type acropodophyllosis, rodreggs acromegaly, acute cholinergic autonomic nerve dysfunction, acute central nervous system demyelinating event, acute disseminated encephalomyelitis acute intermittent porphyria, acute motor and sensory axonal neuropathy syndrome, ADCY 5-related dyskinesia, adenosine monophosphate deaminase 1 deficiency, adenylyl succinate deficiency, addi syndrome, adrenomyeloneuropathy, adult glucanosis, adult onset linear body myopathy, sleep phase advance syndrome, callus hypoplasia, age-related peripheral neuropathy, agnosia, ai Kaer di syndrome, aicare-Goutheres syndrome, AIDS dementia syndrome, al Gazali Aziz Salem syndrome, alanine urine syndrome, albino otopathy, alcohol or sensorineural motor deficiency due to malnutrition, alcoholic neuropathy, alcoholic peripheral neuropathy, alexander disease, ALG11-CDG (CDG-Ip), ALG12-CDG (CDG-Ig), ALG13-CDG, ALG12-CDG, ALG1-CDG (CDG-Ik), ALG2-CDG (CDG-Ii), ALG3-CDG (CDG-Id), ALG6-CDG (CDG-Ic), ALG8-CDG (CDG-Ih), ALG9-CDG (CDG-IL), allen-henden-dadel syndrome, moina alopecia-mental young syndrome, alopecia, epilepsy, sepsis, hypophrenia, alopecia-contracture-dwarfism-mental disorder syndrome, alopecia-mental disorder syndrome, alpepeis syndrome, alpha-ketoglutarate dehydrogenase deficiency, alpha-mannosidosis, alpha-thalassemia x-linked disorder syndrome, childhood alternating hemiplegia, alzheimer's disease type 4, alzheimer's disease without neurofibrillary tangles, aminoacylase 1 deficiency Aminolevulinic acid dehydratase deficient porphyrin, aminomatous microcephalus, A Mi Shen linear myopathy, amyloid neuropathy, myogenic dermatomyositis, amyotrophic lateral sclerosis 6-type amyotrophic lateral sclerosis-Parkinson's disease/dementia syndrome 1, amyotrophic lateral sclerosis, anaplastic astrocytoma, anaplastic ganglioma, anaplastic oligodendroglioma, andermann's syndrome, andersen-Tawil syndrome, iron particle and young cell ataxia, spinocerebellar ataxia, brain deformity, hereditary neuro-cutaneous hemangioma, irises, listless renal dysplasia, anti-synthetase syndrome, aortic arch abnormality, anoxia, the symptoms of ataxia, arachnoid cyst, arachnoiditis, aromatic L-amino acid decarboxylase deficiency, congenital multiple joint bending, distal end type, X-chain type, joint bending renal dysfunction cholestasis syndrome, arts syndrome, asparthaminuria, ataxia telangiectasia, ataxia with ocular nerve loss type 1, ataxia with ocular nerve loss type 2, ataxia with ocular nerve loss type 4, ataxia with vitamin E deficiency, ataxia-telangiectasia, bone hypoplasia type 2, bone hypoplasia type 3, atkin syndrome, atypical Rate syndrome, fresh red nevus autism, autosomal dominant central nuclear myopathy, autosomal dominant cerebellar ataxia/deafness/narcolepsy, autosomal dominant fibular muscle atrophy type 2 autosomal dominant deafness-first dystrophy syndrome, autosomal dominant intermediate fibula muscular dystrophy, autosomal dominant cerebral leukodystrophy with autonomic neuropathy, autosomal dominant neuronal ceroid lipofuscinosis 4B, autosomal dominant nocturnal frontal lobe epilepsy, autosomal dominant very complex intellectual disability, autosomal dominant optic atrophy overlay syndrome, autosomal dominant partial epilepsy with auditory characteristics, autosomal dominant spinal muscular atrophy, autosomal recessive axonal neuropathy with neuromuscular rigidity, autosomal recessive central nuclear myopathy, autosomal recessive fibula muscular atrophy with hoarseness, autosomal recessive intermediate fibula muscular atrophy type a, autosomal recessive intermediate fibula muscular atrophy type B, autosomal recessive young parkinson's disease, autosomal recessive neuronal ceroid lipofuscinosis 4A, adult neuronal ceroid lipofuscinosis, autosomal recessive primary kohlrabi, autosomal recessive spastic ataxia 4, autosomal recessive spastic paraplegia type 49, autosomal recessive spinocerebellar ataxia 9, B4GALT1-CDG (CDG-IId), bannayan-Riley-Ruvalcaba syndrome, bass syndrome, battaglia-Neri syndrome, beck muscular dystrophy, behavioral variant frontotemporal dementia, behcet's disease, bell palsy, benign primary blepharospasm, benign familial neonatal epilepsy, benign familial neonatal-infant seizure, benign hereditary chorea, benign motor epilepsy (BRE), beta-helical protein-related neurodegeneration, bei Sile-mer's disease, bettaglycosis bilateral frontal lobe polynocerebback, bilateral frontal parietal polynocerebback, bilateral lateral sagittal roof occipital polynocerebback, bilateral lateral split polynocerebback, binswangger's disease, biotin enzyme deficiency, biotin-thiamine-reactive basal gangliopathy, berk-barrer's syndrome, bixler Christian Gorlin syndrome, blepharo-nasal-facial malformation syndrome, nodulohead syndrome, bohring-Opitz syndrome, borjeson-forsman-Lehmann syndrome, bowen-Conradi syndrome, carpal reproduction syndrome, short finger-limb mid-mental disorder-heart defect syndrome, brain dopamine-serotonin vesicle transport disease, brain-lung-thyroid syndrome, X-linked gill arch syndrome, brosidopathy, brooks Wisniewski Brown syndrome, brown-Sequard syndrome, bullous dystrophy, syndrome C, ka Bei Zazeng syndrome, CADASIL, trunk pre-bent syndrome, chiral arthropathy-cox-changing heart peritonitis syndrome, CANOMAD syndrome, cantu syndrome, hat myopathy, face skin syndrome, carey-Finerman-ziner syndrome, carney syndrome, cataract ataxia deafness, catel Manzke syndrome, caudal appendage deafness, caudal degeneration, central axopathy, central nervous system germ cell tumor, central nerve cell tumor, central pain syndrome, pontine central myelinolysis, cerebellar ataxia, cerebellar degeneration, cerebellar hypoplasia, cerebellar interstitial disease 3, cerebellar hypoplastic hydrocephalus, brain autosomal recessive arterial disease, cerebral spongiform hemangioma, cerebral hypoplasia, neuropathy, ichthyosis, palmar and plantar keratosis syndrome, folic acid deficiency, cerebral cyst, cerebral palsy cerebral paralysis ataxia, xu Dongxing cerebral palsy, cerebral palsy spastic hemiplegia, cerebral palsy spastic monoplegia, cerebral palsy spastic quadriplegia, cerebral sclerosis, brain-facial-joint syndrome, brain-eye-facial-skeletal syndrome, brain-eye-nasal syndrome, cerebral fluid leakage, brain tendinomatosis, neuronal ceroid lipofuscinosis 1, cervical hirsutism peripheral neuropathy, chanagrin-Dorfman syndrome, fibular muscular atrophy type 1A, chediak-Higashi syndrome, chiari deformity type 1, chiari deformity type 2, chiari deformity type 4, childhood speech loss, childhood morbid rhabdomyopathy, chorea-acanthosis, choriocarcinoma, chorioallantoma, christianson syndrome, chromosome 17p13.1 delectation syndrome, chromosome 17q11.2 delectation syndrome, chromosome 1q13.11 delectation syndrome, chromosome 1p36 deficiency syndrome, chromosome 3 p-syndrome, chronic hiccup, chronic lymphocyte inflammation, chronic progressive extraocular muscle paralysis, chudley Rozdilsky syndrome, cisplatin-induced sensory neuropathy, cleft palate low-cone abnormality, cluster headache, COACH syndrome, COASY protein-related neurodegeneration, kou Cishi disease, cobb syndrome, cockayne syndrome type I, cockayne syndrome type II, cockayne syndrome type III, coenzyme Q10 deficiency, coffin-Lowry syndrome, coffin-Siris syndrome, COG1-CDG (CDG-IIg), COG4-CDG (CDG-IIj), COG5-CDG (CDG-III), COG7-CDG (CDG-IIe), COG8-CDG (CDG-IIh), coan syndrome, cold-induced sweat syndrome, complex regional pain syndrome, congenital deficiency syndrome, central ventilation syndrome, chronic respiratory depression, and the like congenital cytomegalovirus, congenital fiber type imbalance, congenital extraocular muscle fibrosis, congenital systemic lipodystrophy type 4, congenital pain insensitivity with anhidrosis, congenital intrauterine infection-like syndrome, congenital larynx paralysis, congenital mirror dyskinesia, congenital muscular dystrophy, congenital myasthenia syndrome, congenital rubella, congenital toxoplasmosis, persistent spike during diffuse wave sleep syndrome, tics, hypopsia of cornea, cornelia de Lange syndrome, callus hypoplasia, cortical blindness, cortical hypoplasia, cortical basal degeneration, costal-lo syndrome, crane-Heise syndrome, cranio-frontal-nasal dysplasia, craniopharyngeal tube tumor, craniofacial spinal cleft, craniofacial brain dysplasia, creutzfeldt-Jakob disease, cromes syndrome, curry Jones syndrome, cylindrical spiraling myopathy, crepstein facial neuromuscular skeletal syndrome, giant cell inclusion body disease, D-2-hydroxyglutarate urine disease, dandy-Walker cyst, dandy-Walker-like deformity, dandy-Walker deformity, danon disease, dapsone-induced neuropathy, DDOST-CDG (CDG-Ir), DEAF 1-related disorders, dentate nucleus pallidum Louis body atrophy, dermatomyositis, familial developmental language disorder, diabetic neuropathy, dihydrolipoamide dehydrogenase deficiency, dihydropteridine reductase deficiency, diphtheria, distal myopathy with vocal weakness, DOOR syndrome, dopamine beta hydroxylase deficiency, dopamine transporter deficiency syndrome, dopa-responsive dystonia, DOOR syndrome DPAGT1-CDG (CDG-Ij), DPM1-CDG (CDG-Ie), DPM2-CDG, DPM3-CDG (CDG-Io), dravet syndrome, duane syndrome, dubowitz syndrome, dunaliella muscular dystrophy, dykes Markes Harper syndrome, autonomic imbalance-like disorders, dysequilibrium syndrome, congenital hyperkeratosis, autosomal dominant congenital hyperkeratosis, autosomal recessive congenital hyperkeratosis, X-linked congenital hyperkeratosis, myoclonus cerebellar coordination disorder, dystonia 2, DYT-PRKRA, DYT-THAP1, DYT-TOR1A, DYT-TUBB4A, early infant epileptic encephalopathy 25, early infant extracataract, early adult chromosomal Alzheimer's disease, early parkinsonism-mental disorder syndrome, eastern equine encephalitis, empty saddle syndrome, narcolepsy, brain-craniofacial adiposity, encephalopathy, eosinophilic fasciitis, eosinophilic granuloma, ependymoma, simple epidermolysis bullosa with muscular dystrophy, juvenile absence epilepsy, occipital calcified epilepsy, progressive myoclonus epilepsy type 3, epileptic with myoclonus-tone loss, epiphyseal dysplasia hearing loss deformity, paroxysmal ataxia, erythromelalgia, essential tremor, fabricius disease, facial-onset neuronal disease, facial shoulder-arm muscular dystrophy, fallo syndrome, familial amyloidosis, familial bilateral striatal necrosis, familial caudal hypoplasia, familial congenital pulley nerve paralysis, familial autonomic nerve dysfunction, familial encephalopathy, familial exudative vitreoretinopathy, familial focal epilepsy, familial hemiplegic migraine, facial hemiparalysis familial hemophagocytic lymphocytosis, familial infantile convulsive familial infantile paroxysmal chorea, familial perforated brain, familial transthyretin amyloidosis, familial or sporadic hemiplegic migraine, fabert disease, fatal familial insomnia, fatal infantile cerebral myopathy, fatty acid hydroxylase-related neurodegeneration, FBXL 4-related cerebral myopathy mitochondrial DNA depletion syndrome, febrile infection-related epileptic syndrome, feigenbaum Bergeron Richardson syndrome, filippi syndrome, fine-Lubinsky syndrome, fingerprint body myopathy, fitzsimmons Walson Mellor syndrome, fitzsimons-Guilbert syndrome, flotating-Harbor syndrome, flynn Aird syndrome, focal hypoplasia, focal segmental glomerulosclerosis, fountain syndrome, foxG1 syndrome, fragile X syndrome, fragile XE syndrome, redson dermis disorder, the disease may be selected from the group consisting of frontotemporal dysplasia, frontotemporal dementia, frans syndrome, fucosidosis, fushan muscular dystrophy, corydalis enzyme deficiency, galactosialidosis, gallway-Mowat syndrome, gamma aminobutyric acid transaminase deficiency, gangliocytoma, GAPO syndrome, gaucher disease type 1, gaucher disease type 2, gaucher disease type 3, gemignani syndrome, genital patella syndrome, genonaja syndrome, gerstmann-Straussler-schenker disease, megaxius neuropathy, gillespi syndrome, brain glioma disease, glucose transporter type 1 deficiency syndrome, congenital glutamine deficiency, glutarate type I, glutarate type II, glutarate type III, glycogen storage disease type 13, glycogen storage disease type 2, glycogen storage disease type 3 glycogen storage disease type 4, glycogen storage disease type 5, glycogen storage disease type 7, GM1 gangliosidosis type 1, GM1 gangliosidosis type 2, GM1 gangliosidosis type 3, GM3 synthase deficiency, GMs syndrome, goldberg-Shprintzen megacolon syndrome, gomez Lopez Hernandez syndrome, GOSR 2-associated progressive myoclonus ataxia, graham-Cox syndrome, granulomatous polyangiitis, griscelli syndrome type 1, grubben de Cock Borghgraef syndrome, GTP cyclohydrolase I deficiency, GTPCH1 deficient DRD, guanidinoacetic acid methyltransferase deficiency, green-barre syndrome, guriri syndrome, chorionic and retinal cyclotron atrophy, hair defect-photosensitive-intellectual disorder syndrome, hallermann-streilff syndrome, hall-ggs syndrome, hamanishi Ueba Tsuji syndrome, leprosy, harg's disorder, huperzian syndrome, huperzine's syndrome, GTP-mania syndrome, gtch-mania syndrome, gtPCH1 deficient DRD, guan-methyltransferase deficiency syndrome, green-barre syndrome, choriocarcinoma syndrome, and the like, harrod Doman Keele syndrome, hartnup disease, hashimoto's brain disease, angioblastoma, continuous hemilateral headache, hemilateral megabrain disease, hennekam syndrome, hereditary vascular disease, hereditary fecal porphyria, hereditary diffuse white matter brain disease, hereditary fibromatosis with tendon contracture, myopathy and pulmonary fibrosis, hereditary reproductive spasms, hereditary hemorrhagic telangiectasia type 2, hereditary hemorrhagic telangiectasia type 3, hereditary hemorrhagic telangiectasia type 4, hereditary oversvular, hereditary motor and sensory neuropathy type 5, hereditary compression susceptibility neuropathy, hereditary susceptibility to pressure paralysis (localized and symmetrical), hereditary proximal myopathy with early respiratory failure, hereditary sensory motor neuropathy with hyperelastic skin hereditary sensory and autonomic neuropathy type 1e, hereditary sensory and autonomic neuropathy type 2, hereditary sensory and autonomic neuropathy type 7, hereditary sensory and autonomic neuropathy type v, hereditary sensory neuropathy type 1, hereditary spastic paraplegia, hereditary vascular retinopathy, hern's ndez-Aguirre Negrete syndrome, herpes simplex encephalitis, auricular shingles, HIBCH deficiency, homocystinuria, horizontal gaze paralysis with progressive scoliosis, hoyeraal Hreidarsson syndrome, HSD10 disease, HTLV-1-related myelopathy/spastic paraplegia, human HOXA1 syndrome, human immunodeficiency virus-induced neuropathy, huntington's disease, hurle syndrome, hurle-Scheie syndrome, hydropathies, cerebral hydrocephalus (e.g., due to congenital ventricular catheter stenosis), hydrocephalus-palate-contracture syndrome, hydrocephalus, hypercoagulopathy, hyperbetaalaninemia, hyperkali periodic paralysis, hypermethionine blood, hyperphenylalanine blood, hyperproline blood, hyperprolinemia type 2, dejerine-sotttas hypertrophic neuropathy, autosomal dominant hypocalcemia, hypokali periodic paralysis, itana melaninia, insufficient myelination (e.g. basal ganglia and/or cerebellar atrophy), hypoparathyroidism-intellectual impairment-malformation syndrome, hypourethral-intellectual impairment, goldblatt-type syndrome, hypothalamic hamartoma, ichthyosis-alopecia-valgus-eye-valgus-intellectual impairment, idiopathic intracranial hypertension, idiopathic spinal hernia, inclusion body myositis, pigmentary incontinence, infantile axonal neuropathy infant cerebellar retinal degeneration, infant choriocapillaris syndrome, infant myofibromatosis, infant axonal dystrophy, infant-onset spinocerebellar ataxia, infant megalobrama spasticity, infant-onset ascending hereditary spastic paralysis, infectious acute encephalopathy 3, intellectual deficit Buenos-Aires, hand and foot Xu Dongxing intellectual impairment, corpus callosum dysplasia intellectual impairment, intellectual impairment-hypoevolutism syndrome, intellectual impairment-malformation-hypogonadism-diabetic syndrome, intellectual impairment-severe speech retardation-mild malformation syndrome, intellectual impairment-spasticity-fmger malformation syndrome, moderate congenital rhabdomyopathy, internal carotid artery dysplasia, internal nerve fasciculation, IRN syndrome, isaacs syndrome, chromosome 15 syndrome of isobipolar centromere, johanson-Blizzard syndrome, johnson neuroectodermal syndrome, joubert syndrome, juberg Marsidi syndrome, juvenile amyotrophic lateral sclerosis, juvenile dermatomyositis, juvenile Huntington's disease, johnson neuroectodermal syndrome, jouberg Marsidi syndrome, juvenile amyotrophic lateral sclerosis, and combinations thereof adolescent polymyositis, adolescent primary lateral sclerosis, kabuki syndrome, kanzaki disease, kapur Torillo syndrome, kaufman eye-brain-face syndrome, KBG syndrome, KCNQ 2-related disorders, kearns-Sayr syndrome, kennedy disease, dwarfism of follicular keratosis, brain atrophy, nuclear jaundice, keutel syndrome, king deboroough syndrome, kleine Levin syndrome, klumpke paralysis, kosztollanyi syndrome, kozlowski-Krajewska syndrome, kerberkovich disease, kuru, kuzniecky Andermann syndrome, L-2-hydroxyglutarate, rakes encephalitis, laband syndrome, lafora disease, lyne distal myopathy, lambert Eaton muscle weakness syndrome, landau-Kleffner syndrome, I-arginine: glycine guanyltransferase deficiency, delayed distal myopathy, markesbery-Griggs type, lateral meningioma syndrome, laurence-Moon syndrome, LCHAD deficiency, leber hereditary optic neuropathy, leigh syndrome, lennox-Gastaut syndrome, lenz Majewski bone hypertrophy dwarfism, lenz microoculi syndrome, lesch Nyhan syndrome, white matter dystrophy, white matter disease (e.g., with thalamus and brainstem involvement and high lactic acid), levic Stefanovic Nikolic syndrome, lewis-Sumner syndrome, lhemitte-Duclos disease, li-Fraumeni syndrome, limb-girdle muscular dystrophy (e.g., 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, 2P, 2Q, 2S, type 2T), LGI1 antibody limbic encephalitis, localized cutaneous sclerosis, lipoic acid synthetase deficiency, no brain return deformity 1, no brain return deformity 2, X-linked no brain return deformity, localized hypertrophic neuropathy, atresia, logo type progressive aphasia, lowe eye-brain-kidney syndrome, lowry Maclean syndrome, lujan syndrome, lyme, mac Dermot Winter syndrome, giant head-small-paralysis syndrome, giant thrombocytopenia progressive deafness, mal de debarquement syndrome, male pseudobic intellectual disability syndrome, hyperpyrexia, malignant heat, malignant hyperjoint type, infantile hyperdynamic cervical, CDpartial sublevel 1-manoeuve, manoeuvering syndrome 1-Ky (e.g. subjaw 1-subjaw) disorder, with microcephaly), mannosidosis, marchiafava Bignami disease, marden-Walker syndrome, ma Fanti syndrome-autosomal recessive dysnoesia syndrome, marinesco-Sjogren syndrome, martsolf syndrome, mcDonogh syndrome, mcLeod's acanthocytosis syndrome, meckel's syndrome, MECP2 repeat syndrome, medrano roldan syndrome, medulloblastoma, megabrain leukoencephalopathy (e.g., subcortical cyst), megabrain-polynocele-multi-fingered hydrocephalus syndrome, megaloblastic anemia, megacornea-intellectual impairment syndrome, mehes syndrome, MEHMO syndrome, meier-Gorlin syndrome, meige syndrome, melnick-needle syndrome, meningioma, meningitis, menis's disease, paresthesia femoral pain, metaphysis-dysnoesia-conductive deafness syndrome, methionine adenosyltransferase deficiency, mecobalamin deficiency cbl g type, methylmalonic acid homocystinuria type cblc, mg2-cdg (cdg-iia), microlip syndrome, short-head, small-bone development, small-primary dwarfism (e.g., small-primary dwarfism 2-primary, benign dwarfism), montreal, torillo), microcephaly, autosomal dominant microcephaly, microcephaly brain deficiency spastic hypernatremia, microcephaly cervical fusion abnormality, microcephaly deaf syndrome, microcephaly glomerulonephritis Ma Fanti sign, microcephaly microcorneal syndrome, microcephaly-cardiomyopathy, microreplication Xp11.22-p11.23 syndrome, microcephaly syndrome 10, microcephaly syndrome 4, microcephaly syndrome 8, microcephaly with linear skin defect syndrome, microscopic polyangiitis, migraine (e.g., with brainstem precursor), mild phenylketonuria, miller-Dieker syndrome, miller-Fisher syndrome, micro-axin-empty myopathy with extraocular muscle paralysis, mitochondrial complex I deficiency, mitochondrial complex II deficiency, mitochondrial DNA deficiency syndrome, brain myopathy form with methylmalonic urine syndrome, mitochondrial DNA related Leigh syndrome, mitochondrial encephalomyopathy lactic acidosis and stroke-like attacks, mitochondrial membrane protein related neurodegeneration, mitochondrial myopathy and iron-granulomatous anemia, mitochondrial myopathy with diabetes, mitochondrial myopathy with lactic acidosis, mitochondrial neurogastrointestinal type encephalomyopathy syndrome, mitochondrial trifunctional protein deficiency, mixed connective tissue disease, sanhe's far-end muscle lesions, mo Bisi syndrome, MOGS-CDG (CDG-IIb), mohr-Tranebjaerg syndrome, molybdenum cofactor deficiency, monoamine oxidase A deficiency Morse-Rawnsley-Sargent syndrome, morvender syndrome, mousa Al din Al Nassar syndrome, smog, MPDU1-CDG (CDG-If), MPI-CDG (CDG-Ib), MPV 17-related liver and brain mitochondrial DNA depletion syndrome, mucoliposis type 4, mucopolysaccharidosis type III, mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, multifocal motor neuropathy, multiple congenital malformation-hypomyotonia-epileptic seizure syndrome, multiple congenital malformation-hypomyodystonia-epileptic seizure syndrome type 2, multiple myeloma, multiple sulfatase deficiency, multiple system atrophy, multiple system smooth muscle dysfunction syndrome, myoeye-encephalopathy, dystrophy white matter spongiform formation, multiple system atrophy, megacone muscular dystrophy, myophosphorylase kinase deficiency, muscle contracture Ehlers-Danlos syndrome, myasthenia gravis, myelitis, spinal cerebellar disorders, spinal cord meningoectomy, MYH 7-related shoulder fibula myopathy, myhre syndrome, myoclonus epilepsy with broken red fibers, myoclonus cerebellar ataxia deafness, myoclonus-dystonia, myoglobin urosis, myopathy with extrapyramidal signs, myosin-storage myopathy, congenital myotonia, myotonic myodystrophy type 1, myotonic myodystrophy type 2, N syndrome, nance-Horan syndrome, narcolepsy, NBIA/DYT/PARK-PLA2G6, necrotizing autoimmune myopathy, neonatal adrenoleukodystrophy, neonatal meningitis, neonatal early syndrome, neu Laxova syndrome, neuroblastoma, cutaneous melanoma neuro-facial-finger-kidney syndrome, neuroferritin disease, neurofibromatosis type 1, neurofibromatosis type 2, antipsychotic malignancy, neuromyelitis optica spectrum disorder, neuronal ceroid lipofuscinosis 10, neuronal ceroid lipofuscinosis 2, neuronal ceroid lipofuscinosis 3, neuronal ceroid lipofuscinosis 5, neuronal ceroid lipofuscinosis 6, neuronal ceroid lipofuscinosis 7, neuronal ceroid lipofuscinosis 9, neuronal nuclear inclusion body disease, neuropathic pain, neuropathic ataxia retinal pigment degeneration syndrome, remote hereditary motor neuropathy Jerash type, hereditary motor and sensory neuropathy Okinawa type, hereditary motor and sensory neuropathy Russe type, neutral lipid deposition myopathy, neutropenia, nevus basal cell carcinoma syndrome, newly refractory status epilepticus, nicolaides-Baraitser syndrome, niemann-pick disease type a, niemann-pick disease type B, niemann-pick disease type C1, niemann-pick disease type C2, non-sleep-wake disorder, non-dystrophic myotonic, noonan syndrome, nori's disease, northern epilepsy, eye-brain-skin syndrome, eye-face-heart-tooth syndrome, oculopharyngeal muscular dystrophy, oculopharyngeal distal myopathy, okamoto syndrome, olfactory neuroblastoma, oligodendrocytoma, oligodendroglioma, olympic syndrome, olivopogonitis cerebellar atrophy, umbilical cord expansion deadly palate syndrome, OPHN1 syndrome, ocular matrix-myoclonus syndrome, optic atrophy 2, optic access glioma, ornithine carbamoyldeficiency orofacial syndrome 1, orofacial syndrome 10, orofacial syndrome 2, orofacial syndrome 3, orofacial syndrome 4, orofacial syndrome 5, orofacial syndrome 6, orthostatic intolerance caused by NET deficiency, osteopenia and sparse hair, osteoporosis-pseudoglioma syndrome, ear-palate-syndrome type 1, ear-palate-syndrome type 2, ouvrier Billson syndrome, giant brain return-mental disorder-epileptic syndrome, PACS 1-related syndrome, painful orbit and systemic neurofibromatosis-Ma Fanti syndrome, pallidol cone beam syndrome, pallister W syndrome, paris-kenyamine mixed syndrome, pantothenate kinase-related neurodegeneration, tremor paralysis, juvenile paralysis, hunter paralysis, congenital paramyotonic, paraplegia, paraneoplastic/autoimmune (anti-Hu-related) neuropathy, parkinson's disease type 3, parkinson's disease type 9, narcolepsy, paroxysmal severe pain, paroxysmal migraine, narcolepsy, parsonage Turner syndrome, partington syndrome, PCDH 19-related epilepsy limited to women only, pediatric autoimmune neuropsychiatric disease associated with streptococcal infection, PEHO syndrome, peter Li Zawu s-merzbach disease, paraventricular gray ectopic, periventricular leukomalacia, peter syndrome, peters plus syndrome, pfeiffer Mayer syndrome, pfeiffer Palm Teller syndrome, pfeiffer type cranium syndrome, PGM3-CDG, PHACE syndrome, phosphoglycerate kinase deficiency, phosphoglycerate kinase-like material, phosphogypsum, and phosphogypsum phosphoglyceromutase deficiency, phosphoserine transaminase deficiency, photosensitive epilepsy, peter-hopkins syndrome, peter-hopkins like syndrome, plasmacytoma, yellow astrocytoma multiforme, PMM2-CDG (CDG-Ia), POEMS syndrome, poliomyelitis, POLR 3-related leukodystrophy, polyarteritis nodosa, polycystic lipodystrophy with sclerotic leukoencephalopathy, multiple neuropathy-mental retardation-acromegaly-premature menopausal syndrome, capped development of the bridge brain, brain bridge cerebellum hypoplasia type 1, brain bridge cerebellum hypoplasia type 2, brain bridge cerebellum hypoplasia type 3, brain bridge cerebellum hypoplasia type 4, brain bridge cerebellum hypoplasia type 5, brain bridge cerebellum hypoplasia type 6, post-poliomyelitis syndrome, porphyria, post-column ataxia with retinitis pigmentosa, post-partum progressive microcephaly, post-partum epilepsy and post-partum brain atrophy, potassium-aggravated myotonia, potocki-Lupski syndrome, PPM-X syndrome, prader-Willi habit, primary amoebic meningoepithyme encephalitis, primary central nervous system vasculitis, primary calving, primary basal encephalopathy, primary carnitine deficiency, primary central nervous system lymphoma, primary familial cerebral calcification, primary lateral sclerosis, primary central nervous system melanoma, primary erectile tremor, primary progressive aphasia, primose syndrome, progressive bulbar paralysis, progressive encephalomyelitis with tonic and myoclonus, autosomal recessive progressive extraocular myoparalysis 1, progressive hemifacial atrophy, progressive non-fluency aphasia, primary tremor progressive supranuclear palsy, prolyl-peptidase deficiency, proteus syndrome, prof syndrome, pseudoaminopterin syndrome, pseudocholinesterase deficiency, pseudoneonatal adrenoleukodystrophy, pseudopremature senility syndrome, pseudotrisomy 13 syndrome, elastohydropseudoxanthoma, pudendal neuralgia, pure autonomic nerve failure, pyridoxal 5 '-phosphate-dependent epilepsy, pyridoxine-dependent epilepsy, pyruvate dehydrogenase phosphatase deficiency, qazi markouzos syndrome, radioactive brachial plexus neuropathy, ramos Arroyo Clark syndrome, rapid onset dystonia-parkinson's disease, rasmussen encephalitis, reardon Wilson Cavanagh syndrome, reduced body myopathy, raffmm disease, renal dysplasia-limb deficiency syndrome, renier Gabreels Jasper syndrome, restless leg syndrome, retinal large aneurysms with valve-upper pulmonary stenosis, retinal vascular lesions with leukodystrophy, rett syndrome, reversible cerebrovascular contraction syndrome, RFT1-CDG (CDG-In), rhabdoid tumor, punctate cartilage dysplasia type 1 proximal to the limb, riboflavin transporter deficiency, richard-rule syndrome, richeri Costa Da Silva syndrome, ankylosing syndrome, chromosome 10 circulans, chromosome 14 circulans, chromosome 20 circulans, ripple myopathy, RNAse T2-deficient white matter encephalopathy, roussy Levy syndrome, RRM 2B-related mitochondrial DNA depletion syndrome, ruvalcaba syndrome, sala disease, sandhoff disease, sandifer syndrome, sarcoidosis-induced neuropathy, say Barber Miller syndrome, say Meyer syndrome, shoulder fibula syndrome, SCARF syndrome, schaaaf-Yang syndrome, scheie syndrome, scheime immune dysplasia, schindler disease type 1, schinaf-Yang syndrome, cross-species ligation, sham Schwartz Jampel syndrome, scott Bryant Graham syndrome, seaver casbeh syndrome, seckel syndrome, semantic dementia, sensory ataxia neuropathy, septicemia, clear-optic nerve dysplasia, samsame syndrome, SETBP1 disorder, severe congenital linear myopathy, severe intellectual impairment-progressive spastic biparalysis syndrome, gustavs type severe X-linked intellectual impairment, chartaro syndrome, short-chain acyl-coa dehydrogenase deficiency, shprinzen umbilical expansion syndrome, shprinzen-Goldberg craniosylate early closure syndrome, sialidosis type I, sialidosis type II, sickle cell anemia, simpson-Golabi-Behmel syndrome, median maxillary tooth, sjkren-Larsson syndrome, SLC35A1-CDG (CDG-IIf), SLC35A2-CDG, cdc 1-IIc (CDG-IIc), chronic channel congenital muscle weakness syndrome, smith-Finerman-Myers syndrome, smith-Lemli-Opitz syndrome, smith-Magenis syndrome, sneddon syndrome, snyder-Robinson syndrome, sonoda syndrome, spastic dyscrasia, spastic ataxia charlevalox-saguy type, cerebral spastic biparals, spastic paraplegia 1, spastic paraplegia 10, spastic paraplegia 11, spastic paraplegia 12, spastic paraplegia 13, spastic paraplegia 14, spastic paraplegia 15, spastic paraplegia 16, spastic paraplegia 17, spastic paraplegia 18, spastic paraplegia 19, spastic paraplegia 2, spastic paraplegia 23, spastic paraplegia 24, spastic paraplegia 25, spastic paraplegia 26, spastic paraplegia 29, spastic paraplegia 3, spastic paraplegia 31, spastic paraplegia 32, spastic paraplegia 39, spastic paraplegia 4, spastic paraplegia 51, spastic paraplegia 5a, spastic paraplegia 6, spastic paraplegia 7, spastic paraplegia 8, spastic paraplegia 9, spastic paraplegia facial skin lesions, spastic paraplegia-epileptic-intellectual disability syndrome, spastic paraplegia-retinitis-intellectual disability syndrome, spastic quadriplegia-thin corpus callosum-progressive postpartum microcephaly syndrome, recessive spinal column cleft, spinal atrophy ocular palsy cone syndrome, spinal meningioma, spinal muscular atrophy 1, spinal muscular atrophy type 2, spinal muscular atrophy type 3, spinal muscular atrophy-progressive myoclonus syndrome, spinal shock, spinal cerebellar ataxia 1, spinocerebellar ataxia 10, spinocerebellar ataxia 11, spinocerebellar ataxia 12, spinocerebellar ataxia 13, spinocerebellar ataxia 14, spinocerebellar ataxia 15, spinocerebellar ataxia 17, spinocerebellar ataxia 18, spinocerebellar ataxia 19 and 22, spinocerebellar ataxia 2, spinocerebellar ataxia 20, spinocerebellar ataxia 21, spinocerebellar ataxia 23, spinocerebellar ataxia 25, spinocerebellar ataxia 26, spinocerebellar ataxia 27, spinocerebellar ataxia 28, spinocerebellar ataxia 29, spinocerebellar ataxia 3, spinocerebellar ataxia 30, spinocerebellar ataxia 31, spinocerebellar ataxia 34, spinocerebellar ataxia 4, spinocerebellar ataxia 5, spinocerebellar ataxia 7, spinocerebellar ataxia spinocerebellar ataxia 8, spinocerebellar ataxia 9, autosomal recessive spinocerebellar ataxia 3, autosomal recessive spinocerebellar ataxia 4, autosomal recessive spinocerebellar ataxia 5, autosomal recessive spinocerebellar ataxia 6, autosomal spinocerebellar ataxia 7, autosomal spinocerebellar ataxia 8, spinocerebellar ataxia type 6, spinocerebellar ataxia with axonal neuropathy type 1, spinocerebellar ataxia with congenital malformation, spinocerebellar ataxia x-linked type 2, spinocerebellar ataxia x-linked type 3, spinocerebellar degeneration and corneal dystrophy, split hand-urine abnormal spinal column split spinal deformity, spinal epiphyseal dysplasia, SRD5A3-CDG (CDG-Iq), SSR4-CDG, STAC3 disorder, status epilepticus, steinfeld syndrome, stiff man syndrome, stocco dos Santos syndrome, infant striatal degeneration, sturge-Weber syndrome, subacute sclerosant encephalitis, subcortical metatopic, subcortical astrocytoma, subcortical tumor, succinic semialdehyde dehydrogenase deficiency, susac syndrome, symmetrical thalamus calcification, X-linked mental disorder 7 syndrome, dangil disease, TANGO 2-related metabolic encephalopathy and arrhythmia, tarlov cyst, tay-Sachs disease, tel hasher buckling finger syndrome, telfer Sugar Jaeger syndrome, temple-Baraber syndrome, temporal lobe epilepsy, teamy syndrome, spinal cord banding syndrome, dysplastic hydrocephalus syndrome, thoracic outlet syndrome, toxic periodic paralysis, TMEM165-CDG (CDG-IIk) Torillo-Carey syndrome, toriley syndrome, toxic neuropathy (e.g., alcoholic neuropathy, chemotherapy-induced neuropathy), tranebjaerg Svejgaard syndrome, transverse myelitis, trichinosis, hair-nose-phalangeal syndrome type 2, trigeminal neuralgia, triose phosphate isomerase deficiency, triple A syndrome, troyer syndrome, tuberous sclerosis, tubular aggregated myopathy, tumescent multiple sclerosis, typical congenital linear myopathy, tyrosine hydroxylase deficiency, tyrosinemia type 1, wu Erli Hi congenital muscular dystrophy, unvrich t-Lundborg disease, van Benthhem-Driessen-Hanveld syndrome, van Denbosch syndrome, variant Creutzfeld-Jakob disease, uncertain porphyrin disease, vasculitis-induced neuropathy, galen's vein tumor, vici syndrome, vinca Viljoen Kallis Voges syndrome, neoalkali-induced neuropathy, visual snow syndrome, vitamin B6-induced neuropathy, VLCAD deficiency, vogt-Koyanagi-Harada disease, von Hippel-Lindau disease, walker-Warburg syndrome, weaver syndrome, welander distal myopathy, wernicke-Korsakoff syndrome, west syndrome, whipple disease, hypoplasia-callus hypoplasia-intellectual impairment syndrome, wiedemann Oldigs Oppermann syndrome, williams syndrome, wilson disease, wilson-Turner syndrome, wolf-Hirschhorn syndrome, wolchouse Sakati syndrome, wolter Draght syndrome, sabber syndrome, wyburn-Mason syndrome, colored dry skin disease, xia-Gibbs syndrome, XK forebrain, X-linked encephalomyodynia, X-linked chomatosis, X-linked fibular bone atrophy, X-1 fibular bone type 1 and X-linked muscle atrophy type 2, and X-linked muscle atrophy; X-linked fibula muscular dystrophy type 3, X-linked fibula muscular dystrophy type 4, X-linked fibula muscular dystrophy type 5, X-linked fibula muscular dystrophy type 6, X-linked complex callus hypoplasia, X-linked complex spastic paraplegia type 1, X-linked creatine deficiency, X-linked dystonia-Parkinson/Lubag, X-linked hereditary sensory and autonomic neuropathy with deafness, X-linked intellectual impairment-callus hypoplasia-spastic quadriplegia, X-linked intellectual impairment-short-obesity, najm-linked X-intellectual impairment, schimke-linked X-intellectual impairment, siderius-type X-linked intellectual impairment, turner-type X-linked intellectual impairment, X-linked intellectual impairment-congenital malformation-brain atrophy syndrome, X-linked intellectual-oblique head malformation syndrome, X-linked brain return malformation-genital abnormality, X-linked myopathy is accompanied by excessive autophagy, X-linked myotubular myopathy, X-linked nonspecific mental disorder, X-linked paraventricular gray ectopic, X-linked skeletal dysplasia-mental disorder syndrome, zechi Ceide syndrome, zellweger syndrome and ZTTK syndrome. In some embodiments, the disease or condition of the nervous system is an psychotic condition. In some embodiments, the disease or condition of the nervous system is a disease or condition classified according to DSM-V (American Psychiatric Association, & American Psychiatric Association,2013,Diagnostic and statistical manual of mental disorders:DSM-5.Arlington, va.). In some embodiments, the disease or condition of the nervous system is a disease or condition of the central nervous system. In some embodiments, the disease or condition of the nervous system is an inflammatory disease or condition of the nervous system. In some embodiments, the disease or disorder of the nervous system described herein is a disease or disorder selected from the group consisting of: dementia, multiple sclerosis, amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease, huntington's disease, frontotemporal dementia, ataxia-telangiectasia, multiple system atrophy, progressive supranuclear palsy, crabb's disease, corpus callosum hypoplasia associated with peripheral neuropathy, dunaliella muscular dystrophy, guillain-Barre syndrome, fibular muscular atrophy type 1A, hereditary stress-susceptible neuropathy, diabetic neuropathy, toxic neuropathy, age-related peripheral neuropathy, epilepsy, sleep disorders, encephalopathy and neuropathic pain. In some embodiments, the disease or condition of the nervous system is a neurodegenerative disease or condition. As used herein, the term "neurodegenerative disease or disorder" refers to a group of diseases or disorders of the nervous system characterized by injury and/or death of a subtype of neurons. In some embodiments, the neurodegenerative disease or disorder described herein is at least one disease or disorder selected from the group of dementia, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, huntington's disease, and prion disease. In some embodiments, the disease or condition of the nervous system described herein is a toxin and/or drug-induced neuropathy. In some embodiments, the drug-induced neuropathy described herein is induced, partially induced, or suspected of being induced by at least one agent selected from the group consisting of a chemotherapeutic agent, a TNF-alpha inhibitor, an antiretroviral agent, a cardiac drug, a statin, and an antibiotic.

In some embodiments, the drug-induced neuropathy described herein is induced, partially induced, or suspected of being induced by at least one agent selected from the group consisting of: thalidomide, disulfiram, pyridoxine, colchicine, phenytoin, lithium, chloroquine, hydroxychloroquine, cisplatin, oxaliplatin, taxane, vinca alkaloids, bortezomib, suramin, misonazole, infliximab, etanercept, zalcitabine, didanosine, stavudine, amiodarone, perhexiline, metronidazole, dapsone, podophyllotoxin, fluoroquinolones, isoniazid, and nitrofurantoin.

In some embodiments, the toxin-induced neuropathy described herein is induced, partially induced, or suspected of being induced by at least one agent selected from the group consisting of organic solvents, heavy metals, and organophosphates.

In some embodiments, the toxin and/or drug-induced neuropathy described herein is induced, partially induced, or suspected of being induced by alcohol and/or cigarette smoke.

In some embodiments, the toxin and/or drug-induced neuropathy described herein is characterized by at least one selected from the group consisting of dorsal root ganglion toxicity, abnormal microtubule axon transport function, abnormal voltage gating, abnormal sodium channel, and demyelination.

An "effective amount" of an agent (e.g., a therapeutic agent) refers to an amount effective to achieve a desired therapeutic or prophylactic result, at least at the dosages and periods of time necessary. In addition, the effective amount may depend on the individual patient's medical history, age, weight, family history, genetic composition, stage of thyroid-related autoimmune disease, type of previous or concomitant therapy (if any), and other individual characteristics of the subject to be treated.

In some cases, an effective amount of a composition of the invention may be any amount that reduces the severity or occurrence of symptoms of the disease, disorder, and/or condition to be treated without significant toxicity to the subject. In some cases, an effective amount of a pharmaceutical composition of the invention can be any amount that reduces the number of diseased cells (e.g., deregulated immune cells), autoantibodies, and/or other disease markers (e.g., cytokines) without significant toxicity to the subject.

The effective amount of the pharmaceutical composition of the invention (and any additional therapeutic agent) may remain constant or may be adjusted as a sliding ratio or variable dose depending on the subject's response to treatment. In some cases, the frequency of administration can be any frequency that reduces the severity or occurrence of symptoms of the disease, disorder, and/or condition to be treated without significant toxicity to the subject. Various factors can influence the actual effective amount for a particular application. For example, the frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and/or condition may require an increase or decrease in the actual effective amount administered.

The terms "peptide", "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to a series of amino acid residues that are linked to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent amino acid residues.

As used herein, the term "alpha 1-antitrypsin protein" or "AAT" refers to a protein having an amino acid sequence as defined in SEQ ID NO. 1 or a nucleotide sequence encoding a protein having an amino acid sequence as defined in SEQ ID NO. 1. In some embodiments, the AAT described herein is a protein, peptide, or polypeptide. AAT proteins may be obtained by isolation from blood (e.g., human blood), or may be recombinantly produced.

The term "variant" refers to a protein, peptide or polypeptide having an amino acid sequence that differs to some extent from the AAT native sequence peptide, i.e., by amino acid substitution of an amino acid sequence that differs from the AAT native sequence in that one or more amino acids are substituted with another amino acid that has the same characteristic and conformational effects. Preferably, the variants described herein are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acids of SEQ ID NO. 1. Amino acid sequence variants may have substitutions, deletions and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence (e.g., at the N-or C-terminal sequence or within the amino acid sequence). Substitutions may also be conservative, in which case conservative amino acid substitutions are defined herein as exchanges within one of the following five groups:

I. Small aliphatic, non-polar or slightly polar residues: ala, ser, thr, pro, gly

Polar, positively charged residues: his, arg, lys

Iii, polar, negatively charged residues and amides thereof: asp, asn, glu, gln

Large aromatic residues: phe, tyr, trp

V. large aliphatic nonpolar residues: met, leu, ile, val, cys.

As used herein, the term "isoform" refers to splice variants resulting from alternative splicing of AAT mRNA. Isoforms of AAT are known in the art (see, e.g., matsuda, e., ishizaki, r., taira, t., iguchi-Ariga, s.m., and Ariga, h.,2005,Biological&pharmaceutical bulletin,28 (5), 898-901).

As used herein, the term "fragment" refers to a sequence containing amino acids shorter in length than the AAT protein and/or isoforms thereof, particularly a sequence containing fewer amino acids than the AAT sequence shown in SEQ ID No. 1. The fragment is preferably a functional fragment, e.g. a fragment having the same biological activity as the AAT protein shown in SEQ ID NO. 1. The functional fragment is preferably derived from the AAT protein shown in SEQ ID NO. 1. Any AAT fragment may be used as long as it exhibits the same properties or substantially the same properties as the native AAT sequence from which it is derived, i.e., is biologically active. In some embodiments, the disclosure The fragment has the same or substantially the same inhibitory properties as AAT for one or more human neutrophil serine proteases, preferably the fragment has at least about 6.5X10 ⁷ M ^-1 s ^-1 A secondary constant associated with NE of at least about 8.1X10 ⁶ M ^-1 s ^-1 AAT fragment and PR of (C) ₃ Associated secondary constants and/or at least about 4.1X10 ⁵ M ^-1 s ^-1 The second order constant of association of AAT fragments with CG (see, e.g., beatty, K. Et al, 1980,. J.biol. Chem.255,3931-3934.; rao, N.V. et al, 1991,Structural and functional properties.J.Biol.Chem.266,9540-9548.).

Preferably, the (functional) fragment shares about 5 contiguous amino acids, at least about 7 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, at least about 35 contiguous amino acids, at least about 40 contiguous amino acids, at least about 45 contiguous amino acids, at least about 50 contiguous amino acids, at least about 55 contiguous amino acids, at least about 60 contiguous amino acids, at least about 100 contiguous amino acids, at least about 150 contiguous amino acids, at least about 200 contiguous amino acids, at least about 300 contiguous amino acids, or more of the natural human AAT amino acid sequence shown in SEQ ID No. 1. In some embodiments, the (functional) fragments described herein comprise an expression-optimized signaling protein.

In some aspects, the amino acid sequence of AAT, a variant, isoform or fragment thereof is the same as the corresponding amino acid sequence of SEQ ID NO. 1.

To date, a range of natural inhibitors of the protease alpha-1-antitrypsin (AAT) have been successfully used to reduce inflammation in different types of human tissue (Bergin, david a. Et al, 2021,Archivum immunologiae et therapiae experimentalis 60.2:81-97).

The inventors found that AAT could reduce the neuronal pathology pathway (FIGS. 2-4, tables 2-11). This reduction in neuronal pathology pathways is observed in resting cells (fig. 4B) and stimulated cells (fig. 4C), and thus can be used to prevent and/or treat diseases or disorders of the nervous system, and symptoms thereof.

TACE activity modulation is involved in myelin modulation and is a hallmark of inflammation in the acquired form of neuropathy.

The inventors found that AAT was able to inhibit TACE in a dose-dependent manner, without being bound by theory, which rescue myelin production of SCs, and thus subsequently prevent, or slow and/or reverse progression of diseases and/or disorders of the nervous system.

It is exciting that AAT holds some promise for diseases for which there is no curative treatment for the underlying genetic process at the time and for which no treatment is continuously observed that is effective in slowing the progression of the disease process.

Accordingly, the present invention is based, at least in part, on the discovery that AAT can be used to treat the diseases or conditions of the nervous system described herein.

In certain embodiments, the invention relates to a vector for treating and/or preventing a disease or disorder of the nervous system comprising a nucleic acid sequence encoding an AAT protein.

As used herein, the term "vector" refers to a viral vector or nucleic acid (DNA or RNA) molecule, such as a plasmid or other vehicle, that contains one or more heterologous nucleic acid sequences of the invention, and is preferably designed for transfer between different host cells. The terms "expression vector", "gene delivery vector" and "gene therapy vector" refer to any vector that is effective to incorporate and express one or more nucleic acids of the invention in a cell, preferably under the control of a promoter. Cloning or expression vectors may contain additional elements such as regulatory and/or post-transcriptional regulatory elements in addition to the promoter.

The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any kind of deoxyribonucleotide (e.g., DNA, cdna.) or ribonucleotide (e.g., RNA, mrna.) polymer or combined deoxyribonucleotide and ribonucleotide (e.g., DNA/RNA) polymer in either a linear or circular conformation and in single-or double-stranded form. These terms should not be construed as limiting the length of the polymer and may encompass known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and/or phosphate moieties (e.g., phosphorothioate backbones). In general, analogs of a particular nucleotide have the same base pairing specificity; that is, an analog of a will base pair with T.

Protein restriction, such as blood brain barrier penetration and/or enzymatic degradation of AAT, can be reduced using the vectors described herein. Thus, the vector may be implemented in a delivery system, such as in a cell that delivers AAT to a neuronal cell (e.g., a brain neuronal cell).

Accordingly, the present invention is based, at least in part, on the discovery that the vectors described herein are useful for treating and/or preventing diseases or conditions of the nervous system.

In certain embodiments, the invention relates to a genetically modified cell comprising a nucleic acid sequence encoding an AAT protein for use in the treatment and/or prevention of a disease or condition of the nervous system.

As used herein, the term "genetically modified cell" refers to a cell modified by genetic engineering. In some embodiments, the cell is an immune effector cell. As used herein, the term "engineering" and other grammatical forms thereof may refer to one or more alterations of a nucleic acid, such as a nucleic acid within the genome of an organism. The term "engineering" may refer to alterations, additions and/or deletions of a gene. Engineered cells may also refer to cells containing added, deleted and/or altered genes.

In some embodiments, the genetically modified cells described herein include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly the same nucleic acid content as the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the initially genetically modified cell.

In some embodiments, the invention relates to compositions comprising genetically modified cells described herein in place of, or in addition to, an AAT protein, variant or isoform thereof. Thus, the genetically modified cells described herein can be used in cell therapies to deliver AAT into a subject or to a tissue/organ of a subject.

The use of genetically modified cells described herein may reduce protein limitations such as blood brain barrier penetration and/or enzymatic degradation of AAT. Thus, the vector may be implemented in a delivery system, such as in a cell that delivers AAT to a neuronal cell (e.g., a brain neuronal cell).

Accordingly, the present invention is based, at least in part, on the discovery that genetically modified cells as described herein can improve the prevention and/or treatment of diseases or conditions of the nervous system.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the disease or disorder of the nervous system is an inflammatory disease or disorder of the nervous system.

As used herein, the term "inflammatory disease or condition of the nervous system" refers to a disease or condition of the nervous system characterized by increased inflammation. Inflammation is characterized by deregulation of inflammatory markers and/or increased infiltration, activation, proliferation and/or differentiation of immune cells in blood, tissues, organs and/or certain cell types.

Inflammation of a disease or condition of the nervous system may be caused by, for example, physical injury, ionizing radiation, infection (e.g., pathogen infection), immune response resulting from hypersensitivity, cancer, chemical irritants, drugs, toxins, alcohol, nutrients (e.g., nutrient excess), plaque deposits, toxic metabolites, autoimmunity, aging, microorganisms, air pollution, and/or (passive) smoking.

Inflammatory markers are markers that indicate inflammation in a subject. Inflammatory markers include, but are not limited to, CRP, erythrocyte Sedimentation Rate (ESR) and Procalcitonin (PCT), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-33, IL-32, IL-33, IL-35, or IL-36) tumor necrosis factors (e.g., TNF alpha, TNF beta), interferons (e.g., interferon alpha, interferon beta, interferon gamma), MIP-I, MCP, I, RANTES, other chemokines, and/or other cytokines. Inflammatory markers may also be detected indirectly, for example by detecting inhibitors (e.g., binding factors and/or antagonists) of inflammatory markers. In some embodiments, the inflammatory marker is measured in cells involved in inflammation, in cells affected by cells involved in inflammation, in tissue, and/or in blood. In some embodiments, the inflammatory marker is indicative of immune cell infiltration, activation, proliferation, and/or differentiation. Detection of an inflammatory marker or a ratio of two or more inflammatory markers is detected outside of normal range. Whether the normal range of the inflammatory marker(s) and the marker(s) (ratio) must be below or above a threshold value indicative of inflammation is known to those skilled in the art. In some embodiments, the inflammatory marker is a microglial marker, such as a microglial recognition, proliferation, accumulation, and/or activation marker. In some embodiments, the level of gene expression, the level of RNA transcripts, the level of protein expression, the level of protein activity, and/or the level of enzymatic activity of at least one inflammatory marker is detected. In some embodiments, at least one inflammatory marker is detected quantitatively and/or qualitatively.

The inventors found that AAT (or vector/genetically modified cells as described herein) can reduce the neuronal inflammatory pathway (fig. 2-4, tables 2-11). Such a reduction in inflammatory pathways is observed in resting cells (fig. 4B) and stimulated cells (fig. 4C), and thus can be used to prevent and/or treat diseases or disorders of the nervous system and symptoms thereof.

Thus, the present invention is based, at least in part, on the discovery that AAT (or the vector/genetically modified cells described herein) can be used to treat inflammatory diseases or conditions of the nervous system described herein.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the inflammatory disease or disorder of the nervous system is a disease or disorder mediated by bone marrow cells of the nervous system.

As used herein, the term "myeloid-mediated disease or disorder of the nervous system" refers to a disease or disorder of the nervous system characterized by an increase in myeloid-mediated inflammation. Myeloid-mediated inflammation can be detected by any method known in the art, for example by cytokine measurement and/or quantitative and/or qualitative analysis of myeloid cells (see, e.g., davis, b.m., salinas-Navarro, m., cordiro, m.f., et al, 2017,Sci Rep 7,1576).

In some embodiments, the bone marrow cell mediated disease or condition of the nervous system is a disease or condition in which the primary pathology is bone marrow cell mediated inflammation.

In some embodiments, the bone marrow cell-mediated disease or condition of the nervous system is a microglial cell-mediated disease or condition of the nervous system.

The inventors found that AAT (or vector/genetically modified cells as described herein) can reduce neuronal bone marrow cell mediated inflammatory pathways (fig. 2-4, tables 2-11). This reduction in bone marrow cell-mediated inflammatory pathways is observed in resting cells (fig. 4B) and stimulated cells (fig. 4C), and thus can be used to prevent and/or treat diseases or disorders of the nervous system, and symptoms thereof.

Thus, the present invention is based, at least in part, on the discovery that AAT (or the vector/genetically modified cells described herein) can be used to treat microglial-mediated diseases or conditions of the nervous system described herein.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention or a genetically modified cell for use of the invention, wherein the disease or syndrome of the nervous system is a disease or syndrome selected from the group consisting of dementia, multiple sclerosis, amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease and huntington's disease.

As used herein, the term "dementia" refers to a cognitive disorder characterized by dementia (i.e., a general worsening or progressive decline in cognitive ability or dementia-like symptoms). Dementia is often associated with or caused by one or more abnormal processes in the brain or central nervous system (e.g., neurodegeneration). Dementia usually progresses from mild to severe stages and interferes with the ability of the subject to work independently in daily life. Dementia can be classified as cortical or subcortical dementia, depending on the area of the brain affected. Dementia does not include disorders characterized by loss of consciousness (such as delirium) or depression, or other functional psychotic disorders (pseudodementia). Dementia includes irreversible dementias such as those associated with neurodegenerative diseases such as Alzheimer's disease, vascular dementia, lewy body dementia, jacob-Crohn's disease, pick's disease, progressive supranuclear palsy, frontal lobe dementia, idiopathic basal ganglia calcification, huntington's disease, multiple sclerosis and Parkinson's disease, and reversible dementia due to trauma (post-traumatic encephalopathy), intracranial tumors (primary or metastatic), subdural hematoma, metabolic and endocrine disorders (hypothyroidism and hyperthyroidism, wilson's disease, uremic encephalopathy, dialysis dementia, hypoxic dementia and post-hypoxic dementia, and chronic electrolyte disorders), deficiency states (vitamin B12 deficiency and brown-skin diseases (vitamin B6)), infections (AIDS, syphilitic meningoedema, limbic encephalitis, progressive multifocal leukoencephalopathy, fungal infections, tuberculosis) and chronic exposure to alcohol, aluminum, heavy metals (arsenic, lead, mercury, manganese) or drugs (antipyretics, sedative drugs, etc.

As used herein, the term "multiple sclerosis" refers to a disease or disorder characterized by inflammation, demyelination, oligodendrocyte death, membrane damage, and axonal death. In some embodiments, the multiple sclerosis described herein refers to relapsing/remitting multiple sclerosis or progressive multiple sclerosis. In some embodiments, multiple sclerosis is at least one of the four major multiple sclerosis types defined in the international neurological survey (Lublin and reingled, 1996,Neurology 46 (4): 907-11), i.e., relapsing/remitting multiple sclerosis, secondary progressive multiple sclerosis, progressive/relapsing multiple sclerosis, or Primary Progressive Multiple Sclerosis (PPMS).

In some embodiments, the multiple sclerosis described herein refers to symptoms of multiple sclerosis, including vision problems, dizziness, sensory dysfunction, weakness, impaired coordination ability, loss of balance, fatigue, pain, neurocognitive deficits, mental health deficits, bladder dysfunction, bowel dysfunction, sexual dysfunction, heat sensitivity.

As used herein, the term "huntington's disease" refers to a neurodegenerative disease caused by repeated amplification of trinucleotides in the HTT gene (e.g., CAG, which is translated into polyglutamine or PolyQ), which results in the production of pathogenic mutant huntingtin (HTT or mHTT). In some embodiments, the mutant huntingtin accelerates the rate of neuronal cell death in certain areas of the brain. In some embodiments, huntington's disease as described herein refers to symptoms of huntington's disease, including impaired motor function, cognitive dysfunction, depression, anxiety, dyskinesia, chorea, stiffness, muscle contractures (dystonia), slow or abnormal eye movement, impaired gait, altered posture, impaired balance, unexpected weight loss, sleep rhythm disorders, circadian rhythm disorders, and autonomic nervous system dysfunction.

As used herein, the term "amyotrophic lateral sclerosis" refers to a progressive neurodegenerative disease that affects upper motor neurons (motor neurons in the brain) and/or lower motor neurons (motor neurons in the spinal cord) and results in the death of motor neurons. In some embodiments, amyotrophic lateral sclerosis includes all classifications of amyotrophic lateral sclerosis known in the art, including, but not limited to, classical amyotrophic lateral sclerosis (typically affecting both lower motor neurons and upper motor neurons), primary lateral sclerosis (PLS typically affecting only upper motor neurons), progressive bulbar paralysis (PBP or bulbar onset, one form of amyotrophic lateral sclerosis typically beginning with dysphagia, mastication, and speaking difficulties), progressive amyotrophy (PMA, typically affecting only lower motor neurons), and familial amyotrophic lateral sclerosis (inherited form of amyotrophic lateral sclerosis).

In some embodiments, the term "amyotrophic lateral sclerosis" refers to symptoms of amyotrophic lateral sclerosis, including, but not limited to, progressive weakness, atrophy, muscle beam tremor, hyperreflexia, dysarthria, dysphagia, and/or paralysis of respiratory function.

As used herein, the term "alzheimer's disease" (AD) refers to mental decline associated with a particular degenerative brain disease characterized by age spots, neuroinflammatory tangles, and progressive neuronal loss, which are clinically manifested by progressive memory deficits, confusion, behavioral problems, failure to care for oneself, and/or progressive physical deterioration.

In some embodiments, the nicds-ADRDA (national institute of neurological and communication disorders and alzheimer's disease and related disease association) standard is used to identify subjects with alzheimer's disease:

1) Clinical dementia grade (CDR) =1; simple mental state examination (MMSE) was 16 to 24 minutes, and medial temporal lobe atrophy (determined by Magnetic Resonance Imaging (MRI)) was >3 minutes in the Scheltens scale. In some embodiments, the term alzheimer's disease includes all stages of the disease, including the following stages as defined by the NINCDS-ADRDA alzheimer's disease diagnostic criteria in 1984.

2) Definite alzheimer's disease: patients meet the criteria for likely alzheimer's disease and have histopathological evidence of AD via autopsy or biopsy.

Alzheimer's disease, which may be suffering from or precursor to: dementia has been demonstrated by clinical and neuropsychological examination. Cognitive disorders must also be progressive and exist in two or more areas of cognition. The age of onset of the defect is between 40 and 90 years, and finally there must be no other diseases that can produce dementia syndromes.

3) May have or not be precursor Alzheimer's disease: there is a dementia syndrome with atypical onset, manifestations; and no known etiology; but there are no diseases that are considered to be the origin of dementia that can produce co-morbidities of dementia. In some embodiments, the term alzheimer's disease refers to a stage of alzheimer's disease. In some embodiments, the term alzheimer's disease refers to two stages of alzheimer's disease. In some embodiments, the term "alzheimer's disease" refers to symptoms of alzheimer's disease including, but not limited to, memory loss, confusion, mental difficulties, language changes, behavioral changes, and/or personality changes.

As used herein, the term "parkinson's disease" refers to a neurological syndrome characterized by dopamine deficiency, which is caused by degenerative, vascular or inflammatory changes of the substantia nigra basal ganglia. Symptoms of parkinson's disease include, but are not limited to, the following: resting tremor, gear-like rigidity, bradykinesia, postural reflex disorders, good response to 1-dopa treatment without obvious motor paralysis, cerebellar or pyramidal signs, muscular atrophy, movement disorders and/or speech disorders. In a specific embodiment, the invention is used for the treatment of dopaminergic dysfunction related syndrome. In some embodiments, parkinson's disease includes any stage of parkinson's disease. In some embodiments, the term parkinson's disease includes the early stages of parkinson's disease, which refers broadly to the first stage of parkinson's disease, wherein a person suffering from the disease exhibits mild symptoms that are non-disabling, such as paroxysmal tremors of a single limb (e.g., a hand), and which affects only one side of the body.

In some embodiments, the term parkinson's disease includes the advanced stages of parkinson's disease, which refers to the more severe stages of progression of parkinson's disease, wherein a person suffering from the disease exhibits symptoms that are typically severe and may lead to some disabilities (e.g., including tremors on both sides of the body, balance problems, etc.). Symptoms associated with advanced stages of parkinson's disease may vary significantly among individuals and may not manifest until years after the initial appearance of the disease.

In some embodiments, the term "parkinson's disease" refers to symptoms of parkinson's disease, including, but not limited to tremors (e.g., most pronounced tremors at rest), tremors (e.g., tremors of hands, arms, legs, jaws, and faces), muscle stiffness, lack of postural reflex, slow autonomous movement, retroversion, mask-like facial expression, humpback posture, poor balance, poor coordination, bradykinesia, postural instability, and/or gait abnormalities.

Thus, the present invention is based, at least in part, on the discovery that AAT (or the vector/genetically modified cells described herein) is particularly useful for treating certain diseases or conditions of the nervous system described herein.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the disease or disorder of the nervous system is at least one symptom of a disease or disorder of the nervous system selected from the group consisting of: tremor, memory loss, slurred speech, dizziness, vision changes, and headache.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the disease or disorder of the nervous system is a schwann cell mediated disease or disorder.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the disease or disorder of the nervous system is a TACE-mediated disease or disorder.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the disease or disorder of the nervous system is a disease or disorder of the peripheral nervous system.

As used herein, the term "disease or condition of the peripheral nervous system" is meant to include any disease or condition that significantly affects the peripheral nervous system, preferably any disease or condition that primarily affects the peripheral nervous system.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the disease or condition of the peripheral nervous system is a motor and sensory neuropathy of the peripheral nervous system.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention or a genetically modified cell for use of the invention, wherein the sensory neuropathy of the peripheral nervous system is acquired motor and sensory neuropathy of the peripheral nervous system.

Acquired motor and sensory neuropathy of the peripheral nervous system such as acquired demyelinating diseases including, but not limited to, nerve injury, diabetic peripheral neuropathy, drug-related peripheral neuropathy, leprosy and inflammatory neuropathy. These neuropathies can affect both myelinated schwann cells and peripheral axons/neurons.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the sensory neuropathy of the peripheral nervous system is guillain-barre syndrome.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the sensory neuropathy of the peripheral nervous system is hereditary motor and sensory neuropathy of the peripheral nervous system.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the hereditary motor and sensory neuropathy of the peripheral nervous system is fibula muscular atrophy or a symptom thereof.

As used herein, the term "fibular muscular dystrophy" refers to hereditary motor and sensory neuropathy of the peripheral nervous system, characterized by progressive loss of muscle tissue and/or touch of various parts of the body. In some embodiments, the fibular muscular dystrophy described herein is at least one subtype selected from the group of: CMT1, CMTX, CMT4, CMT2, severe, early CMT, CMT 5, CMT 6, CMT 7 and mid-term CMT.

Symptoms of fibular muscular dystrophy include, but are not limited to, leg, ankle and/or foot weakness, reduced leg and/or foot muscle mass, high arch, toe bending (hammer toe), reduced running ability, difficulty in lifting the foot at the ankle (drop foot), abnormal gait, frequent trips or falls, and reduced or unconscious leg and/or foot feel

In a mouse model of diseases and conditions of the peripheral nervous system (such as CMT 1A), the inventors confirmed that AAT is a finding of effective therapeutic background.

AAT is expected to improve axonal myelination disorders, allowing myelination to form normally around axons, thus potentially allowing CMT1A patients to live in normal healthy life.

In certain embodiments, the invention relates to compositions for use of the invention, wherein the AAT protein, variant, isoform and/or fragment thereof is human plasma extracted.

In certain embodiments, the invention relates to a composition for use of the invention, wherein the alpha 1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof is recombinant alpha 1-antitrypsin (rhAAT), variant, isoform and/or fragment thereof.

In certain embodiments, the present invention relates to a composition for use according to the present invention, wherein the composition comprises at least one pharmaceutical carrier.

As used herein, the term "drug carrier" refers to an agent (e.g., a molecule or cell) that improves the drug delivery properties of the composition, carrier, and/or genetically modified cell for use in the present invention. In some embodiments, the drug delivery properties described herein include at least one property selected from the group of: penetration ability (e.g., cell membrane and/or blood brain barrier), site-specific delivery (e.g., brain-specific delivery), controlled release delivery, and stability (e.g., reduced enzymatic degradation). In some embodiments, the pharmaceutical carrier described herein is at least one agent selected from the group consisting of: delivery cells, liposomes, nanoparticles, fusion proteins, vesicles, nanospheres, micelles, nanocapsules, nanoshells, lipid particles, and dendrimers.

In some embodiments, the pharmaceutical carrier described herein is a pharmaceutically acceptable diluent or carrier.

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the pharmaceutical carrier is a blood brain barrier permeability enhancer.

As used herein, the term "blood brain barrier permeability enhancer" refers to an agent that can be used to deliver a composition, carrier, and/or genetically modified cell for use in the present invention to the nervous system (including the brain) and across the blood brain barrier. Any strategy known in the art may be used to achieve an enhancement of blood brain barrier permeability (see, e.g., salamah, t.s., & Banks, w.a.; 2014,Advances in pharmacology,71,277-299; tashima, t.; 2020, receptor-Mediated transduction. Chemical and Pharmaceutical Bulletin,68 (4), 316-32; pardridge, w.m.,2020,Frontiers in aging neuroscience,11,373;Upadhyay,R.K, 2014,BioMed research international).

In some embodiments, the composition for use of the invention is fused to a blood brain barrier enhancing protein. In some embodiments, the blood brain barrier enhancing protein described herein is at least one intact protein selected from the group of transferrin, insulin, an insulin-like growth factor, a low density lipoprotein, a variant, an isoform and/or a fragment of the protein.

Trojan horse strategies may also be used (see, e.g., pardridge, W.M.,2017,BioDrugs 31,503-519). In some embodiments, the compositions for use of the invention are linked to an antibody or fragment thereof that binds an endogenous BBB receptor transporter (such as an insulin receptor or transferrin receptor).

The composition for use in the present invention may also be modified to increase lipophilicity and subsequently improve BBB penetration characteristics (see e.g. Upadhyay, r.k.,2014, # BioMed research international, article ID 869269,37). In some embodiments, the compositions for use of the invention comprise a modification that increases lipophilicity. In some embodiments, the lipophilicity-increasing modifications described herein comprise adding at least one hydrophilic peptide, replacing a sequence portion with at least one hydrophilic peptide, adding a lipid portion, and/or replacing a non-lipid portion with a lipid portion.

In certain embodiments, the invention relates to a composition for use of the invention, a vector for use of the invention, or a genetically modified cell for use of the invention, wherein the composition, vector, or genetically modified cell is formulated for intra-cerebral administration, intravenous injection, intravenous infusion, quantitative pump infusion, inhalable nasal spray, eye drops, skin patches, sustained release formulations, ex vivo gene therapy, or ex vivo cell therapy.

The pharmaceutical compositions of the invention may also be delivered to patients by a variety of techniques, including DNA injection (also known as DNA vaccination) with and without the use of in vivo electroporation of nucleic acids encoding the AAT proteins, variants, isoforms and/or fragments thereof of the invention, liposome-mediated nanoparticle-facilitated recombinant vectors, such as recombinant lentiviruses, recombinant adenoviruses and recombinant adeno-associated viruses described herein.

The composition may be injected intravenously or topically into the brain or spinal cord or electroporated into the tissue of interest.

As used herein, the terms "subject"/"subject in need thereof" or "patient"/"patient in need thereof" are well known in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some cases, the subject is a subject in need of treatment or a subject suffering from a disease or disorder. However, in other embodiments, the subject may be a normal subject. The term does not indicate a particular age or gender. Thus, adult, pediatric, and neonatal subjects, both male and female, are contemplated. Preferably, the subject is a human. Most preferably, the human suffers from a disease or syndrome associated with a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection). In some embodiments, the subject has a neurological disorder independent of viral infection.

The term "about", especially when referring to a given amount, is intended to cover deviations of plus or minus ten (10)%, preferably 5%, even more preferably 2% and most preferably 1%.

The present invention relates to a composition for treating and/or preventing a disease or syndrome associated with any viral infection in a subject in need thereof, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin protein, variants, isoforms and/or fragments thereof.

The α1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof may be plasma extracted AAT, variant, isoform and/or fragment thereof, in particular human plasma extracted AAT, variant, isoform and/or fragment thereof; or recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof, preferably alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof are recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof. Viral infection may be caused by DNA viruses (double-stranded or single-stranded), RNA viruses (single-stranded or double-stranded, whether positive or negative), retroviruses, or any newly emerged virus (whether enveloped or non-enveloped).

In some embodiments of the invention, the compositions are for use in treating and/or preventing a disease or syndrome associated with a respiratory viral infection in a subject in need thereof. In some embodiments, the respiratory viruses described herein are viruses selected from the group of: rhinoviruses, RSV, parainfluenza, metapneumoviruses, coronaviruses, enteroviruses, adenoviruses, bocaviruses, polyomaviruses, herpes simplex viruses, and cytomegaloviruses.

In some embodiments of the invention, the composition is for use in treating and/or preventing a disease or syndrome associated with a DNA virus infection in a subject in need thereof.

In some embodiments, the DNA viruses described herein are selected from the group consisting of: adenovirus, rhinovirus, RSV, influenza virus, parainfluenza virus, metapneumovirus, coronavirus, enterovirus, adenovirus, bocavirus, polyomavirus, herpes simplex virus, cytomegalovirus, bocavirus, polyomavirus and cytomegalovirus.

In some embodiments of the invention, the compositions are for use in treating and/or preventing a disease or syndrome associated with an RNA viral infection in a subject in need thereof. The RNA virus may be an enveloped or coated virus or a non-enveloped or naked RNA virus. The RNA virus may be a single stranded RNA (ssRNA) virus or a double stranded RNA (dsRNA) virus. The single stranded RNA virus may be a positive sense ssRNA virus or a negative sense ssRNA virus.

In some embodiments, the RNA viruses described herein are selected from the group consisting of: rhinoviruses, RSV, influenza viruses, parainfluenza viruses, metapneumoviruses, coronaviruses, enteroviruses, adenoviruses, bocaviruses, polyomaviruses, herpes simplex viruses and cytomegaloviruses.

In some embodiments of the invention, the composition is for use in treating and/or preventing a disease or syndrome associated with a coronavirus infection in a subject in need thereof. In some embodiments, the coronaviruses described herein are coronaviruses from a genus selected from the group of α -CoV, β -CoV, γ -CoV, or δ -CoV. In another specific embodiment, the coronaviruses described herein belong to the genus α -CoV or β -CoV. In some embodiments, the coronaviruses described herein are selected from the group consisting of: human coronavirus OC43 (HCoV-OC 43), human coronavirus HKU1 (HCoV-HKU 1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL 63, new Haven coronavirus), middle east respiratory syndrome related coronavirus (MERS-CoV or "novel coronavirus 2012"), severe acute respiratory syndrome coronavirus (SARS-CoV or "SARS-typical"), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or "novel coronavirus 2019"). Preferably, the viral infection is an RNA viral infection, most preferably a coronaviral infection caused by a coronavirus selected from the non-limiting group comprising MERS-CoV, SARS-CoV and SARS-CoV-2. Most preferably, the viral infection is a SARS-CoV-2 infection.

It is to be understood that the present invention includes all diseases or syndromes associated with viral infections, preferably coronavirus infections, more preferably SARS-CoV-2 infections. A disease or syndrome associated with SARS-CoV-2 infection is also known as COVID-19. In one embodiment, the disease or syndrome associated with a viral infection is an inflammation, such as inflammation of systemic blood vessels (e.g., kawasaki disease), an immune disease (e.g., graves' disease), and/or a respiratory syndrome, particularly severe acute respiratory syndrome. The disease or syndrome may be associated with a viral infection, as a viral infection is associated with the disease or syndrome prior to the disease or syndrome, contributing to the disease or syndrome and/or causing the disease or syndrome. For example, viral infection may induce inflammation that directly or indirectly induces a disease or syndrome. The inflammation associated with a viral infection may be acute or chronic. Inflammation associated with viral infection may then persist throughout the body and/or specific organs (e.g., brain) and/or cause sustained damage. In some embodiments of the invention, the composition is for use in treating and/or preventing an inflammatory disease or syndrome of the nervous system associated with a viral infection in a subject in need thereof.

As used herein, the term "inflammatory disease or syndrome of the nervous system" refers to a disease, syndrome, and/or disorder characterized by increased inflammation in the nervous system as compared to a healthy reference subject. The diseases or syndromes described herein (including those of the nervous system) are associated with viruses. Inflammation is characterized by deregulation of inflammatory markers and/or increased infiltration, activation, proliferation and/or differentiation of immune cells in the blood and/or brain. Inflammatory markers are markers that indicate inflammation in a subject. In certain embodiments, the inflammatory marker described herein is a marker selected from the group consisting of: CRP, erythrocyte Sedimentation Rate (ESR) and Procalcitonin (PCT), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-33, IL-32, IL-33, IL-35, or IL-36), tumor necrosis factors (e.g., TNFα, TNFβ), interferons (e.g., interferon γ) MIP-I, MCP-I, RANTES, other chemokines, and/or other cytokines. Inflammatory markers may also be detected indirectly, for example by detecting inhibitors (e.g., binding factors and/or antagonists) of inflammatory markers. In some embodiments, the inflammatory marker is measured in cells involved in inflammation, in cells affected by cells involved in inflammation, in cerebrospinal fluid, and/or in blood. In some embodiments, the inflammatory marker is indicative of immune cell infiltration, activation, proliferation, and/or differentiation. Detection of an inflammatory marker or a ratio of two or more inflammatory markers is detected outside of normal range. Whether the normal range of the inflammatory marker(s) and the marker(s) (ratio) must be below or above a threshold value indicative of inflammation is known to those skilled in the art. In some embodiments, the level of gene expression, the level of RNA transcripts, the level of protein expression, the level of protein activity, and/or the level of enzymatic activity of at least one inflammatory marker is detected. In some embodiments, at least one inflammatory marker is quantitatively and/or qualitatively detected to determine inflammatory diseases or syndromes of the nervous system of a subject in need of treatment and/or prevention.

In some embodiments, an inflammatory disease or syndrome of the nervous system described herein is characterized by acute inflammation, i.e., the duration of the inflammatory symptoms is typically on the order of a few minutes (e.g., 2, 5, 10, 15, 30, 45 minutes) to a few days (e.g., 2, 3, 5, 7, 10, or 14 days). Acute inflammation usually occurs as a direct result of irritation (e.g., viral infection). In some embodiments, the inflammatory disease or syndrome of the nervous system is characterized by chronic inflammation, i.e., the duration of the inflammatory symptoms is typically at least about a few days (e.g., 2, 3, 5, 7, 10, or 14 days) or the inflammatory symptoms recur at least once (e.g., one or more times, two or more times, or three or more times). In some embodiments, the inflammatory disease or syndrome of the nervous system is characterized by chronic low-grade inflammation. Chronic low grade inflammation can occur without clinical symptoms.

In certain embodiments, a subject in need of treatment and/or prophylaxis has a history of viral infection. Thus, a subject in need of treatment and/or prophylaxis is infected at least once with a virus. In certain embodiments, a subject in need of treatment and/or prophylaxis is infected at least once with a virus. In certain embodiments, a subject in need of treatment and/or prophylaxis is infected at least once with a virus during childhood. In certain embodiments, a subject in need of treatment and/or prophylaxis is infected at least once with a virus. In certain embodiments, a subject in need of treatment and/or prophylaxis has been infected at least once with a virus in the past 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 years. In certain embodiments, the viral infection is active (e.g., detectable) at a diagnostic time point of an inflammatory disease or syndrome of the nervous system of a subject in need of treatment and/or prevention.

Methods for detecting viral infections are known to those skilled in the art. In some embodiments, the invention relates to a method for detecting a virus selected from the group of: virus isolation, nucleic acid-based methods, microscope-based methods, host antibody detection, electron microscopy, and host cell phenotype.

In some embodiments, the viral infection described herein is detected in a sample, such as in a sample selected from the group of: nasopharyngeal swabs, blood, tissue (e.g., skin), sputum, gargle, bronchial wash, urine, semen, stool, cerebrospinal fluid, dry blood spots, nasal mucus.

In some embodiments, the viral infections described herein are obtained as information retrieved from a patient's medical history.

Examples of detection of (previous) SARS-CoV-2 infection include human IFN-. Gamma.SARS-CoV-2 ELISPot ^PLUS Kit (ALP), dipstick (Mabtech, 3420-4 AST-P1-1) or determination of T cell response (Zuo, J., dowell, A.C., pearce, H.et al, 2021, nat Immunol).

In some embodiments, the inflammatory disease or syndrome of the nervous system described herein is an inflammatory disease or syndrome of the sympathetic nervous system. In some embodiments, the inflammatory disease or syndrome of the nervous system described herein is an inflammatory disease or syndrome of the parasympathetic nervous system. In some embodiments, the inflammatory disease or syndrome of the nervous system described herein is an inflammatory disease or syndrome of the central nervous system. In some embodiments, the inflammatory disease or syndrome of the nervous system described herein is an inflammatory disease or syndrome of the peripheral nervous system.

In certain embodiments, the viral-associated inflammatory disease or syndrome of the nervous system is selected from the group of: multiple sclerosis, amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease and huntington's disease.

Examples of established links between inflammatory diseases or syndromes of the nervous system and viral infections:

the disease or syndrome is preferably associated with a coronavirus infection, and more preferably the disease or syndrome is associated with a SARS-CoV-2 infection. The disease or syndrome associated with SARS-CoV-2 disease or syndrome is preferably at least one selected from the group consisting of: fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, weakening or changing of smell or taste, loss of appetite, weight loss, stomach pain, conjunctivitis, rash, lymphoma, apathy and somnolence, preferably fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, weakening or changing of smell or taste.

The present invention relates to a composition for treating and/or preventing a disease or syndrome associated with a viral infection, preferably a coronavirus infection, in a subject in need thereof, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof. The coronavirus is preferably SARS-CoV-2.

The inventors found that AAT and rhAAT inhibit viral entry of various viruses and reduce inflammation (particularly in microglia of the nervous system). In addition, SH-SY5Y cells are of neuronal origin and are often used to study neurodegenerative diseases, including Parkinson's disease (Xicoy, H., wiringa, B. & Martens, G.J.the SH-SY5Y cell line in Parkinson's disease research: a-system review.mol neurogenesis 12,10,2017), which cells exhibit relatively high copy numbers of spike protein-initiated protease mRNA, i.e., trypsin and cathepsin B. These nervous tissues (Bielarz V, willemart K, avalose N et al, susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2infection.Brain Res.2021, day 5, month 1) and similarly derived cells, combined with relatively high levels of ACE2 expression in SH-SY5Y, become susceptible to SARS-CoV-2 virus infection.

Thus, the present invention is based, at least in part, on the broad role of AAT in diseases or syndromes associated with viral infections.

The subject may be particularly suitable for treatment and/or prophylaxis with AAT and/or rhAAT proteins.

Accordingly, the present invention also relates to a composition for the treatment and/or prevention of a disease or syndrome associated with a viral infection, preferably a coronavirus infection, more preferably a SARS-CoV-2 infection, in a subject in need thereof, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof, wherein the subject in need thereof has at least one altered level compared to at least one reference subject selected from the group consisting of: i) Endogenous Alpha Antitrypsin (AAT), at least one spike protein-triggering protease, angiotensin converting enzyme 2 (ACE 2 receptor) and interferon-gamma (IFN-gamma). The α1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof may be plasma extracted AAT, variant, isoform and/or fragment thereof, in particular human plasma extracted AAT, variant, isoform and/or fragment thereof; or recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof, preferably alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof are recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof.

A "subject in need thereof" is also referred to as a subject of interest. The subject in need thereof may be a subject (i.e., an infected subject) during or suffering from a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) having a disease or syndrome associated with the viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection). An infected subject may be in need of treatment and/or prevention of a disease or syndrome associated with a viral infection, preferably a coronavirus infection, more preferably a SARS-CoV-2 infection. Treatment with AAT and/or rhAAT may inhibit or reduce entry of a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 virus) into a cell, reduce transmission of a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 virus) in vivo, and/or reduce inflammation in response to a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) by inhibiting spike protein-triggered proteases. The subject in need of treatment and/or prophylaxis may have respiratory syndrome, more preferably acute respiratory syndrome, even more preferably severe acute respiratory syndrome. A subject in need of treatment and/or prophylaxis may have at least one symptom selected from the group consisting of: fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, weakening or changing of smell or taste, anorexia, weight loss, gastralgia, conjunctivitis, rash, lymphoma, apathy and somnolence, preferably selected from the group consisting of: fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, and weakening or changing of smell or taste. A subject in need of treatment and/or prophylaxis may require intensive care and/or artificial ventilation. A subject in need of treatment and/or prophylaxis may be defined by one or any combination of the above definitions.

The subject in need thereof may also be a subject prior to a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), which is particularly susceptible to a disease or syndrome following a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection). Such subjects may need to be protected from diseases or syndromes associated with viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) prior to infection.

The at least one reference subject may be a reference subject group. Preferably, the reference (reference) subject is a subject suffering from or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) or having mild symptoms (i.e. an infected subject), more preferably the subject is a subject suffering from or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection).

According to the invention, asymptomatic means that the (reference) subject is asymptomatic, preferably free of symptoms selected from the group consisting of: fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, weakening or changing of smell or taste, anorexia, weight loss, gastralgia, conjunctivitis, rash, lymphoma, apathy and somnolence, more preferably without symptoms selected from the group consisting of: fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, and weakening or changing of smell or taste. Asymptomatic (reference) subjects preferably do not have respiratory syndrome, more preferably do not have acute respiratory syndrome, even more preferably do not have severe acute respiratory syndrome. Asymptomatic (reference) subjects do not require intensive care and/or artificial ventilation. Asymptomatic (reference) subjects may be defined by one of the above definitions or any combination thereof.

The (reference) subject with mild symptoms according to the invention preferably does not suffer from respiratory syndrome, more preferably does not suffer from acute respiratory syndrome, even more preferably does not suffer from severe acute respiratory syndrome. The (reference) subject with mild symptoms preferably does not require intensive care and/or artificial ventilation. A (reference) subject with mild symptoms may have at least one symptom selected from the group consisting of: fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, weakening or changing of smell or taste, loss of appetite, weight loss, gastralgia, conjunctivitis, rash, lymphoma, apathy and somnolence, more preferably without fever, cough, fatigue, dyspnea, chills, joint or muscle pain, expectoration, phlegm production, wheezing, myalgia, joint pain or sore throat, headache, nausea, vomiting, diarrhea, sinus pain, nasal obstruction, weakening or changing of smell or taste, wherein a (reference) subject with mild symptoms does not need intensive care and/or artificial ventilation. A (reference) subject with mild symptoms may be defined by one of the above definitions or any combination thereof.

The reference subject may be a child, in particular a child aged less than 10 years, preferably less than 5 years. The age of the reference subject may be 1 to 10 years, preferably 2 to 5 years.

The (reference) subject during or suffering from a viral infection, in particular a coronavirus infection, more in particular a SARS-CoV-2 infection, is a subject which is preferably a (reference) subject infected with a virus, in particular a coronavirus, more in particular a SARS-CoV-2 infection. An infected (reference) subject means that a virus, in particular a coronavirus, more in particular a SARS-CoV-2 virus, has entered, preferably proliferated in, cells of the body of the (reference) subject.

After infection with a virus, particularly a coronavirus, more particularly SARS-CoV-2, the level of interferon-gamma in the infected subject increases. An increase in interferon-gamma levels in turn leads to an increase in angiotensin converting enzyme 2 (ACE 2 receptor) levels. An increase in the level of angiotensin converting enzyme 2 (ACE 2 receptor) stimulates an increase in spike protein activity and the triggering of spike proteins by proteases. The endogenous level of AAT is then reduced in response to an increase in the level of activity of the at least one spike protein-initiated protease. Thereafter, as AAT binds (and inhibits) active spike protein-triggered proteases, the endogenous levels of AAT are further depleted. A subject having at least one selected from the group consisting of: i) a lower level of endogenous alpha-antitrypsin (AAT), ii) a higher level of at least one spike-protein-triggering protease, iii) a higher level of angiotensin converting enzyme 2 (ACE 2) receptor, and iv) a higher level of interferon-gamma (IFN- γ) compared to at least one reference subject. Thus, these subjects of interest are particularly relevant and suitable for receiving treatment and/or prophylaxis with a composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof. The α1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof may be plasma extracted AAT, variant, isoform and/or fragment thereof, in particular human plasma extracted AAT, variant, isoform and/or fragment thereof; or recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof, preferably alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof are recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof. Most preferably, the AAT protein is a recombinant alpha 1-antitrypsin (rhAAT) protein produced in Chinese Hamster Ovary (CHO) cells and/or Human Embryonic Kidney (HEK) cells.

The present invention also relates to a composition for use in the treatment and/or prevention of a disease or syndrome associated with a viral infection, preferably a coronavirus infection, more preferably a SARS-CoV-2 infection, in a subject in need thereof, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein and/or a recombinant alpha 1-antitrypsin (rhAAT) protein, variants, isoforms and/or fragments thereof, wherein the subject in need thereof has at least one selected from the group consisting of:

1. a lower level of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. a higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

3. Higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

4. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

At least one asymptomatic or mildly symptomatic subject is also referred to as at least one reference subject. The reference subject is defined as described herein.

A "subject in need thereof" is also referred to as a subject of interest. There is a need for the treatment and/or prevention of disease syndromes associated with viral infections (preferably coronavirus infections, more preferably SARS-CoV-2 infections) as defined herein.

i) The level defined in iv) may be a protein level and/or an mRNA level, preferably a protein or mRNA level. Protein levels are measured using antibody-based assays such as enzyme-linked immunosorbent assay (ELISA) and/or Biological Layer Interferometry (BLI) based on fiber optic biosensors (ForteBio octets).

The level of spike protein-triggering protease can be determined by measuring the activity of spike protein protease using a fluorescent-producing peptide derived from SARS-CoV-2spike protein. The method is described in Jaimes et al (Javier A. Jaimes, jean K. Milet, gary R. Whittaker Proteolytic Cleavage of the SARS-CoV-2Spike Protein and the Role of the Novel S1/S2 Site, CELL, iScience 23,101212,2020, 6 months, 26 days). Transparent method peptide: the sequences HTVSLLRSTSQ (SEQ ID NO: 3) and TNSPRRARS, respectively, which are derived from the S1/S2 site of SARS-CoV-2spike (S)The fluorogenic peptide, which is composed of the VA (SEQ ID NO: 4) sequence and carries a (7-methoxycoumarin-4-yl) acetyl/2, 4-dinitrophenyl (MCA/DNP) FRET pair, is synthesized by biomatrik (Wilmington, DE, USA). Recombinant furin is available from New England Biolabs (Ipswich, MA, USA). Recombinant L-1-toluenesulfonamide-2-phenylethylchloromethyl ketone (TPCK) -treated trypsin is available from Sigma-Aldrich (St Louis, MO, USA). Recombinant PC1, proteolytic enzyme, cathepsin B and cathepsin L can be derived from R &D Systems (Minneapolis, MN, USA).Peptide assay to generate fluorescence:for each fluorogenic peptide, the reaction was performed in a volume of 100. Mu.L containing buffer and peptide diluted to 50. Mu.M, the buffer for furin (diluted to 10U/mL) consisting of 100mM Hepes, 0.5% Triton X-100, 1mM CaCl2 and 1mM 2-mercaptoethanol (pH 7.5); the buffer for PC1 (diluted to 2.2 ng/. Mu.L) consisted of 25mM MES, 5mM CaCl2, 1% (w/v) Brij-35 (pH 6.0); PBS for trypsin (diluted to 8 nM); the buffer for the proteolytic enzyme (diluted to 2.2 ng/. Mu.L) was prepared from 50mM Tris, 50mM NaCl, 0.01% (v/v)20 (pH 9.0); the buffer for cathepsin B (diluted to 2.2 ng/. Mu.L) consisted of 25mM MES (pH 5.0); the buffer for cathepsin L (diluted to 2.2 ng/. Mu.L) consisted of 50mM MES, 5mM DTT, 1mM EDTA, 0.005% (w/v) Brij-35 (pH 6.0). The reaction was performed in triplicate at 30 ℃ and fluorescence emission was measured once per minute using a spectromax fluorometer (Molecular Devices, sunnyvale, CA, USA) for 45 minutes with wavelengths set to λex 330nm and λem 390nm, enabling tracking of the fluorescence intensity over time as well as the calculation of the reaction Vmax. The assay should be performed in triplicate and the results represent the average of Vmax from three independent experiments.

IFN-gamma protein levels can be measured using flow cytometry, particle-based immunoassays. The method may be used with the BD human Th1/Th2 cytokine or Chemokine Bead Array (CBA) kit from Huang et al (Huang KJ, su IJ, theron M et al, an interface-gamma-related cytokine stormin SARS parameters.J Med virol.2005;75 (2): 185-194.Doi: 10.1002/jmv.20255). The BD human Th1/Th2 cytokine CBA kit (BD PharMingen, san Diego, CA) measures IFN- γ levels in particle-based immunoassays by flow cytometry. The kit allows simultaneous measurement of six cytokines in a serum sample from a 50ml patient. These immunoassays have a limit of detection for IFN-gamma of 7.1pg/ml.

Preferably, the endogenous level of AAT described herein is protein level. The level of at least one spike protease described herein is preferably mRNA level. The ACE2 receptor levels described herein are preferably mRNA levels. The IFN-gamma levels described herein are preferably protein levels. Protein and/or mRNA levels may be measured in blood, urine or saliva (preferably in blood, more preferably in plasma, most preferably in human plasma).

Several factors have been known for the entry and spread of viruses (particularly coronaviruses, more particularly SARS-CoV-2 virus) into cells, while others remain the subject of investigation. The entry of the SARS-CoV-2 virus is mediated by spike proteins, spike protein-initiated proteases and the ACE2 receptor. SARS-CoV-2 spike protein is also known as spike protein S. The spike protein triggers the protease to cleave the spike protein of SARS-CoV-2, thereby triggering the entry of SARS-CoV-2 into the cell. SARS-CoV-2 enters cells through the evoked interaction of spike protein with ACE receptor. Thus, the SARS-CoV-2 virus enters the cell more easily and faster if at least one or more elicitor proteases are present in the subject of interest. In addition, the more ACE2 receptor present, the easier and faster SARS-CoV-2 enters the cell. Infection with SARS-CoV-2 results in inflammation and thus in increased IFN-gamma expression. IFN-gamma expression in response to SARS-CoV-2 infection can in turn lead to increased ACE2 receptor expression. During the proliferation of SARS-CoV-2, IFN- γ levels are further increased, thereby stimulating an increase in ACE2 interaction with spike proteins and subsequent initiation of spike proteins, a decrease in AAT levels and an increase in inflammation after the virus enters the cell, resulting in even higher levels of IFN- γ. After infection with SARS-CoV-2, IFN-gamma levels are first increased, then AAT levels are decreased and cathepsin L levels are increased and/or other spike-protein-initiated (S-initiated) proteases are increased.

Accordingly, the present invention is based, at least in part, on the following findings: AAT and rhAAT simultaneously reduce viral entry and viral-related inflammation, particularly due to high IFN- γ levels. Such a combined effect is particularly useful for the patient populations described herein.

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the spike protein causing protease is at least one selected from the group consisting of: transmembrane protease serine subtype 2 (TMPRSS 2), transmembrane protease subtype 6 (TMPRSS 6), cathepsin L, cathepsin B, proprotein convertase 1 (PC 1), trypsin, elastase, neutrophil elastase, proteolytic enzymes and furin.

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the spike protein-induced protease is cathepsin L and/or furin.

AAT is expressed endogenously in humans. AAT is also known as an alpha-1-proteinase inhibitor. AAT is capable of inhibiting proteases, in particular spike proteases, such as transmembrane protease serine subtype 2 (TMPRSS 2), transmembrane protease subtype 6 (TMPRSS 6/proteolytic enzyme-2), cathepsin L, cathepsin B, proprotein convertase 1 (PC 1), trypsin, elastase, neutrophil elastase, proteolytic enzyme and furin. If the four participants of the invention (AAT, spike protein-triggered protease, ACE2 receptor, and IFN- γ) are altered in an infected subject of interest as compared to a reference subject, the infected subject of interest may particularly benefit from treatment and/or prevention of a disease or syndrome associated with a SARS-CoV-2 infection. Low endogenous AAT levels may lead to a subject of interest being more susceptible to diseases or syndromes associated with SARS-CoV-2, particularly to covd-19.

In certain embodiments, the invention relates to compositions for use according to the invention, wherein the lower level of endogenous AAT prior to or during viral infection is caused by AAT deficiency.

Alpha 1-antitrypsin(hereinafter "AAT") is a protein naturally occurring in the human body and produced in the liver (preferably in hepatocytes). According to Janciauskiene et al (Janciauskiene SM, bals R, koczella R, vogelmeier C,t, welte T.the discovery of α1-antitrypsin and its role in health and disease. Respir Med.2011;105 1129-1139. Doi:10.1016/j.rmed.2011.02.002), AAT has a normal plasma concentration in the range of 0.9 to 1.75g/L. MW was considered to be 52,000 (Branly M, nukiwa T, crystal RG. Molecular basic of alpha-1-antippsin prescribing. Am J Med.1988;84 (6A): 13-31.Doi:10.1016/0002-9343 (88) 90154-4), which corresponds to normal plasma concentrations of 16 to 32. Mu.M. Crystal1990 (Crystal RG. Alpha 1-antitrypsin deficiency, emphysema, and lever treatment. Genetic basis and strategies for treatment. J Clin invest 1990May;85 (5): 1343-52.Doi:10.1172/JCI114578.PMID:2185272; PMCID: PMC 296579) describes that 11. Mu.M is a threshold level for the clinical manifestation of AAT deficiency. For most healthy individuals, 2g of AAT expression per day in the liver is sufficient to reach a critical serum level of 11 μm, then endogenous AAT levels are sufficient to protect the lower respiratory tract from Neutrophil Elastase (NE) and inhibit progressive destruction of alveoli, ultimately leading to emphysema. Crystal1990 further indicated that normal endogenous levels of AAT vary between 20-53. Mu.M in healthy individuals. Prior to viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection), the endogenous level of AAT protein in the plasma of a healthy human subject ranges between 5 and 60. Mu.M, preferably between 10 and 40. Mu.M, more preferably between 25 and 30. Mu.M, even more preferably between 16 and 32. Mu.M. Alternatively, the endogenous level of AAT protein in the plasma of a healthy human subject is preferably above 30 μm, more preferably above 40 μm, most preferably above 50 μm, prior to viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection). The AAT protein may be a plasma AAT protein or a recombinant AAT (rhAAT) protein. In some embodiments, the plasma AAT protein is derived from plasma. In some embodiments, the recombinant AAT protein is recombinantly produced in, for example, HEK cells, CHO cells, or E.coli (E.coli) cells . Pharmaceutical companies around the world extract AAT protein from human plasma for the treatment of AAT deficiency, a genetic disorder. AAT of plasma origin is approved by the united states Food and Drug Administration (FDA) and European Medicines Administration (EMA), respectively, in the united states and the european union. Preferably, the AAT proteins of the invention have a human amino acid sequence, most preferably the amino acid sequence shown in SEQ ID NO. 1.

In a549 cells overexpressing ACE2 receptor, 10 μm AAT reduced pseudovirus entry by 20-30%.

Dose-dependent inhibition of TMPRSS2 proteolytic activity was achieved by AAT at a concentration of 1-100. Mu.M according to Azouz et al (Nurit P.Azouz, andrea M.Klingler and Marc E.Rothenberg, alpha1-Antitrypsin (AAT) is an Inhibitor of the SARS-CoV-2-Priming Protease TMPRSS2, (bioRxiv preprint online, https:// doi.org/10.1101/2020.05.04.077826, release 5.5.2020).

In a549 cells that overexpress only ACE2 receptor, 100 μm AAT reduced pseudoviral entry by 50-75%.

Only in a549 cells overexpressing both the ACE2 receptor and the spike protein priming protease TMPRSS2, 100 μm AAT reduced pseudoviral entry by up to 45%.

Notably, viral entry was observed independent of TMPRSS2. It has been demonstrated that a trigger protease such as furin and/or cathepsin L may be particularly capable of replacing TMPRSS2. In this regard, AAT and rhAAT reduce the activity of protease cathepsin B, cathepsin L, trypsin, furin, PC1, proteolytic enzymes, elastase and neutrophil elastase.

Thus, the inhibition of viral entry by AAT and rhAAT exceeds that of other TMPRSS2 inhibitors, as AAT and rhAAT are effective in inhibiting several trigger proteases (e.g., proteases that may replace TMPRSS2 function) and subsequent ACE2 mediated viral entry.

Thus, AAT as well as rhAAT reduces viral entry by reducing the elicitation protease activity, particularly by widely and effectively reducing the elicitation protease activity.

The present invention is therefore based, at least in part, on the surprising discovery that: compositions comprising AAT and/or rhAAT, variants, isoforms and/or fragments thereof are particularly effective in treating and/or preventing diseases or syndromes associated with viral infections (preferably coronavirus infections, more preferably SARS-CoV-2 infections), particularly in subjects having one or more of the prerequisites described herein.

In the present invention, the lower level of endogenous AAT according to i) is preferably the protein level in human plasma. The level of endogenous AAT protein in human plasma according to i) is preferably less than 200 μm, preferably less than 150 μm, 100 μm, less than 90 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 25 μm, less than 20 μm, less than 15 μm, less than 11 μm or less than 10 μm. More preferably less than 200. Mu.M, less than 100. Mu.M, less than 25. Mu.M, less than 15. Mu.M, or less than 11. Mu.M. Low levels of AAT are insufficient to successfully inhibit spike-protein-induced proteases. Thus, low levels of endogenous AAT can promote the development of viral proliferation (particularly coronavirus proliferation, more particularly SARS-CoV-2 proliferation) and/or diseases or syndromes associated with viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection). Lower levels of endogenous AAT may be caused by AAT deficiency prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), preferably prior to a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection). AAT deficiency is a disorder inherited in an autosomal co-dominant mode. Co-dominant means that two different versions of the gene may be active (expressed) and both versions contribute to the genetic trait. The most common version (allele) of the SERPINA1 gene is called M, producing normal levels of alpha-1 antitrypsin. There are two copies of the M allele (MM) per cell in most humans in the general population. Other versions of the SERPINA1 gene result in reduced alpha-1 antitrypsin levels. For example, the S allele produces moderately low levels of this protein, and the Z allele produces very little alpha-1 antitrypsin. Individuals with two copies of the Z allele (ZZ) in each cell may suffer from alpha-1 antitrypsin deficiency. Individuals with SZ combinations have an increased risk of developing pulmonary diseases (such as emphysema), especially those who smoke. It is estimated that there are 1.61 million people worldwide with one copy of the S or Z allele and one copy of the M allele (MS or MZ) in each cell. Individuals with MS (or SS) combinations will typically produce sufficient alpha-1 antitrypsin to protect the lungs. However, the risk of impaired lung or liver function in humans with MZ allele is slightly increased. The subject suffering from AAT deficiency in the present invention preferably has a ZZ mutation, SZ mutation, MS mutation, MZ mutation or SS mutation, preferably ZZ mutation, of the SERPINA1 gene. The low level of AAT secretion in ZZ mutations in the SERPINA1 gene is due to AAT misfolding and its subsequent accumulation in the hepatocyte Endoplasmic Reticulum (ER) (Crystal 1990), as the accumulation of misfolded AAT negatively affects hepatocyte health causing progressive liver disease, leading to its eventual death.

Lower levels of endogenous AAT during viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) may also be caused by viral infection, coronavirus infection or SARS-CoV-2 infection, respectively. That is, during viral infection, the endogenous AAT level of the reference subject may temporarily increase because healthy hepatocytes attempt to overcompensate for a decrease in endogenous AAT level, while the endogenous AAT level of a subject with hereditary AAT deficiency decreases due to an increase caused by a lack or incomplete viral infection (lack of healthy hepatocytes).

Lower levels of endogenous AAT may also be caused by liver diseases such as non-alcoholic fatty liver disease, type 1 or type 2 diabetes (preferably type 1), obesity and cardiovascular disorders. Accumulation of fatty acid deposits in the liver can lead to increased stress (increased IFN- γ), tissue inflammation and subsequent damage to hepatocytes, resulting in reduced healthy secretion levels of AAT. As with other types of liver disease, it is notable that over time AAT deficiency can lead to liver disease, as accumulation of misfolded AAT in the hepatocyte ER ultimately leads to liver failure.

In the present invention, the spike protein-causing protease may be any protease capable of causing spike protein of a virus (preferably coronavirus, more preferably SARS-CoV-2). Preferably, the spike protein-triggering protease is at least one selected from the group consisting of: transmembrane protease serine subtype 2 (TMPRSS 2), transmembrane protease subtype 6 (TMPRSS 6), cathepsin L, cathepsin B, proprotein convertase 1 (PC 1), trypsin, elastase, neutrophil elastase, proteolytic enzyme and furin, more preferably TMPRSS2, cathepsin L and furin, even more preferably cathepsin L or furin. The spike-protein-initiated protease is furin, also known as a paired basic amino acid cleavage (PACE) enzyme.

The higher level of at least one spike protein-triggering protease, preferably cathepsin L, described herein is preferably an mRNA level in human plasma or a protein level in human plasma. The plasma concentration of cathepsin L in healthy subjects is 0.2 to 1ng/mL (i.e.10 to 50pM, molecular weight of about 23-24kDa; kirschke 1977https:// febs. Onlineibrary. Wiley. Com/doi/10.1111/j.1432-1033.1977.Tb11393. X)). In addition, immune cells are known to be a major source of extracellular cysteine cathepsins in inflammation, including in the brain (Hayashi et al, 2013, von Bernhardi et al, 2015, wendt et al, 2008, wendt et al, 2007). The level of at least one spike protein-triggered protease protein described herein is preferably above 0.2, 0.5 or 1ng/ml in human plasma. The level of at least one spike protein-triggering protease protein described herein is preferably higher than 10, 20, 30, 40, 50, 75 or 100pM, preferably higher than 10 or 50pM, even more preferably higher than 50pM. In this embodiment, the at least one spike protein-triggering protease is preferably cathepsin L. In certain embodiments, at least one spike protein trigger protease described herein is at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine spike protein trigger proteases. In certain embodiments, the at least one spike protein-triggering protease described herein is at least one protease selected from the group consisting of TMPRSS2, cathepsin B, cathepsin L, trypsin, furin, PC1, proteolytic enzyme, elastase, and neutrophil elastase. In certain embodiments, at least one spike protein-triggering protease described herein is at least one protease selected from the group consisting of TMPRSS2, cathepsin B, cathepsin L, trypsin, furin, PC1, and proteolytic enzymes.

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the higher level of at least one spike protein-triggering protease is caused by age and/or genetic predisposition.

The higher level of at least one spike protein-triggering protease may be caused by age and/or genetic predisposition. In particular, higher levels of cathepsins B and L (preferably cathepsin B) may be caused by age. Higher levels of spike protease proteins (particularly cathepsins B and L, more preferably cathepsin B) may accumulate in lysosomes. The level of spike-protein-triggering protease is higher in a subject of interest who is over 50 years old, preferably over 60 years old, more preferably over 70 years old, even more preferably over 80 years old, and most preferably over 90 years old. Subjects of african american origin may have a higher genetic predisposition to spike protein-induced proteases, particularly furin. The relative mRNA ratios of furin and cathepsin L were different for HeLa cells compared to a549 cells. HeLa cells are of non-American origin. A549 cells are airway epithelial cells of caucasian origin. HeLa cells are more susceptible to SARS-CoV-2 entry than A549 cells and are less responsive to AAT treatment. Genetic predisposition may be determined by genetic analysis of the individual subject of interest.

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the higher level of ACE2 receptor is caused by at least one selected from the group of infection, inflammation, age and genetic predisposition.

The higher levels of ACE2 receptor described herein are preferably mRNA levels in human plasma. Higher levels of ACE2 receptor may be caused by at least one selected from the group of infection, inflammation (e.g., IFN- γ), age, and genetic predisposition. The infection may be a viral infection and/or a bacterial infection, preferably a viral infection, more preferably a coronaviral infection, even more preferably a SARS or SARS-CoV-2 infection. Another example of an infection is leishmaniasis. ACE2 receptor expression is upregulated in response to higher levels of IFN- γ, which in turn increases with immune response to inflammation. Inflammation may be caused by, for example, bacterial and/or viral infection, cancer, delayed type hypersensitivity; autoimmune diseases such as autoimmune encephalomyelitis, rheumatoid arthritis, autoimmune insulitis (also known as type 1 diabetes), allograft rejection and graft versus host reaction, non-specific inflammation and cytokine release. The age is preferably greater than 50 years old, preferably greater than 60 years old, more preferably greater than 70 years old, even more preferably greater than 80 years old, and most preferably greater than 90 years old. An example of a genetic predisposition to high levels of IFN-gamma is familial mediterranean fever. Genetic predisposition may be determined by genetic analysis of the individual subject of interest. Higher levels of ACE2 receptor described herein may also be present in tissues selected from the group consisting of lung (Calu 3), colon (CaCo 2), liver (HEPG 2), kidney (HEK-293T) and brain (SH-SY 5Y), as demonstrated by qPCR.

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the higher level of IFN- γ is caused by at least one selected from the group of infection, inflammation, age and genetic predisposition.

The higher level of interferon-gamma (IFN-gamma) according to iv) is preferably the level of protein or mRNA in human plasma, more preferably the level of protein in human plasma. In healthy humans, IFN-gamma levels are below or near the detection limit of the assay, e.g., below 30 to 50pg/mL (Billau 1996; kimura 2001). IFN-gamma is almost entirely produced by Natural Killer (NK) cells, CD4+ and some CD8+ lymphocytes. NK or T cells produce IFN-gamma requiring the cooperation of helper cells (principally mononuclear phagocytes) which also need to be in some activated state (Billiau A. Interferon-gamma: biology and role in pathogensis. Adv immunol.1996;62:61-130.Doi:10.1016/s0065-2776 (08) 60428-9). Thus, inflammatory conditions leading to NK cell activation and T cell activation lead to increased IFN-gamma levels. Inflammatory states involving circulating NK or T cells (infection, cancer) are expected to lead to higher plasma levels.

The following table provides values for IFN-gamma plasma levels under several conditions.

TABLE 1

The level of IFN-gamma protein in human plasma according to iv) is preferably higher than 1, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 300, 400 or 500pg/ml. The higher level of IFN-gamma is preferably caused by at least one selected from the group of infection, inflammation, age and genetic predisposition, more preferably by inflammation. The infection may be a viral infection and/or a bacterial infection, preferably a viral infection, more preferably a coronaviral infection, even more preferably a SARS or SARS-CoV-2 infection. Another example of an infection is leishmaniasis. Inflammation may be caused by, for example, bacterial and/or viral infection, cancer, delayed type hypersensitivity; autoimmune diseases such as autoimmune encephalomyelitis, rheumatoid arthritis, autoimmune insulitis (also known as type 1 diabetes), allograft rejection and graft versus host reaction, non-specific inflammation and cytokine release. The age is preferably greater than 50 years old, preferably greater than 60 years old, more preferably greater than 70 years old, even more preferably greater than 80 years old, and most preferably greater than 90 years old. An example of a genetic predisposition to high levels of IFN-gamma is familial mediterranean fever. Genetic predisposition may be determined by genetic analysis of the individual subject of interest.

The present invention is therefore based, at least in part, on the surprising discovery that: compositions comprising AAT and rhAAT, variants, isoforms and/or fragments thereof reduce viral proliferation and inflammation, particularly IFN-gamma related inflammation.

In one embodiment, a "subject in need thereof," i.e., a subject of interest has two selected from the group consisting of:

1. lower levels of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. a higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

3. Higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

4. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

Thus, a "subject in need thereof" may have

1. A lower level of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

2. The at least one spike protein priming protease is present at a higher level prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

Preferably, in this example, the AAT protein level in human plasma described herein is less than 52. Mu.M, and the cathepsin L protein level in human plasma is greater than 10pM.

In another embodiment, a "subject in need thereof" may have

1. A lower level of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

2. higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection).

In yet another embodiment, a "subject in need thereof" may have

1. A lower level of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

2. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

In these embodiments, 1 and 4 may be at least one disease or condition selected from the group consisting of: AAT deficiency, liver disease (such as non-alcoholic fatty liver disease), diabetes, obesity, and cardiovascular disorders. In some embodiments, the invention relates to a composition for use according to the invention, wherein the higher level of IFN- γ is caused by at least one disease or disorder selected from the group consisting of: AAT deficiency, liver disease (such as non-alcoholic fatty liver disease), diabetes, obesity, and cardiovascular disorders. In some embodiments, the invention relates to a composition for use according to the invention, wherein the lower level of AAT is caused by at least one disease or disorder selected from the group consisting of: AAT deficiency, liver disease (such as non-alcoholic fatty liver disease), diabetes, obesity, and cardiovascular disorders. Diabetes may be type 1 or type 2 diabetes. Liver disease may be acetaminophen-induced liver injury, severe chronic hepatitis, alcoholic Liver Disease (ALD), which encompasses a variety of phenotypes including simple steatosis, steatohepatitis, liver fibrosis and cirrhosis or even HCC (hepatocellular carcinoma). The cardiovascular condition may be any condition caused by sudden decrease or blockage of cardiac blood flow, myocardial infarction, acute Coronary Syndrome (ACS), and is also present in patients with acute myocardial infarction in which the left ventricular ejection fraction is inversely related to AAT concentration in serum, indicating that systolic dysfunction is associated with an inflammatory response.

Preferably, in this example, the AAT protein level in human plasma described herein is less than 52. Mu.M, and the IFN-gamma protein level in human plasma described herein is greater than 0.19pM.

In a further embodiment, a "subject in need thereof", i.e. a subject of interest has two selected from the group consisting of:

1. a higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

3. A higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

In yet another embodiment, a "subject in need thereof" may have

1. A higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

2. higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection).

In yet another embodiment, a "subject in need thereof" may have

1. A higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

2. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

In yet another embodiment, a "subject in need thereof" may have

1. Higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

2. A higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

In a further embodiment, a "subject in need thereof", i.e. a subject of interest has three selected from the group consisting of:

1. lower levels of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. a higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

3. Higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

4. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

Thus, a "subject in need thereof" may have

1. Lower levels of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. A higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

3. higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection).

In a further embodiment, a "subject in need thereof" may have

1. Lower levels of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. A higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

3. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

Preferably, in this example, the AAT protein level in human plasma described herein is less than 36. Mu.M, the cathepsin L protein level in human plasma described herein is greater than 10pM, and the IFN-gamma protein level in human plasma described herein is greater than 0.19mM.

More preferably, in this embodiment, the AAT protein level in human plasma described herein is less than 52. Mu.M, the cathepsin L protein level in human plasma described herein is greater than 10pM, and the IFN-gamma protein level in human plasma described herein is greater than 0.19mM.

In a further embodiment, a "subject in need thereof" may have

1. A higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

3. a higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

In a further embodiment, a "subject in need thereof", i.e., a subject of interest has

1. Lower levels of endogenous alpha-antitrypsin (AAT) prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

2. a higher level of at least one spike protein-eliciting protease prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) than at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

3. higher levels of angiotensin converting enzyme 2 (ACE 2 receptor) in a subject prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), compared to at least one subject that is asymptomatic or has mild symptoms during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), and

4. A higher level of interferon-gamma (IFN-gamma) in the subject prior to or during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection) than in at least one subject that is asymptomatic or has mild symptoms during the viral infection (preferably coronavirus infection, more preferably SARS-CoV-2 infection).

The AAT protein in the composition for use of the invention may be a plasma AAT protein, a variant, isoform and/or fragment thereof; or recombinant AAT proteins, variants, isoforms and/or fragments thereof. Preferably, the AAT protein, variant, isoform and/or fragment thereof is a recombinant AAT protein, variant, isoform and/or fragment thereof. The plasma AAT protein is preferably derived from plasma AAT protein, more preferably from human plasma (also known as human plasma extracted AAT). In certain embodiments, the invention relates to compositions for use according to the invention, wherein the α1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof is recombinant α1-antitrypsin (also known as rhAAT), variant, isoform and/or fragment thereof.

Recombinant AAT proteins are recombinantly produced in CHO cells, HEK cells (HEK 293 and/or HEK 293T) or e.coli cells, for example. Pharmaceutical companies around the world extract AAT protein from human plasma for the treatment of AAT deficiency, a genetic disorder. AAT of plasma origin has been approved by FDA and EMA. Preferably, the AAT proteins of the invention have a human amino acid sequence, most preferably the amino acid sequence shown in SEQ ID NO. 1. The recombinant AAT protein, variant, isoform and/or fragment thereof preferably does not contain an Fc domain and/or a histidine tag (His tag).

The inventors found that recombinant AAT (rhAAT produced in CHO) binds with different affinities to AAT antibodies and has a more pronounced biological effect than plasma-derived AAT. In particular, recombinant AAT (rhAAT) inhibits ACE 2/spike protein mediated cell fusion more effectively than plasma-derived AAT and has different enzymatic inhibition properties.

The present invention is therefore based, at least in part, on the surprising discovery that: recombinant AAT (rhaAT) produced in CHO cells is particularly effective for the treatment and/or prevention of diseases or syndromes associated with viral infections, preferably coronavirus infections, more preferably SARS-CoV-2 infections.

In certain embodiments, the invention relates to compositions for use according to the invention, wherein the α1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof is recombinant α1-antitrypsin produced in human cells (e.g., HEK293 or HEK293T cells).

The inventors found that recombinant AAT (rhAAT without His tag) produced in human cells, in particular HEK293, was particularly effective in reducing the enzymatic activity of cathepsin L, trypsin, furin and neutrophil elastase compared to recombinant AAT (rhAAT) produced in CHO cells as well as plasma-derived AAT.

The present invention is therefore based, at least in part, on the surprising discovery that: recombinant AAT produced in human cells, particularly in HEK293, is particularly effective for use in the treatment and/or prevention of diseases or syndromes associated with viral infections, preferably coronavirus infections, more preferably SARS-CoV-2 infections.

In certain embodiments, the invention relates to compositions for use according to the invention, wherein the α1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof is recombinant α1-antitrypsin (rhAAT produced in CHO) with a more non-human glycan profile. In certain embodiments, the non-human glycan profile described herein is a mammalian cell-derived glycan profile and/or a glycoengineered glycan profile. For example, glycosylation engineering strategies for reducing fucosylation of glycoproteins and/or enhancing sialylation of glycoproteins are known to those skilled in the art. In certain embodiments, the non-human glycan profile described herein is a CHO cell-derived glycan profile. CHO cells express a different glycosylation machinery than human cells, which results in a different composition of glycans on the surface of the recombinant protein (lande, m.e. & Durocher, y., & 2017,Journal of biotechnology,251,128-140).

In certain embodiments, the invention relates to a composition for use according to the invention, wherein the AAT protein, variant, isoform and/or fragment thereof is recombinant AAT produced by pXC-17.4 (GS System, lonza), variant, isoform and/or fragment thereof.

The inventors found that recombinant AAT (recombinant AAT 1) produced in CHO by pXC-17.4 (GS System, lonza) induced a more pronounced inhibition of elastase and neutrophil elastase activity than plasma-derived α1-protease inhibitors (plasma-derived AAT) and recombinant AAT (recombinant AAT 2) produced in CHO by PL136/PL137 (pCGS 3, merck).

The present invention is therefore based, at least in part, on the surprising discovery that: certain forms of recombinant AAT (rhaAT) are particularly useful for the treatment and/or prevention of diseases or syndromes associated with viral infections, preferably coronavirus infections, more preferably SARS-CoV-2 infections.

As used herein, a "fragment" of an AAT protein, peptide or polypeptide of the invention refers to a sequence that contains fewer amino acids in length than an AAT protein, peptide or polypeptide of the invention, particularly a sequence that contains fewer amino acids than an AAT sequence as set forth in SEQ ID No. 1. The fragment is preferably a functional fragment, e.g. a fragment having the same biological activity as the AAT protein shown in SEQ ID NO. 1. The functional fragment is preferably derived from the AAT protein shown in SEQ ID NO. 1. Any AAT fragment may be used as long as it exhibits the same properties as the native AAT sequence from which it is derived, i.e., is biologically active.

The functional AAT fragment can comprise or consist of the C-terminal fragment of AAT as shown in SEQ ID NO. 2. The C-terminal fragment of SEQ ID NO. 2 consists of amino acids 374 to 418 of SEQ ID NO. 1.

More preferably, the AAT fragment is a fragment comprising fewer amino acids than the C-terminal AAT sequence 374-418 (SEQ ID NO: 2). Alternatively, the AAT fragment consists essentially of SEQ ID NO. 2.

"homology" refers to the percent identity between two polynucleotide or two polypeptide portions. Two nucleic acid or two polypeptide sequences are "substantially homologous" to each other when they exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% or at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% -98% sequence identity over a defined length of the molecule. As used herein, substantially homologous also refers to sequences that exhibit complete identity to the specified sequence.

In general, "identity" refers to the exact nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. The percent identity can be determined by directly comparing sequence information between two molecules by aligning the sequences, calculating the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.

Alternatively, homology may be determined by readily available computer programs, or by hybridizing polynucleotides under conditions that form stable duplex between homologous regions, then digesting with single strand specific nucleases, and sizing the digested fragments. Substantially homologous DNA sequences can be identified in Southern hybridization experiments under stringent conditions, for example, as defined for this particular system. Defining appropriate hybridization conditions is within the skill of the art.

In some embodiments, the invention relates to a composition for use according to the invention, wherein the alpha 1-antitrypsin fragment is a C-terminal sequence fragment or any combination thereof.

The peptide variant may be a linear peptide or a cyclic peptide and may be selected from the group comprising short cyclic peptides derived from the C-terminal sequence as shown in SEQ ID No. 2. Preferably, the short cyclic peptide derived from the C-terminal sequence of alpha 1-antitrypsin will be selected from the non-limiting group comprising loop- (CPFVFLM) -SH, loop- (CPFVFLR) -SH and loop- (CPEVFLM) -SH or any combination thereof.

As used herein, an "isoform" of an AAT protein, peptide or polypeptide of the invention refers to a splice variant resulting from alternative splicing of AAT mRNA.

In some embodiments, the amino acid sequence of AAT, variant, isoform or fragment thereof as described herein is at least 80% identical to the corresponding amino acid sequence of SEQ ID NO. 1. In some embodiments, the amino acid sequence of AAT, variant, isoform or fragment thereof is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding amino acid sequence of SEQ ID NO. 1.

The peptides, isoforms, fragments or variants of the invention may preferably be conjugated to an agent that increases accumulation of the peptide, isoforms, fragments or variants thereof in a target cell, preferably a respiratory cell. Such agents may be compounds that induce receptor-mediated endocytosis, such as, for example, membrane transferrin receptor-mediated endocytosis of transferrin conjugated to therapeutic drugs (Qian z.m. et al, "Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway" Pharmacological Reviews,54,561,2002), or cell membrane permeable carriers that may be selected, for example, from the group of fatty acids such as capric acid, myristic acid and stearic acid, which have been used for intracellular delivery of peptide inhibitors of protein kinase C (ioanides c.g. et al, "inhibit of IL-2receptor induction and IL-2production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C"Cell Immunol, 131,242,1990) and protein tyrosine phosphatases (Kole h.k. Et al, "a peptide-based protein-tyrosine phosphatase inhibitor specifically enhances insulin receptor function in intact cells" j.biol. Chem.271,14302, 1996) or cell membrane permeable carriers that may be selected from peptides. Preferably, a carrier permeable to cell membranes is used. More preferably, a carrier peptide permeable to the cell membrane is used.

If the cell membrane permeable carrier is a peptide, it is preferably a positively charged amino acid rich peptide.

Preferably, such positively charged amino acid-rich peptides are arginine-rich peptides. Futaki et al (Futaki s et al, "ranging-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery" j. Biol. Chem.,276,5836,2001) have demonstrated that the number of Arginine residues in a cell membrane permeable carrier peptide has a significant effect on the internalization process and that it appears that there is an optimal number of Arginine residues for internalization, preferably they contain more than 6 arginines, more preferably they contain 9 arginines. The arginine-rich peptide preferably comprises at least 6 arginines, more preferably at least 9 arginines.

The peptide, isoform, fragment or variant thereof may be conjugated to a cell membrane permeable carrier via a spacer (e.g., two glycine residues). In this case, the cell membrane permeable carrier is preferably a peptide.

Typically, the arginine-rich peptide is selected from the non-limiting group consisting of: HIV-TAT 48-57 peptide (GRKKKRRQRRR; SEQ ID NO. 5), FHV-coat 35-49 peptide (RRRRNRTRRNRRRVR; SEQ ID NO. 6), HTLV-II Rex 4-16 peptide (TRRQRTRRARRNR; SEQ ID NO. 7) and BMV gag 7-25 peptide (SEQ ID NO. 8).

Any cell membrane permeable carrier may be used, as determined by one of skill in the art.

Since the natural peptide (in L-form) is inherently subject to degradation by natural proteases, the peptides of the invention, isoforms, fragments or variants thereof, as well as cell membrane permeable peptides, can be prepared to include the D-form and/or the "reverse-inverted isomers" of the peptide. In this case, the reverse-inverted isomers of the peptides of the present invention and fragments and variants of the cell membrane permeable peptides are prepared.

The peptides of the invention, isoforms, fragments or variants thereof, optionally conjugated to agents that increase peptide accumulation in cells, may be prepared by a variety of methods and techniques known in the art, such as, for example, chemical synthesis or recombinant techniques, as described by Maniatis et al 1982,Molecular Cloning,A laboratory Manual,Cold Spring Harbor Laboratory.

Preferably, the peptides of the invention, isoforms, fragments or variants thereof, optionally conjugated to agents that increase peptide accumulation in cells, as described herein, are recombinantly produced in a cell expression system. A variety of single cell host cells may be used to express the DNA sequences of the present invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of: coli, pseudomonas, bacillus, streptomyces, fungi such as yeast, and animal cells such as CHO, YB/20, NSO, SP2/0, rl.1, B-W and L-M cells, african green monkey kidney cells (e.g., COS1, COS 7, BSCl, BSC40 and BMTlO), insect cells (e.g., sf 9), and human cells and plant cells in tissue culture.

The "therapeutically effective dose or amount" of the α1-antitrypsin protein, variants, isoforms and/or fragments thereof of the invention means an amount that, upon administration, brings about a positive therapeutic or prophylactic response in terms of treating a disease or syndrome associated with a coronavirus infection in a subject.

The term "coronavirus infection" may refer to an infection caused by a coronavirus selected from the group comprising MERS-CoV, SARS-CoV and SARS-CoV-2 and any variant thereof. In some embodiments, the SARS-CoV-2 variant described herein is a SARS-CoV-2 variant selected from the group of lineage b.1.1.207, lineage b.1.1.7, cluster 5, 501.v2 variant, lineage p.1, lineage b.1.429/cal.20c, lineage b.1.427, lineage b.1.526, lineage b.1.1.317, lineage b.1.1.318, lineage b.1.351, lineage b.1.617, and lineage P.3. In some embodiments, the SARS-CoV-2 variants described herein are SARS-CoV-2 variants described by the Nextstrain clade selected from the group 19A, 20C, 20G, 20H, 20B, 20D, 20F, 20I, and 20E. In some embodiments, the SARS-CoV-2 virus described herein is a mutant SARS-CoV-2 variant comprising at least one mutation selected from the group of D614G, E484K, N501Y, S477G/N, P681H, E484Q, L452R and P614R. In some embodiments, the SARS-CoV-2 variant described herein is a SARS-CoV-2 variant derived from a variant described herein. In some embodiments, the SARS-CoV-2 virus described herein is a SARS-CoV-2 variant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity to the viral genomic sequence of the last SARS-CoV-2 variant described herein.

Coronavirus infection can cause respiratory tract infections, resulting in a disease or syndrome that is a respiratory syndrome. The respiratory syndrome may be Severe Acute Respiratory Syndrome (SARS). In some embodiments, SARS-CoV-2 infection is distinguishable by at least one of the following three clinical courses of infection: (1) mild disease with upper respiratory manifestations, (2) non-life threatening pneumonia, and (3) severe conditions including pneumonia, acute Respiratory Distress Syndrome (ARDS), severe systemic inflammation, organ failure, cardiovascular complications.

The composition for use of the present invention may further comprise one or more pharmaceutically acceptable diluents or carriers.

"pharmaceutically acceptable diluent or carrier" refers to a carrier or diluent that can be used to prepare generally safe, non-toxic, and desirable pharmaceutical compositions, and includes carriers or diluents that are acceptable for human pharmaceutical use.

Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Pharmaceutically acceptable diluents or carriers include starch, dextrose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The pharmaceutical composition may further contain one or more pharmaceutically acceptable salts such as, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and organic acid salts such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances (such as wetting or emulsifying agents, pH buffering substances, gels or jellifying materials, flavoring agents, colorants, microspheres, polymers, suspending agents, and the like) may also be present herein. In addition, one or more other conventional pharmaceutical ingredients may also be present, such as preservatives, wetting agents, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, and the like, especially if the dosage form is in a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethyl cellulose, polysorbate 80, phenethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, p-chlorophenol, gelatin, albumin, and combinations thereof. A complete discussion of pharmaceutically acceptable excipients can be found in REMINGTON' S PHARMACEUTICAL SCIENCES (Mack Pub.Co., N.J.1991), which is incorporated herein by reference.

Alternatively, the pharmaceutical composition of the invention further comprises one or more additional therapeutic agents. Preferably, the one or more therapeutic agents comprise a therapeutically effective amount of one or more nucleoside analogues, protease inhibitors, immunosuppressants (e.g., sha Lilu mab or tolizumab), chloroquine, hydroxychloroquine antibiotics, antibodies to structural components of the virus or fragments thereof (e.g., passive immunotherapy), interferon beta (e.g., interferon beta-1 a), and/or vaccines.

In certain other embodiments, the pharmaceutical composition of the invention and one or more additional therapeutic agents will be administered substantially simultaneously or concurrently. For example, a pharmaceutical composition for use in the present invention may be administered to a subject while the subject is undergoing a course of treatment with one or more additional therapeutic agents. In addition, it is contemplated that the subject has or may be concurrently receiving other forms of antiviral therapy.

In some embodiments, additional therapeutic agents may be used to reduce possible side effects associated with administration of the antibodies or antigen binding fragments thereof of the invention.

In some embodiments, additional therapeutic agents may be used to support the effects associated with administration of the antibodies or antigen binding fragments thereof of the invention.

In some embodiments, administration of the additional therapeutic agent and the antibody or antigen-binding fragment thereof of the invention produces a synergistic effect with respect to the desired effect and/or side effect.

In some embodiments, the additional therapeutic agent described herein is at least one agent selected from the group of nucleoside analogs, protease inhibitors, and immunomodulators (such as immunosuppressants).

In some embodiments, the additional therapeutic agent described herein is at least one agent selected from the group of nucleoside analogs, protease inhibitors, immunosuppressives (e.g., sha Lilu mab or tolizumab), chloroquine, hydroxychloroquine antibiotics, antibodies to structural components of viruses or fragments thereof (e.g., passive immunotherapy), interferon beta (e.g., interferon beta-1 a), and/or vaccines.

Non-limiting examples of nucleoside analogs include ribavirin, adefovir, beta-d-N4-hydroxycytidine, BCX4430, gemcitabine hydrochloride, 6-azauridine, mizoribine, acyclovir flexer, and combinations of one or more thereof.

Non-limiting examples of protease inhibitors include HIV and/or HCV protease inhibitors.

In some embodiments, the immunomodulatory agent described herein is interferon beta. In some embodiments, the immunomodulatory agent described herein is interferon beta-1 a. Non-limiting examples of immunosuppressants include interleukin inhibitors such as, for example, IL-6 (e.g., sha Lilu mab or tolizumab), IL-1, IL-12, IL-18, and TNF-alpha inhibitors.

Additional therapeutic agents may improve or supplement the therapeutic effects of the compositions and methods described herein.

Accordingly, the present invention is based, at least in part, on the following findings: certain combinations (such as IFN- β -1a with AAT) improve the effects of AAT on viral entry and inflammation.

The invention also encompasses gene delivery vectors and pharmaceutical compositions containing the vectors. Preferably, the gene delivery vector is in the form of a plasmid or vector comprising one or more nucleic acids encoding the AAT proteins, variants, isoforms and/or fragments thereof of the invention. Examples of gene delivery vectors include, for example, viral vectors, non-viral vectors, particulate vectors, and liposomes. Gene delivery is preferably performed in vitro or ex vivo.

Accordingly, the present invention is based, at least in part, on the following findings: AAT gene delivery (gene therapy) reduces the effects of inflammation.

In one embodiment, the viral vector is a vector suitable for ex vivo and in vivo gene delivery (preferably ex vivo gene delivery). More preferably, the viral vector is selected from the group comprising: adeno-associated virus (AAV) and lentiviruses (e.g., 1 st, 2 nd and 3 rd generation lentiviruses), other viral vectors such as adenovirus vectors, herpes virus vectors, and the like are not excluded. Other delivery means or vehicles are known (such as yeast systems, microbubbles, gene guns/means of attaching the vector to gold nanoparticles), and in some embodiments, one or more viral or plasmid vectors may be provided via liposomes, nanoparticles, exosomes, microbubbles or gene guns.

In other embodiments of the invention, the pharmaceutical compositions of the invention are sustained release formulations, or formulations that are administered using a sustained release device. Such devices are well known in the art and include, for example, transdermal patches and mini-implant pumps, which can provide drug delivery in a continuous, steady state manner in multiple doses over time to achieve a sustained release effect with a non-sustained release pharmaceutical composition.

The pharmaceutical compositions of the present invention may be administered to a subject by various routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, via buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal and intra-articular or combinations thereof. For human use, the compositions may be administered as a suitably acceptable formulation in accordance with normal human practice. The skilled artisan will readily determine the dosing regimen and route of administration that best suits a particular patient. The compositions of the present invention may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment guns" or other physical methods such as electroporation ("EP"), "hydrodynamic methods" or ultrasound. The composition may also be administered by intravenous injection, intravenous infusion, infusion with a dosage pump, inhalation nasal spray, eye drops, skin patches, slow release formulations, ex vivo gene therapy or ex vivo cell therapy, preferably by intravenous injection.

The composition may be injected intravenously or topically into the lungs or respiratory tract or electroporated into the tissue of interest.

The invention further provides a method of treating and/or preventing a disease or syndrome associated with a coronavirus infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of i) an α1-antitrypsin protein, variants, isoforms and/or fragments thereof as described herein, or ii) a pharmaceutical composition for use in the invention as described herein.

Further provided are methods of modulating the onset of a coronavirus infection in a subject exposed or suspected of being exposed to a coronavirus, comprising administering to a subject in need of such treatment a therapeutically effective amount of i) an α1-antitrypsin protein, variant, isoform and/or fragment thereof as described herein, or ii) a pharmaceutical composition for use in the invention as described herein.

The invention also relates to determining the susceptibility of a subject of interest to treatment and/or prevention of a disease or syndrome associated with a viral infection, preferably a coronavirus infection, more preferably a SARS-CoV-2 infection, using a composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein as defined herein, variants, isoforms and/or fragments thereof, comprising the steps of:

a) Determining the level of at least one of the group consisting of endogenous alpha 1-antitrypsin, at least one spike protein-eliciting protease, ACE2 receptor and interferon-gamma in a subject of interest prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

b) Determining the level of at least one of the group consisting of endogenous alpha 1-antitrypsin, at least one spike protein-eliciting protease, ACE2 receptor and interferon-gamma in at least one reference subject during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection), wherein the reference subject is asymptomatic or has mild symptoms,

c) Comparing the level of interest determined in step a) with the reference level determined in step b),

wherein a subject of interest is more susceptible to treatment and/or prevention of a disease or syndrome associated with a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection) if the subject of interest has at least one selected from the group consisting of:

1. lower levels of endogenous alpha-antitrypsin (AAT) of interest compared to reference levels of AAT,

2. A higher level of at least one spike protein-triggered protease of interest compared to a reference level of at least one spike protein-triggered protease,

3. higher levels of ACE2 receptor of interest than reference levels of angiotensin converting enzyme 2 (ACE 2 receptor), and

4. higher levels of IFN-gamma of interest compared to a reference level of interferon-gamma (IFN-gamma).

All definitions and combinations provided herein apply to this embodiment, if applicable and unless otherwise indicated.

The invention further relates to a method for determining a therapeutically effective amount of alpha 1-antitrypsin (AAT) for the effective treatment and/or prevention of a disease or syndrome associated with a viral infection, preferably a coronavirus infection, more preferably a SARS-CoV-2 infection, using a composition for use according to the invention, the method comprising the steps of:

a) Determining the level of endogenous alpha 1-antitrypsin in a subject of interest prior to or during a viral infection (preferably a coronavirus infection, more preferably a SARS-CoV-2 infection),

b) The amount of AAT in the composition is determined, which is required to achieve an AAT level of at least 10 μm, preferably at least 20 μm, more preferably at least 50 μm, even more preferably at least 100 μm and most preferably at least 200 μm in the subject.

All definitions and combinations provided herein apply to this embodiment, if applicable and unless otherwise indicated.

In some embodiments, the application relates to a method according to the application, wherein the virus is a coronavirus. In some embodiments, the application relates to methods according to the application, wherein the virus is SARS-CoV-2. In some embodiments, the application relates to a method according to the application, wherein the disease or syndrome is respiratory syndrome or severe acute respiratory syndrome. In some embodiments, the application relates to a method according to the application, wherein the disease or syndrome is an inflammatory disease or syndrome of the nervous system. In some embodiments, the application relates to a method according to the application, wherein the inflammatory disease or syndrome of the nervous system is a disease or syndrome selected from the group of multiple sclerosis, amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease and huntington's disease.

All definitions and combinations provided herein apply to these embodiments, if applicable and unless otherwise indicated.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter herein belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "at least one" means "one or more," "two or more," "three or more," and the like.

"or" should be understood to mean either, both, or any combination thereof, of the alternatives.

"and/or" should be understood to mean either or both of the alternatives.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The terms "comprising" and "including" are used synonymously. "preferred" means one of a series of options, not excluding others. "such as" means an example, but is not limited to the examples mentioned. "consisting of" means including and being limited to any content following the phrase "consisting of".

Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "other embodiments," "some embodiments," "a particular embodiment," or "further embodiments" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be appreciated that a positive recitation of a feature in one embodiment serves as a basis for excluding that feature in a particular embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, the general methods and techniques described herein may be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual,2d ed., cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (1989) and Ausubel et al Current Protocols in Molecular Biology, greene Publishing Associates (1992), and Harlow and Lane antibodies: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (1990).

While embodiments of the invention have been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It is to be understood that changes and modifications may be made by one of ordinary skill in the art within the scope and spirit of the following claims. In particular, the invention encompasses further embodiments having any combination of features from the different embodiments described above and below.

Drawings

Fig. 1: IFNgamma mediated microglial activation

Fig. 2: AAT reduces IFNgamma mediated microglial activation

Fig. 3: verification of AAT anti-inflammatory action against RNA sample extraction

Fig. 4: verification of microglial activation and AAT anti-inflammatory action by GSEA analysis

Fig. 5: AAT (Sigma Aldrich, batch A6150) inhibited TACE activity in a cell-free assay, showing no relative fluorescence units (A) or control activity percentage (B), IC ₅₀ 15.3. Mu.M (C)

Fig. 6: sciatic nerve Electrophysiology (EMG) results, (a) amplitude, (B) conduction velocity

Fig. 7: grip test results, (A) absolute value, (B) percentage of 6 week time point

Fig. 8: results of the rotating stick test (A) absolute value (B) percentage of 6 week time point

Fig. 9: DNAJB9 and PLA2G4B gene expression in response to AAT treatment

Fig. 10: number of axons

Fig. 11: axon diameter

Fig. 12: g ratio

Fig. 13: individual histological images of sciatic nerve semi-thin cross sections. Scale bar 10 μm, a) group 1: WT control, B) cmt1a+ mediator, C) cmt1a+ hAAT

Fig. 14: plasma IL-6 concentration

Fig. 15: plasma TNFα concentration

Fig. 16: study protocol for CMT1A mouse model and AAT administration

Fig. 17: morphology and count of cells after treatment: SH-SY5Y morphological analysis and cell counting after 6-OHDA administration and AAT treatment. The bright field panels (20 x) and cell counts (D0 and D4 of cultures) show the effect of treatment on SH-SY5Y cell phenotype and proliferation and AAT positive effects. A) Example image, B) quantization

Fig. 18: cell viability: the graph shows cell viability measured by absorbance (450 nm) of the control and samples

Fig. 19: quantification of IL-6 in cell supernatants

Examples

It will be appreciated by persons skilled in the art that the invention described herein is susceptible to variations and modifications other than those specifically described. It will be understood that the present invention includes all such changes and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. Accordingly, the present disclosure is to be considered as illustrative and not restrictive in all embodiments, the scope of the invention being indicated by the appended claims, and all changes that come within the meaning and range of equivalency are intended to be embraced therein. Throughout this specification, various references are cited, each of which is incorporated by reference in its entirety. The foregoing description will be more fully understood with reference to the following examples.

Example 1

A) HMC3-MHCII of human microglial cells ^Luc Cells were plated on day 0, activated with ifnγ on days 1 to 2, and luciferase activity and cell viability were measured on day 4.

B) Activation was measured by MHCII driven luciferase activity and normalized to cell viability. Luciferase activity under all conditions was expressed as fold-over of untreated control. The potential effect of the highest concentration buffer for drug presentation (ifnγ, AAT) was excluded. All conditions (FIG. 1) were performed in triplicate and error bars represent standard deviation.

Example 2

A) HMC3-MHCII of human microglial cells ^Luc And HMC3-MHCII ^Luc ；Ubi ^AAT Cells were plated on day 0, ifnγ was presented on days 1 to 2, and luciferase activity and cell viability were measured on day 4.

B) HMC3-MHCII from day 0 to day 4 ^Luc AAT is applied to the cells. Activation was measured by MHCII driven luciferase activity and normalized to cell viability. Luciferase activity under all conditions was expressed as a percentage of ifnγ control. All conditions were performed in triplicate, errorThe difference bars represent standard deviation.

Example 3

A) HMC3-MHCII of human microglial cells ^Luc Cells were seeded on day 0, ifnγ was presented on days 1 to 2, and luciferase activity and cell viability were measured on day 4.

B) HMC3-MHCII from day 0 to day 4 ^Luc AAT is applied to the cells. Activation was measured by MHCII driven luciferase activity and expressed as a percentage of ifnγ control under all conditions. All conditions were performed in triplicate and error bars represent standard deviation.

C) In the same HMC3-MHCII ^Luc Batch RNA extraction was performed on the cultures. RNA was Quality Controlled (QC) prior to sequencing. Sequencing QC was done prior to mapping the human genome. The mapped reads were counted and differential gene expression between different conditions was measured (see tables 2-11).

Example 4

RNAseq data from plasma sources and recombinant AAT were pooled, normalized and analyzed by Genetic Set Enrichment Analysis (GSEA) (https:// www.gsea-msigdb. Org/GSEA/index. Jsp; subramannian, A., et al Proc. Natl. Acad. Sci. USA,102 (43): 15545-15550,2005.). Up-regulated gene families (grey bars) and down-regulated gene families (black bars) are shown according to the normalized enrichment score.

A) The ifnγ -induced inflammatory profile is confirmed by upregulation of several processes associated with inflammation (bold italics).

B-C) AAT treatment was able to significantly down-regulate some of these pathways in the absence (B) or presence (C) of ifnγ (bold italics). Importantly, AAT inhibits both ifnγ and inflammatory responses. However, the down-regulation of the inflammatory response gene in (C) was not significant.

For other gene families, KRAS signaling is very important as it is involved in oncogenic processes and immunomodulation (Dias Carvalho et al 2018). Also, the p53 pathway is notable as a regulator of stress response.

Example 5

AAT treatment balances the expression of inflammatory genes

Gene tables associated with antigen presentation (table 2), cytokine signaling (table 3), interferon signaling (table 4) and complement activation (table 5). The major symptomatic genes (FC >2;p values <0.05; left panel) are defined by differential expression between untreated and IFNγ treated cells (inflammation; middle panel). The major genes significantly and inversely regulated by AAT treatment are highlighted (right panel; bold underlined).

It was found that a large number (about 35%) of the major inflammatory genes were affected by AAT treatment (6 out of 23 genes associated with antigen presentation, 14 out of 33 genes associated with cytokine signaling, 1 out of 7 genes associated with complement activation, 5 out of 13 genes associated with interferon signaling), showing their anti-inflammatory potential. For example, in HMC3-MHCII ^Luc The promoter of the HLA-DRA gene used as a driver of luciferase expression in the line was continuously up-and down-regulated (bold italics) in inflammatory and AAT treatments, respectively. Notably, FC shown in AAT-treated inflammatory conditions should not be directly compared to FC found in inflammation, as fc=2 for the former represents that the inflammation inducing/inhibiting genes have been at 50% balance.

The table bottom lists the secondary inflammatory genes (p-values <0.05; FC < 2) that are significantly and inversely regulated by AAT treatment under inflammatory or resting conditions (regulation in AAT).

TABLE 2

TABLE 3 Table 3

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TABLE 4 Table 4

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TABLE 5

Example 6 AAT treatment enhances expression of marker genes associated with M2 anti-inflammatory microglia

M2 microglial gene expression was promoted after M2 type induction (FC study; satoh 2017). AAT treatment in resting microglia (FC AAT) and activated microglia (FC inflammation + AAT) can similarly enhance expression of the M2 gene, but at a lower magnitude. About 60% of the modified genes are common under AAT treatment conditions (bold underlined).

TABLE 6

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Example 7-AAT treatment affects expression of neurodegenerative disease Risk Gene

Risk genes (Timmerman, strickland, and Zuchner 2014; parnell and Booth 2017;Nikolac Perkovic and Pivac 2019; blauwendraat, nalls, and Singleton 2020; chang et al 2012) associated with PD (21 genes), AD (15 genes), MS (53 genes), MCT (50 genes), PN (88 genes), and GBS (30 genes; FC) were extracted from the public library and evaluated for expression in AAT-treated resting microglia (FC AAT) and activated microglia (FC inflammation+AAT). The modification genes common under AAT treatment conditions are highlighted (bold underlined). Notably, the expression of several risk genes in all of the above diseases is altered by AAT.

TABLE 7

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TABLE 8

TABLE 9

Table 10

TABLE 11

Example 8

Further experiments included CSF-1 treatment optimized for (mΦ) to M1 macrophage transition, dose dependence of ifnγ on M1 macrophage activation, AAT treatment of resting and ifnγ -activated macrophages, and RNA extraction and RNAseq and analysis. Wherein the cells are human primary resting (MΦ) or M1 differentiated macrophages. Treatment included pretreatment with AAT for 24 hours followed by activation (or non-activation) of cells with CSF-1 or IFN in the presence of AAT for 24 hours. Cells were further treated in AAT for 48 hours and finally tested for pro-inflammatory properties using culture supernatantCytokine release (IL-6, TNF. Alpha., IL-1. Beta., IL-8; multiplex reads) or alternatively luciferase activity (MHCII) ^Luc Line) and simultaneously extracting RNA for microarray analysis. Cultures with consistent cytokine release or luciferase assay readings will be used as samples for RNA extraction and microarray analysis.

Experimental conditions:each condition was performed in triplicate,

untreated (AAT-free, CSF-1-free) resting macrophage control

Ifnγ: activated macrophage control

AAT (plasma-derived; sigma, or recombinant; lonza): effect of AAT on resting macrophages

Ifnγ and AAT (plasma-derived; sigma or Lonza): anti-inflammatory effect of AAT on activated macrophages

And (3) outputting:

resting macrophage gene expression

Pro-inflammatory differential regulatory genes (untreated fold; significant p-value; fold up/down threshold)

AAT-driven changes in gene expression on resting and activated macrophages

Differences between recombinant and plasma-derived AAT gene regulation

Method

Human microglial cell line culture

HMC3-MHCII encoding Renilla luciferase under the major histocompatibility Complex II promoter (HLA-DRA) ^Luc Cell lines have been described as a valuable tool for studying human microglial activation and are available from prof.karl-Heinz krausen, university of Geneva. Transduction of the vector with lentiviral vector to obtain HMC3-MHCII ^Luc ；Ubi ^AAT Cell lines (see FIG. 3). HMC3-MHCII ^Luc And HMC3-MHCII ^Luc ；Ubi ^AAT Cell culture dish treated by TCGreiner, 7.664160) on a cow supplemented with 10% (v/v) fetusesSerum (FBS, gibco, 10270106) and 100. Mu.g/ml penicillin/streptomycin (Pen/Strep, thermoFisher, 15070063) in DMEM high glucose+glutamine (Gibco, 41965039). The culture was maintained at 37℃in a 5% CO2 atmosphere. Passaging was accomplished by flash washing the cells in PBS1X, trypsinizing for 3 minutes at RT (Tryple Express, thermoFisher, 12604021), followed by centrifugation (5 minutes, 1000 RPM) and resuspension in the supplemented DMEM described above. Cells were counted and plated at the desired concentration.

Human microglial cell line transduction

Lentiviruses encoding human AAT under the ubiquitin promoter and GFP under the human PGK promoter were obtained according to the protocols described in Marc Giry-Latererire, els Verhoeyen, and Patrick Salmon,2011,Methods in molecular biology. Briefly, will be 4.5x10 ⁶ Spreading of HEK cells onAAT, 10. Mu.g of packaging plasmid (psPAX 2 from Didier Troo [ Addge plasmid 12260) were used in dishes and after 16 hours with 15. Mu.g of pCWXPG-UBI-SP:]is the gift of (a) and 5 μg of an envelope (pMD 2G from Didier Trono [ Addgene plasma 12259)]Gift of (a). The medium was changed 8 hours after transfection. After 48 hours, the virus supernatant was collected and filtered using a 45 μm PVDF filter and stored at-80 ℃. Determining viral titer and selecting HMC3-MHCII with approximately 100% and 50% AAT expressing cells ^Luc ；Ubi ^AAT Cell lines were used for experimental conditions.

Interferon gamma mediated activation of human microglia

HMC3-MHCII ^Luc Cell lines were seeded into 96-well plates at a density of approximately 2500 cells/well. After 24 hours, their activation was induced by presentation with different concentrations (0.1; 1;10 or 100 ng/ml) of IFNγ (Sigma, SRP 3058) for 24 hours. Ifnγ was then removed and the cells were cultured for 48 hours, then cell viability and activation were assessed (see fig. 2).

Exogenous/endogenous AAT treatment on IFNγ -activated human microglia

HMC3-MHCII ^Luc And HMC3-MHCII ^Luc ；Ubi ^AAT The (endogenous AAT) cell line was seeded into 96-well plates at a density of about 2500 cells/well. HMC3-MHCII ^Luc Cell lines were plated and plasma-derived AAT and recombinant AAT (produced in CHO cells, AAT 1 and AAT 2) were added at different concentrations (1, 10 or 25 μm) after 3 hours. After 24 hours, exogenous or endogenous AAT was still present, microglial activation was induced by ifnγ presentation (10 ng/ml) for 24 hours duration. IFNγ was then removed and HMC3-MHCII was used ^Luc And HMC3-MHCII ^Luc ；Ubi ^AAT Cells were cultured in the presence of exogenous or endogenous AAT for 48 hours, and then cell viability and activation of the cell cultures were assessed (see fig. 3 and 4).

Cell viability and activation measurement of human microglia

Measurement of HMC3-MHCII according to manufacturer's protocol ^Luc And HMC3-MHCII ^Luc ；Ubi ^AAT Viability (cell count kit-8, sigma, 96992) and activation of cell culturesLuciferase Assay System,Promega,E2710)。

RNA harvesting, sequencing and differential expression analysis

RNA extraction was performed using the RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. RNA samples from plasma-derived AAT and recombinant AAT Nr.2 were checked for quality (2100 bioanalyzer, agilent) and libraries were prepared using the Truseq RNA library kit (Illumina, RS-122-2001). The library was sequenced (HiSeq 4000, illumina), the sequencing quality was controlled (FastQC), the human genome was mapped (STAR v.2.7.0f; UCSC hg 38), reads were counted (HTSeq v0.9.1) and differential expression analysis was performed using the R/Bioconductor package (edge 1.30.1).

RNA harvesting, sequencing and differential expression analysis

Human monocyte-derived M1 macrophages (GM-CSF, promocell, C-12916) were cultured in M1-macrophage generating medium XF in a fibronectin-coated cell culture dish and the culture was maintained at 37℃under 5% CO2 atmosphere according to the manufacturer's protocol CSF-1 (50 ng/ml, sigma, SRP 3058) activation.

Multiple cytokine assays

Cell culture supernatants were collected and IL-6, TNFα, IL-1β, and IL-8 were measured using a bead-based Luminex assay according to the manufacturer's protocol.

Cell-free TACE/ADAM17 Activity

TACE activity and its inhibition of human AAT (AAT) was performed in black 96-well immunoplates (437111,ThermoFisher Scientific) using recombinant human TACE/ADAM17 kit (930-ADB and ES003, R & D Systems). The enzymatic activity of TACE/ADAM17 was measured by mixing 0.005. Mu.g of rhTACE with 10. Mu.MMca-PLAQAV-Dpa-RSSSR-NH 2 fluorescing peptide substrate III in assay buffer (25 mM Tris, 2.5. Mu.MZnCl 2,0.005% Brij-35 (w/v), pH 9.0) to a final volume of 100. Mu.l. AAT (Sigma Aldrich, batch A6150) was resuspended in water (vehicle) and the control TACE/ADAM17 activity was assessed in the presence of vehicle (AAT was used in an amount of 100. Mu.M). Different concentrations (0, 6.25, 12.5, 25, 50 and 100 μm) of AAT were added to evaluate their dose-dependent inhibition of TACE/ADAM 17. All conditions were performed in triplicate. The activity in the form of Relative Fluorescence Units (RFU) was measured in kinetic mode (9 time points within 5 minutes) using a SpectraMax iD3 microplate reader (low PMT gain, 1 second exposure, top reading 1mm, wavelength: excitation 320nm, emission 405 nm). Bar graphs were obtained as a percentage of control activity by averaging the values obtained over 5 minutes for each condition.

Animals

As a murine model of CMT1A, we used C3-PMP22 transgenic mice (B6. Cg-Tg (PMP 22) C3Fbas/J, the Jackson Laboratory) that expressed three copies of the wild-type human perimyelin protein 22 (PMP 22) gene (Verhamme, king et al 2011). Mice were kept in macrolon cages with filter housing and placed in a room with continuous air filtration to avoid contamination. During the experiment, paired animals will be kept in a thermostated cage with a day/night period of 12/12 hours. Animals were fed ad libitum (control of tap water and nutrition). Animal protocols were approved by the langidok Lu Xiyong animal research committee. This protocol and our laboratory procedure are in compliance with French legislation (reference number: D3417223, APAFIS #23920-2020020320279696 v3) executing European instructions. Animals were followed daily for health to ensure that only animals in good health entered the test procedure and follow-up study.

In vivo study paradigm

Animals were divided into 3 groups (wild type control (0.9% NaCl subcutaneously), CMT 1A-vehicle (0.9% NaCl subcutaneously), CMT 1A-human alpha-1 antitrypsin (50 mg/kg per injection twice a day subcutaneously), 3 mice per group (3 weeks old, male, 18±2.5g at study start), and after 7 days of on-site adaptation, all mice underwent the following protocol.

From 4 weeks of age, animals were blood sampled to determine interleukin-6 (IL-6) and tumor necrosis factor alpha (tnfa) levels, as depicted in fig. 16.

hAAT plasma levels were assessed every 5 days from the first day of treatment to the last day of treatment.

On the first and last days of treatment, animals were tested for neuromuscular properties by the rotarod test, grip test and sciatic nerve electrophysiology test. After the last treatment for 8 weeks, these tests were repeated and animals were sacrificed and left sciatic nerve sampled for histological evaluation of neuron number and size.

Example 9

TACE activity was assessed in kinetic mode with no or different AAT concentrations according to the manufacturer's instructions (recombinant human TACE/ADAM17 kit, 930-ADB, R & D Systems). All conditions were performed in triplicate and shown as mean ± SD (fig. 5).

Example 10

The most common type of CMT is CMT1A, which is characterized by a repetition of the PMP22 gene, resulting in PMP22 protein accumulation and progressive demyelination in schwann cells. PMP22 is a four-transmembrane glycoprotein contained in dense myelin of the peripheral nervous system. The repetition of PMP22 is associated with the onset of fibular atrophy of type 1A (CMT 1A). The C3-PMP22 transgenic mice (B6. Cg-Tg (PMP 22) C3 Fbas/J) expressed three copies of the wild-type human peripheral myelin protein 22 (PMP 22) gene. The causal relationship between the additional PMP22 gene and CMT1A has not been clear to date and remains elusive. However, several plausible hypotheses may relate genetic abnormalities (i.e., duplications of the PMP22 gene) to pathological manifestations. Without being bound by theory, PMP22 overexpression may negatively affect myelination in the Peripheral Nervous System (PNS). These mice exhibit age-dependent demyelinating neuropathy, characterized primarily by loss of distal strength and sensation. C3-PMP mice showed no obvious clinical signs at 3 weeks and developed progressive and observable neuromuscular injury after 4 weeks. As with adults with human CMT1A, these mice have a stable low nerve conduction velocity. These mice had delayed myelination and at 3 weeks of age they contained a reduced number of myelinated fibers. The mouse model was used to study the effect of AAT in different paradigms.

After two weeks, positive efficacy of increased rotarod delay, grip and nerve conduction performance of AAT administration was observed in CMT1A mice compared to untreated control groups. Furthermore, no weight loss was observed in the AAT treated group compared to the vehicle group, indicating that no systemic toxicology was present for the compound under these experimental conditions.

Table 12 body weight

Sciatic nerve Electrophysiology (EMG) provides a sensitive and quantitative method to measure the composite muscle action potential and nerve conduction velocity amplitude of an animal and is accomplished by stimulating the sciatic nerve. Similar Composite Muscle Action Potential (CMAP) amplitudes were observed between groups at baseline (6 weeks of age). As expected, a strong and significant decrease in CMAP amplitude was observed in the cmt1a+ vehicle group compared to the 8 week old wild type control group. The results show an improvement in EMG parameters in the CMT1A mice treated with AAT compared to the control. (FIG. 6) demonstrates the positive efficacy of hAAT on axonal degeneration caused by CMT1A disorders.

A lower Nerve Conduction Velocity (NCV) was observed in both CMT1A groups compared to the baseline (6 week old) wild type control group. At baseline, the NCV differences between groups were not statistically significant. As expected, a strong and significant decrease in NCV was observed in cmt1a+ vehicle group compared to the 8 week old wild type control group. The cmt1a+haat treated group showed an increase in NCV compared to the vehicle treated group. Since nerve conduction velocity is dependent on myelin integrity, these data also indicate the positive efficacy of hAAT on demyelination of schwann cells caused by CMT disease.

TABLE 13 sciatic nerve electrophysiology

TABLE 14 average composite muscle action potential

Two-way ANOVA with repeated measures and Bonferroni t-test

***：p<0.001, compared to WT control;p<0.05 compared to CMT1A+ vehicle

Table 15: average nerve conduction velocity

Two-way ANOVA with repeated measures and Bonferroni t-test

* A.x; * **: p <0.01; p <0.001, compared to WT control

Grip strength test neuromuscular strength was measured by assessing the grip of an animal on a metal grid. A lower grip strength was observed in the CMT group compared to the baseline (6 week old) wild type control group. At baseline, the grip strength difference between groups was not statistically significant. As expected, a strong and significant decrease in grip strength was observed in the cmt1a+ vehicle group compared to the 8 week old wild type control group. The results showed improved grip in the AAT treated CMT1A mice compared to the control group (fig. 7).

Watch 16 grip strength

TABLE 17 average grip strength

Two-way ANOVA with repeated measures and Bonferroni t-test

* A.x; * **: p <0.01; p <0.001, compared to WT control

The rotarod test measures neuromuscular coordination by assessing the ability of an animal to maintain balance on a rotating cylinder. At baseline (6 weeks of age), similar rotarod delays were observed between groups. As expected, a strong and significant decrease in the rotarod delay was observed in the cmt1a+ vehicle group compared to the 8 week old wild type control group. The results showed an improvement in the rotarod delay of the AAT treated CMT1A mice compared to the control group (fig. 8).

Table 18 delay of turning bar

Table 19 average stick turning delay

Two-way ANOVA with repeated measures and Bonferroni t-test

* The method comprises the steps of carrying out a first treatment on the surface of the * **: p <0.05; p <0.001, compared to WT control

Example 11

PMP22 protein appears to be particularly important for protecting nerves from physical stress, helping the nerves to recover structure after being kneaded or squeezed (pressed). Compression can interrupt nerve signaling, resulting in a sensation commonly referred to as "sleeping" on the limb. The ability of nerves to recover from normal daily compression (e.g., when sitting for a long period of time) can prevent the extremities from continually losing sense. In CMT1A patients, the myelination process is not completed correctly, and pathological symptoms associated with the disease most often become apparent after the second decade of life.

The PMP22 gene also plays a role in the growth of schwann cells and in the maturation of cells to perform specific functions (differentiation). The newly produced PMP22 protein is processed and packaged in a specific cellular structure called the endoplasmic reticulum and golgi apparatus before becoming part of the myelin. Completion of these processing and packaging steps is critical to proper myelin function. The pathomechanical feature of CMT1A is myelin deficiency due to the additional PMP22 gene, which results in abnormally high concentrations of peripheral myelin proteins 22 (PMP 22) in schwann cells. GSEA analysis of human microglia showed that AAT treatment had up-regulated genes associated with the Unfolded Protein Response (UPR) pathway and cell survival (anti-apoptosis) (fig. 4C, fig. 9).

Example 12

ADAM17, also known as TACE, is a transmembrane protein that includes an extracellular zinc-dependent protease domain. In the context of CMT1A, the inhibition of SC-mediated myelination by neuregulin type 1III (NRG 1-III) by ADAM17 is known. It is speculated that AAT is able to cross the Blood Nerve Barrier (BNB) and interact with ADAM17 to successfully inhibit its activity, and doing so allows SC to "manually" overcome the distress signal generated by overloaded ER containing PMP22 and promote the formation of myelin sheath around axons.

EXAMPLE 13 plasma hAAT levels

At the time points of analysis (day 14, day 19, day 24 and day 29), hAAT was not detected in plasma of wild type control and vehicle-treated CMT1A mice. The mean values of hAAT detected in the CMT1A+hAAT plasma on days 14, 19, 24 and 29 were 6.07. Mu.g/mL, 6.99. Mu.g/mL, 8.14. Mu.g/mL and 5.22. Mu.g/mL, respectively.

EXAMPLE 14 sciatic nerve histology

As expected, a significant increase in total number of axons, decrease in axon diameter, and g-ratio per surface was observed in cmt1a+ vehicle group compared to the 8 week old wild type control group (table 20, fig. 10-13).

A slight increase in the total number of axons per surface was observed in cmt1a+haat treated group compared to vehicle group. Furthermore, a significant increase in axon diameter and a decrease in g-ratio (equal to the ratio of inner diameter to outer diameter of myelin axons) were observed in the hAAT treated CMT1A animals compared to vehicle treated groups. Even so, cmt1a+haat animals exhibited statistically different numbers of axons, axon diameters, and g ratios than the wild type control group (table 20, fig. 10 to 13). Taken together, these data demonstrate the positive but partial efficacy of hAAT on CMT1A disorder-induced histopathology when administered twice daily at 50mg/kg via the subcutaneous route.

TABLE 20 ischial nerve histology

Single factor ANOVA and Tukey test: p is p<0.001, compared to WT control;p<0.001 compared to CMT1A+ vehicle

Example 14-IL-6

Similar plasma IL-6 concentrations were observed between groups at baseline (day 1) and at day 8.

As expected, a significant increase in plasma IL-6 concentration was observed in the cmt+ vehicle group on days 14 and 29.

The cmt+haat treated group also exhibited significantly increased plasma IL-6 concentrations compared to the baseline concentration. However, on day 29, the plasma IL-6 concentration was lower in animals treated with hAAT than in animals treated with vehicle (table 21 and fig. 14), indicating a direct or indirect effect of hAAT on the inflammatory cytokine.

Table 21: plasma IL-6 levels

Student t-test; * A.x; * **: p is p<0.05；p<0.01；p<0.001, at this time point compared to WT control;p<0.05, at this point in time compared to CMT1A+ vehicle

EXAMPLE 15 TNF alpha

As expected, a significant increase in plasma TNF- α concentration was observed in cmt1a+ vehicle groups on days 14 and 29.

The cmt1a+haat treatment group also exhibited significantly increased plasma TNF- α concentrations at days 14 and 29 compared to baseline concentrations (table 22 and fig. 15), indicating that hAAT had no effect on the inflammatory cytokine levels in the CMT1A model.

Table 22: plasma TNFalpha levels

Student t-test; * A.x; * **: p <0.05; p <0.01; p <0.001, at this time point compared to WT control.

Example 16

The effect of AAT on SH-SY5Y of 6-OHDA treatment was quantitatively assessed by cell morphology and cell proliferation followed by cell viability.

Cells and treatments

SH-SY5Y cells, which are commonly used to model neurodegenerative disorders, are used to generate in vitro models of Parkinson's disease (Que R et al, front. Immunol 2021). Cells were cultured in DMEMF12/Glutamax supplemented with 10% FBS.

Cells were treated with neurotoxin 6-hydroxydopamine (6-OHDA, sigma Aldrich 162957) at a concentration of 50 μm, 100 μm alone or in combination with AAT 25 μm (AAT plasma source, sigma Aldrich batch a 9024) for 24 hours. Cells were treated with AAT alone or in combination with 6OHDA for 24 hours and then incubated with fresh AAT for another 24 hours. Control cells were treated with PBS.

Cell growth and viability assays

On day 0, the next day after treatment of cells as described above, cells were plated in equal numbers in 24-well plates and final total cell counts were performed on day 4 of culture (cell growth). Cell viability was assessed using Cell Counting Kit-8 (CCK-8;Sigma Aldrich 96992). After treatment, the cells were incubated with 10. Mu.l of CCK-8 solution for 2 hours in an incubator. Absorbance was measured at 450nm using a SpectraMax iD3 microplate reader. Experiments were performed in triplicate.

Human IL-6 immunoassays

Cell supernatants were subjected to human Il-6 immunoassays (R & D D6050). Briefly, 40000 cells were plated and treated in 24 well plates the next day after as described in the "cells and treatments" paragraph. At the end of the treatment, the cells were washed with PBS and incubated with 2% FBS medium for 24 hours, then the cell culture supernatant was collected and centrifuged to remove particles. The assay procedure was performed on standard and sample replicates as described in the manufacturing instructions. Absorbance was measured at 450nm using a SpectraMax iD3 microplate reader. A standard curve was prepared from seven IL-6 standard dilutions and IL-6 sample concentrations were determined.

50-100. Mu.M 6-OHDA for 24 hours induced cell proliferation injury by inducing cell death and reducing the number of cells after 4 days of culture (FIG. 17). In contrast, treatment with AAT in combination significantly increased the number of cells counted compared to 6-OHDA alone (fig. 17), indicating a positive effect of AAT on cell survival/proliferation. The T-test p-value is significant for the following cases: p=0.001 compared to 6 ohda; 6ohda compared to aat+6ohda, p=0.003. The control was not significant compared to AAT. Error bars are s.e.m.

The treated cells were then challenged in a cell viability assay. SH-SY5Y treated with 6-OHDA alone showed a strong decrease in cell viability compared to control cells and AAT-alone treated cells (FIG. 18).

Cell counts and cell viability were significantly enhanced when 6-OHDA treatment was combined with AAT compared to 6-OHDA treatment alone (fig. 18). P-values were calculated by t-test, no significant difference was observed between control and AAT alone. All conditions were performed in triplicate. Based on these results, we can conclude that administration of AAT in the PD cell model (6-OHDA induced SH-SY 5Y) has a beneficial effect on cell growth and viability, possibly protecting cells from 6-OHDA induced death.

In addition to exploring the effect of AAT on pro-inflammatory cytokines, we also quantified IL-6 in cell supernatants. Media from cells induced by 6-OHDA had increased IL-6 levels compared to control media, whereas media collected from cells treated with AAT showed lower concentrations of IL-6 (fig. 19). The T-test p-value was significant for the control compared to 6-OHDA, p=0.05, and not for the other comparisons.

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Sequence listing

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His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro

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Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val

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Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys

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Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala

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20 25 30

Leu Phe Met Gly Lys Val Val Asn Pro Thr Gln Lys

35 40

<210> 3

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> fluorescence-producing peptide derived from SARS-CoV-2 spike

<400> 3

His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln

1 5 10

<210> 4

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> fluorescence-producing peptide derived from SARS-CoV-2 spike

<400> 4

Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

1 5 10

<210> 5

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HIV-TAT 48-57 peptide

<400> 5

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210> 6

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> FHV-coat 35-49 peptide

<400> 6

Arg Arg Arg Arg Asn Arg Thr Arg Arg Asn Arg Arg Arg Val Arg

1 5 10 15

<210> 7

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> HTLV-II Rex 4-16 peptide

<400> 7

Thr Arg Arg Gln Arg Thr Arg Arg Ala Arg Arg Asn Arg

1 5 10

<210> 8

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> BMV gag 7-25 peptide

<400> 8

Lys Met Thr Arg Ala Gln Arg Arg Ala Ala Ala Arg Arg Asn Arg Trp

1 5 10 15

Thr Ala Arg

Claims (16)

1. A composition for use in the treatment and/or prevention of a disease or condition of the nervous system, the composition comprising a therapeutically effective amount of an alpha 1-antitrypsin (AAT) protein, variants, isoforms and/or fragments thereof.

2. A vector for treating and/or preventing a disease or condition of the nervous system comprising a nucleic acid sequence encoding an AAT protein.

3. A genetically modified cell comprising a nucleic acid sequence encoding an AAT protein for use in the treatment and/or prevention of a disease or condition of the nervous system.

4. The composition for use according to claim 1, the vector for use according to claim 2 or the genetically modified cell for use according to claim 3, wherein the disease or disorder of the nervous system is a disease or disorder of the peripheral nervous system.

5. The composition for use according to claim 4, the vector for use according to claim 4 or the genetically modified cell for use according to claim 4, wherein the disease or condition of the peripheral nervous system is a motor and sensory neuropathy of the peripheral nervous system.

6. The composition for use according to claim 5, the vector for use according to claim 5 or the genetically modified cell for use according to claim 5, wherein the sensory neuropathy of the peripheral nervous system is hereditary motor and sensory neuropathy of the peripheral nervous system.

7. The composition for use according to claim 6, the vector for use according to claim 6 or the genetically modified cell for use according to claim 6, wherein the hereditary motor and sensory neuropathy of the peripheral nervous system is fibula muscular dystrophy or a symptom thereof, preferably at least one symptom selected from the group consisting of: weakness of the legs, ankle and/or foot, reduced muscle mass of the legs and/or foot, high arch, bending of the toes, reduced running ability, difficulty in lifting the foot at the ankle, abnormal gait, frequent trips or falls, hypoesthesia or unconsciousness of the legs and/or foot.

8. The composition for use according to claim 1, the vector for use according to claim 2 or the genetically modified cell for use according to claim 3, wherein the disease or disorder of the nervous system is an inflammatory disease or disorder of the nervous system.

9. The composition for use according to claim 8, the vector for use according to claim 8 or the genetically modified cell for use according to claim 8, wherein the inflammatory disease or disorder of the nervous system is a disease or disorder mediated by bone marrow cells of the nervous system.

10. The composition for use according to any one of claims 1, 8 or 9, the vector for use according to any one of claims 2, 8 or 9 or the genetically modified cell for use according to any one of claims 3, 8 or 9, wherein the disease or syndrome of the nervous system is a disease or syndrome selected from the group consisting of parkinson's disease, dementia, multiple sclerosis, amyotrophic lateral sclerosis, alzheimer's disease and huntington's disease.

11. The composition for use according to any one of claims 1, 8 to 10, the vector for use according to any one of claims 2, 8 to 10 or the genetically modified cell for use according to any one of claims 3, 8 to 10, wherein the disease or disorder of the nervous system is at least one symptom of a disease or disorder of the nervous system selected from the group consisting of: tremor, memory loss, slurred speech, dizziness, vision changes, and headache.

12. The composition for use according to any one of claims 1, 4 to 11, wherein the AAT protein, variant, isoform and/or fragment thereof is human plasma extracted.

13. The composition for use according to any one of claims 1, 4 to 11, wherein the alpha 1-antitrypsin (AAT) protein, variant, isoform and/or fragment thereof is recombinant alpha 1-antitrypsin (rhAAT), variant, isoform and/or fragment thereof.

14. The composition for use according to any one of claims 1, 4 to 13, wherein the composition comprises at least one pharmaceutical carrier.

15. The composition for use according to claim 14, wherein the pharmaceutical carrier is a blood brain barrier permeability enhancer.

16. The composition for use according to any one of claims 1, 4 to 11, the vector for use according to any one of claims 2, 4 to 6 or the genetically modified cell for use according to any one of claims 3 to 7, wherein the composition, the vector or the genetically modified cell is formulated for intra-cerebral administration, intravenous injection, intravenous infusion, quantitative pump infusion, inhalative nasal spray, eye drops, skin patches, sustained release formulations, ex vivo gene therapy or ex vivo cell therapy.