Inhibition of vascular leakage

Abstract

The present invention relates to the use of proline / arginine-rich end leucine-rich repeat protein (PRELP) in the prevention of vascular leakage and the consequent treatment of various conditions characterised by such leakage. The present invention further relates to the use of PRELP in the prevention of vascular leakage associated with immunotherapy.

Description

[0001] INHIBITION OF VASCULAR LEAKAGE

[0002] Field of the invention

[0003] The present invention relates to the use of proline / arginine-rich end leucine-rich repeat protein (PRELP) in the prevention of vascular leakage and the consequent treatment of various conditions characterised by such leakage. The present invention further relates to the use of PRELP in the prevention of vascular leakage associated with immunotherapy.

[0004] Background of the invention

[0005] In most organs, endothelial cells form a dynamic barrier between the blood and the tissue, controlling the passage of molecules from the vasculature into the tissue. In the brain, the vasculature forms a particularly strong barrier, the blood-brain barrier (BBB), to protect the brain. In many diseases, the vascular barrier can become dysfunctional and lead to an increase in leakage.

[0006] The BBB is a tight functional barrier composed of capillary endothelial cells, astrocytes, pericytes, and neurons, which prevents neurotoxic plasma components, blood cells, and pathogens from entering the brain (Sweeney et al., 2019; Weiss et al., 2009). Defects in the BBB results in the accumulation of toxic molecules from the vasculature. This leakage is associated with a range of central nervous system (CNS) diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, stroke, and epilepsy (Sweeney et al., 2019; Weiss et al., 2009).

[0007] The BBB is characterized by two major adhesion complexes: adherens junctions and tight junctions. These dynamic structures are governed by diverse proteins secreted from the components of the neurovascular unit and trigger downstream signalling events involved in gene expression, cytoskeletal reorganization, and endothelial permeability. Among these signalling events, the endothelial to mesenchymal transition (EndMT) has been recognized as a major biological event that controls vascular leakage by affecting cell-cell adhesion between brain vascular endothelial cells (Cho et al., 2018; Man et al., 2019; Piera-Velazquez and Jimenez, 2019).

[0008] EndMT describes a transition whereby endothelial cells dedifferentiate into mesenchymal cells. The endothelium acquires mesenchymal and stem cell-like characteristics, which is accompanied by degradation of the vascular basement membrane, loss of cell-cell contact, and acquisition of a migratory phenotype. A reduction in the expression of specific endothelial markers and a gain in the expression of mesenchymal markers is observed. Vascular endothelial cells can also show partial transition to mesenchymal cells, called pEndMT (Kovacic et al., 2019). The contribution of EndMT to human disease has been demonstrated in an increasing number of pathologies. TGF-P is a strong activator of EndMT. As observed in many other biological systems, TGF-P has complex and dual roles in vascular biology, such as an activator and an inhibitor of BBB function in context dependent manners (Diniz ct al.. 2019: Li ct al.. 201 I ). TGF-P applied to Human umbilical vein endothelial cell (HUVEC) culture causes damage of endothelial cell-cell adhesion through activation of pEndMT (Guo et al., 2015). Increased TGF-P signalling has been suggested as a common underlying mechanism in several EndMT- associated disorders.

[0009] BBB dysfunction is found in many life-limiting diseases. Mortality due to neurological disorders accounts for over 0.5 million worldwide (WHO: 2019) and one of the major risk factors associated with neurological disorders is a vascular dysregulation.

[0010] Proline / arginine-rich end leucine-rich repeat protein (PRELP), is a small secreted proteoglycan expressed by pericytes and vascular smooth muscle cells surrounding the brain vasculature. It is a class II member of the small leucine rich proteoglycan (SLRP) family (Dellett et al. 2012, lozzo and Schaefer 2015). SLRP family members bind various extracellular proteins such as TGF-p, BMP, EGF, IGF, Wnt, and collagens and can regulate multiple signalling pathways in context dependent manners (Chacon-Solano et al. 2022, Dellett et al. 2012, Lopez and Bonassar 2022, Morris et al. 2007). They are involved in various biological processes such as cancer, inflammation, and development (Birke et al. 2014, Luehders et al. 2015, Papadaki et al. 2020). It is naturally expressed in the body and it is soluble due to its rich glycan modifications.

[0011] The present invention is based on the discovery that a secreted small proteoglycan, proline / arginine-rich end leucine-rich repeat protein (PRELP) is a regulator of endothelial cell barrier permeability.

[0012] The present inventors found that PRELP influences BBB permeability and neuro-vasculature leakage in the brain. The present inventors found that PRELP is expressed in pericytes and vascular smooth muscle cells surrounding brain vasculature in adult mouse brain, and that PRELP knockout mice show leakage from the neuro-vasculature. They also found that PRELP application to endothelial cells is able to enhance cell-cell integrity / adhesion of endothelial cells in association with activation of transition from mesenchymal to endothelial like state (MEndT) and inhibits TGF-P mediated damage of cell-cell adhesion.

[0013] A range of disease pathologies are associated with abnormal permeability of an endothelial cell barrier and the resultant vascular leakage. This abnormal permeability may result from the loss of cell-cell integrity related to the endothelial to mesenchymal transition (mEndT). In some cases, this is mediated by TGF-p. PRELP can therefore be used in the treatment of diseases characterised by these factors, by acting as an inhibitor of vascular leakage across the endothelial cell barrier.

[0014] Although PRELP has previously been suggested to have a role in maintaining vascular function (Davaapil, 2017), the present inventors have shown for the first time that PRELP application to endothelial cells enhances cell-cell integrity, induces mesenchymal-endothelial transition and inhibits TGF- mediated damage to cellcell adhesion. Davaapil, 2017 found PRELP- / - animals exhibit signs of blood-brain barrier breakdown, however, the mechanisms for this are not fully understood. Furthermore, Davaapil, 2017 found that PRELP could not decrease permeability of a normal endothelial cell layer without inducing damage. The present inventors surprisingly found that the application of PRELP can inhibit leakage from a TGF- damaged endothelial cell layer, indicating that PRELP might be more effective in such damaged vasculature. The data in Davaapil, 2017 shows a large number of genes were differentially regulated in PRELP- / - mice, including genes involved in immune processes, basal membrane (BM) components, other SLRPs and signalling molecules. Further attempts to elucidate the pathways involved in blood-brain barrier breakdown in cultured cells proved inconclusive, in part due to the complexity of the signalling pathways involved . The skilled person could have no reasonable expectation of success in using PRELP as therapeutic candidate on the basis of these data.

[0015] Summary of the invention

[0016] In a first aspect, the invention provides Proline / arginine-rich end leucine-rich repeat protein (PRELP) for use in a method of treating a disease characterized by increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage.

[0017] In some embodiments, the increased neurovascular permeability is increased permeability of the blood-brain barrier, the blood-spinal cord barrier, the blood-ocular barrier, and / or the blood-retinal barrier. In particular embodiments, the increased neurovascular permeability is increased permeability of the blood-brain barrier.

[0018] In some embodiments, the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction. The disease may be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

[0019] In some embodiments, the increased neurovascular permeability is caused by an increase in endothelial cell to mesenchymal cell transition events at the blood-brain barrier. In some embodiments, the increased endothelial cell to mesenchymal cell transition events are caused by TGF-P mediated damage to cell-cell adhesion.

[0020] In certain embodiments, the increased neurovascular permeability is caused by or associated with administration of an immunotherapeutic agent.

[0021] In a second aspect, the invention provides PRELP for use in combination with an immunotherapeutic agent for treating increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage. In some embodiments, PRELP is for administration simultaneously, separately or sequentially with an immunotherapeutic agent. In some embodiments, the increased neurovascular permeability is caused by or associated with administration of the immunotherapeutic agent. In certain embodiments, the immunotherapeutic agent is an anti-Amyloid-P immunotherapeutic agent. In certain embodiments, the anti- Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

[0022] In a third aspect, the invention provides a combination comprising PRELP and an immunotherapeutic agent for use in treating increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage associated with the administration of the immunotherapeutic agent, wherein PRELP is for administration before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent. The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. The wherein the anti- Amyloid-P immunotherapeutic agent may be lecanemab or donanemab.

[0023] In a fourth aspect, the invention provides PRELP for use in a method of preventing blood-brain barrier breakdown and / or reducing blood-brain barrier leakage. The blood-brain barrier breakdown or blood-brain barrier leakage may be mediated by TGF-p.

[0024] In a fifth aspect, the invention provides PRELP protein for use in a method of maintaining or enhancing bloodbrain barrier integrity.

[0025] In a sixth aspect, the invention provides PRELP for use in a method of treating TGF-P mediated neurovascular permeability or blood-brain barrier leakage.

[0026] In some embodiments, PRELP is administered in the form or a pharmaceutical composition comprising a therapeutically effective amount of PRELP and a pharmaceutically acceptable carrier.

[0027] There is also provided a pharmaceutical composition comprising PRELP and an immunotherapeutic agent. The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. The wherein the anti- Amyloid-P immunotherapeutic agent may be lecanemab or donanemab. The PRELP may be for simultaneous, separate or sequential administration with the immunotherapeutic agent.

[0028] In other embodiments, the production of PRELP is induced in a subject to be treated by gene therapy or cell therapy.

[0029] In a seventh aspect, the invention provides a method of treating a subject suffering from a disease characterised by increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage, comprising administering a therapeutically effective amount of PRELP to the subject.

[0030] In an eighth aspect, the invention provides a method of treating or preventing increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage, the method comprising administering a therapeutically effective amount of PRELP to a subject in need thereof. In some embodiments, the increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage is caused by or associated with immunotherapy, the method comprising administering an effective amount of PRELP to a subject in need thereof before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent.

[0031] In a ninth aspect, the invention provides a method of treating or preventing increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage caused by or associated with an the anti-Amyloid-P immunotherapeutic agent, wherein the method comprises administering PRELP in combination with the anti-Amyloid-P immunotherapeutic agent. PRELP may be administered before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent.

[0032] The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. In certain embodiments, the anti-Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

[0033] In a further aspect of the invention, there is provided PRELP for use in a method of treating a disease characterized by abnormal neurovascular permeability in combination with an immunotherapeutic agent. The PRELP may be for administration before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent. The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. The wherein the anti-Amyloid-P immunotherapeutic agent may be lecanemab or donanemab. In some embodiments, the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction. In some embodiments, the disease may be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

[0034] In a further aspect of the invention, there is provided an immunotherapeutic agent for use in a method of treating a disease characterized by abnormal neurovascular permeability in combination with PRELP. The immunotherapeutic agent may be for administration before, simultaneously with, separately to, or sequentially with PRELP. The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. The wherein the anti-Amyloid-P immunotherapeutic agent may be lecanemab or donanemab. In some embodiments, the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction. In some embodiments, the disease may be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

[0035] In a further aspect of the invention, there is provided the combination of an immunotherapeutic agent and PRELP for use in a method of treating a disease characterized by abnormal neurovascular permeability. The immunotherapeutic agent and PRELP may be for simultaneous, separate or sequential administration. The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. The wherein the anti-Amyloid- P immunotherapeutic agent may be lecanemab or donanemab. In some embodiments, the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction. In some embodiments, the disease may be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

[0036] In a further aspect of the invention, there is provided PRELP for use in treating or reducing abnormal neurovascular permeability caused by an immunotherapeutic agent that has been administered prior to the administration of PRELP. The immunotherapeutic agent may be an anti-Amyloid-P immunotherapeutic agent. The wherein the anti-Amyloid-P immunotherapeutic agent may be lecanemab or donanemab.

[0037] In a further aspect of the invention, there is provided PRELP for use in preventing abnormal neurovascular permeability caused by an immunotherapeutic agent. The immunotherapeutic agent may be an anti-Amyloid- P immunotherapeutic agent. The wherein the anti-Amyloid-P immunotherapeutic agent may be lecanemab or donanemab. The PRELP may be for simultaneous, separate or sequential administration with the immunotherapeutic agent.

[0038] In some embodiments, the subject to be treated is a human subject.

[0039] Brief description of the Figures

[0040] Figure 1. PRELP is expressed in mural cells around brain vasculatures and ependymal cells in mouse brain. (A-C) Embryonic PRELP expression of head. Sections of Prelp+ / LacZ embryos were stained for X-gal. (A) Embryonic day 12.5 (E12.5). Arrowhead indicate cortical hem. (B) Embryonic day 17.5 (E17.5). Arrow indicate the nasal septum and arrowhead indicate the sites of bone formation (C) Embryonic day 15.5 (E15.5). (D-I) Whole-mount X-gal staining of adult Prelp + / LacZ brains. (D-E) Dorsal and ventral views. X-gal staining is observed in the blood vessels. Scale bar: 2 mm. (F) Magnified from (E) around the ventral-posterior area. G, Magnified from (E) around ventral lateral. (H) Magnified from (D) around cerebrum. (I) Sagittal section of X-gal stained brain around cerebellum. Scale bar: 500 pm. (J-K) Anti-PRELP antibody staining of rat brain. Staining is visible in large arterioles / venules (J) and (K) capillaries Scale bar: 20 pm. (L) Section around lateral ventricle (LV). ependymal layer (EL). Choroid plexus (CP). Scale bar: 50 pm. (M) Section around 4th ventricle (4V). Scale bar: 50 pm. N, Section of retina around ciliary body (CB). Scale bar: 50 pm.

[0041] Figure 2. PRELP is expressed in vSMCs around vascular vessels and pericyte around capillaries. (A) Single cell analysis data of PRELP expression in cortical and hippocampal cells were obtained from previously published studies (Zeisel et al. 2015). Figure was generated by tool provided by the paper (Zeisel et al. 2015). (B-D) Double staining images with X-gal (B) and PECAM-1 (endothelial cell 584 marker) antibody (C) of Prelp- / + large vascular. (D) Merged image. (E-F) Double staining with X-gal (E) and a-smooth muscle actin (a-SMA) antibody (F; vSMC marker) of Prelp- / + intermediate vasculature. (G) Merged image. (H-J) Double staining images with b-galactosidase (b-gal) antibody (H) and GFAP antibody (I; astrocyte marker) of Prelp- / + brain capillaries. (J) Merged image. Scale bar: 25 pm (J,M,P). (K-M) Double staining images with p-gal antibody (K) and NG2 antibody (L; pericyte marker) of Prelp- / + brain capillaries. (M) Merged image. (N-P) Double staining images with P-gal antibody (N) and Isolectin IB4 (O; endothelial cell marker) of Prelp- / + brain capillaries. (P) Merged image. Pericyte bodies are marked by arrows. Pericyte processes are marked by arrowheads.

[0042] Figure 3. PRELP deletion results in leakage from the BBB. (A-D) Assessment of BBB integrity in the cerebellum by IgG staining in wild-type (A), Omd -I- (B) and Prelp -I- (C). Immunofluorescence was performed using anti -mouse IgG conjugated with Alexa Fluor fluorophore. DAPI was used as a nuclear stain. Scale bar: 10 pm. (D) IgG signal outside blood vessels was quantified (n = 4). (E-G) BBB disruption in Prelp -I- is more apparent in the cerebellum. IgG and DAPI staining of Prelp -I- cerebellum (E) and Prelp -I- cortex (F). Scale bar: 10 pm. G, Quantified result (n = 3). (H-M) 70 kDa 599 Dextran injection confirms BBB leakage in Prelp -I- cerebellum. Wild-type (H-J) and Prelp -I- (K-M) mice were injected with 70 kDa Dextran-Texas Red (H,K). Tissues were processed and stained 601 with tomato lectin (TL, vascular vessel and microglia marker) (I,L). Merged image (J,M). Scale bar: 20 pm.

[0043] Figure 4. Cell -cell adhesion of cerebellum vasculature is downregulated in Prelp- / - mice. (A-J) 605 Reduced VE-cadherin coverage of Prelp -I- vessels. Sections of wild-type (A-C), Omd -I- (D-F) and Prelp -I- (G-I) cerebellums were stained with VE-cadherin (A,D,G) and TL (B,E,H). Staining of Prelp -I- vessels was found to be uneven, inconsistent (arrows). Scale bar 15 pm. J, VE-cadherin staining intensity was quantified (n = 3). (K-T) Weaker claudin-5 staining in Prelp -I- vessels. Claudin-5 and TL staining was performed in wild-type (K-M), Omd -I- (N-P) and Prelp -I- (Q-S) cerebellum. Staining 610 in Prelp -I- vessels was found to be more discontinuous (arrow). (T) Quantification of staining intensity (n = 3). (U-DD) ZO-1 staining is reduced in Prelp -I- . Wild-type (U-W), Omd -I- (X-Z) and Prelp -I- 612 (AA-CC) sections were stained with ZO-1 and TL. Punctated staining along vessels were found (arrow). (DD) Quantification of staining intensity (n = 3). TL; Tomato lectin. Figure 5. Expression profiling analysis of wild type and Prelp- / - mouse meninges. (A) Schematic draw of meninges. (B) Top 10 GO Biological Processes. (C) Top 10 Cellular components over representative analysis. (D) GSEA of genes up-regulated in Prelp- / -. (E) GSEA of genes down regulated in Prelp- / - .

[0044] Figure 6. Microglia is activated in Prelp- / - mouse cerebellum. (A-G) Iba-1 staining of wild-type (A), Omd - / -

[0045] (B), and Prelp -I- (C) cerebellum sections. Microglia were manually counted based on Iba-1 and nuclear DAPI staining (arrows). Scale bar 25 pm. (D) Microglial density was quantified. E-F, Skeletonizing images of Iba- 1 staining to quantify microglial branch length. G, Quantification of branch length per microglial density in wild-type, Omd -I- and Prelp -I- brains (n = 3). (H-L) No differences in astrocyte number, morphology, and staining intensity were observed between wild-type, Omd- / - and Prelp- / - mice. (M) No differences in water content between the wild type and Prelp- / - brain were observed.

[0046] Figure 7. Effect of PRELP on leakage from endothelial cell monolayer. (A-B) Effect of PRELP on human umbilical vein endothelial cells (HUVEC) monolayer Trans-Epithelial Electrical Resistance (TEER). (A) Schematic drawing of the assay. (B) Confirmation of HUVECs monolayer confluency by PECAM-1 staining.

[0047] (C) TEER measurement. (D) Permeability assay was performed using HUVEC monolayer.

[0048] Figure 8. BBB components are damaged in Prelp" mouse cerebellum. (A-J) Reduction of laminin in Prelp'1' mouse. Wild-type (A-C), Omd~ ~ (D-F) and Prelp~ ~ (G-I) cerebellum were stained for laminin (A,D,G) and IgG for leakages (B,E,H) (C,F,I) Merged image. (J) Quantification of Laminin staining (n = 3). Areas of IgG leakage are indicated by arrows. Scale bar: 20 mm. (K-S) Cerebellum from Wild-type (K-M), Omd~ ~ (N-P) and Prelp~ ~ (Q-S) were stained with perlecan antibody (K,N,Q) and TL (L,O,R). (M,P,S) Merged image. T, Quantification of Perlecan staining (n = 3). Cerebellum from Wild-type (U-W), Omd / (X-Z) and Prelp (AA-CC) were stained with collagen IV antibody (U,X,AA) and IgG antibody (V,Y,BB). Scale bar: 20 mm. (W,Z,CC) Merged, image. (DD) Quantification of collagen IV staining (n = 3). Areas of IgG leakage are indicated by arrows. Scale bar: 20 mm. (EE-MM) double-Immunostaining. Double staining of AQA4 (EE, HH, KK) and IgG (FF,II,LL) in wild-type (EE-GG), Omd^' (HH-JJ) and Prelp~ ~ (KK-MM) cerebellum were performed. (GG,JJ,MM) Merged image. Arrows show areas of IgG leakage. (NN) Quantification of aquaporin-4 staining (n = 3). Scale bar: 20 mm. Quantification reveals that there is reduced aquaporin-4 staining around blood vessels in Prelp~^~. Scale bar: 20 mm. (OO-XX) PDGFR-P (OO,RR,UU) and TL (PP,SS,VV) double stain allows us to visualize pericytes and blood vessels in wild-type (OO-QQ), Omd / (RR-TT) and Prelp~ ~ (UU-WW). QQ,TT,WW, Merged. Pericytes detaching from blood vessel can be observed (arrow). (XX) Quantification of pericytes per vessel (n = 3). Scale bar: 20 mm.

[0049] Figure 9. (A-F) Wild-type (A), Omd^~ (B) and Prelp~ ~ (C) sections were stained with GFAP. Scale bar 25 mm. (D,E) Quantification. Astrocyte count (D) and mean grey value (E) were quantified. (F) Brain water contents of wild type and Prelp ' mice were determined. Figure 10. (A) Coomassie Brilliant Blue (CBB) staining of purified recombinant PRELP (prPRELP) expressed in Mimic Sf9 insect cells. The prPRELP is stable for frozen storage. (B) Western blotting of control CM and PRELP CM. There is no detectable PRELP protein in control CM. Doxycycline induced significant amount of PRELP protein expression. PRELP CM after 24 h induction was used as there is degraded protein at 48 h. (C) Quantification of PRELP protein in PRELP CM. 1.2 mg / ml PRELP protein was included in PRELP CM.

[0050] Figure 11. Expression profiling of PRELP application on HUVECs. (A-D) The effect of purified recombinant PRELP protein on HUVEC monolayer culture was examined by mRNA expression profiling. After ontological analysis using Ingenuity software, significantly affected Ingenuity Canonical pathways were classified into four categories: EndMT / Cell adhesion (A), Cancer (B), Inflammation (C), and EMT related Signalling pathways (D).

[0051] Figure 12. Mechanism of Epithelial-mesenchymal transition. Schematic drawing of “Regulation of the EMT pathway” Ingenuity Canonical Pathway of expression profiling data obtained from purified PRELP application vs control in HUVEC monolayer culture. The image was created by Ingenuity Pathway Analysis according to their rule.

[0052] Figure 13. Effect of PRELP on cell-cell adhesion of HUVEC monolayer. (A-F) Control CM (A), PRELP CM (B), TGF-P (C), or TGF-P + PRELP CM (D) was applied to HUVECs and P-catenin antibody staining was performed. Scale bar: 30 pm. (E) p-catenin staining intensity at cell-cell contacts was determined. (F) p-catenin staining intensity in the nuclei was determined. (G-J) Control CM (G), PRELP CM (H), TGF-P (I), or TGF- P + PRELP CM (J) was applied to HUVECs and ZO-1 antibody staining was performed. Arrows indicate segregation of ZO-1 from cell-cell contacts. Scale bar: 25 pm. (K-N) Control CM (K), PRELP CM (L), TGF- P (M), or TGF-P + PRELP CM (N) was applied to HUVECs and claudin-5 antibody staining was performed.

[0053] Brief description of the sequences

[0054] SEQ ID NO: 1 - PRELP

[0055] HGNC: 9357; NCBI Entrez Gene: 5549; Ensembl: ENSG00000188783; OMIM®: 601914;

[0056] UniProtKB / Swiss-Prot: P51888

[0057] 10 20 30 40 50

[0058] MRSPLCWLLP LLILASVAQG QPTRRPRPGT GPGRRPRPRP RPTPSFPQPD

[0059] 60 70 80 90 100

[0060] EPAEPTDLPP PLPPGPPSI F PDCPRECYCP PDFPSALYCD SRNLRKVPVI

[0061] 110 120 130 140 150

[0062] PPRIHYLYLQ NNFITELPVE SFQNATGLRW INLDNNRIRK IDQRVLEKLP

[0063] 160 170 180 190 200

[0064] GLVFLYMEKN QLEEVPSALP RNLEQLRLSQ NHISRI PPGV FSKLENLLLL

[0065] 210 220 230 240 250

[0066] DLQHNRLSDG VFKPDTFHGL KNLMQLNLAH NILRKMPPRV PTAIHQLYLD

[0067] 2 60 270 280 2 90 300

[0068] SNKIET IPNG YFKS FPNLAF IRLNYNKLTD RGLPKNSFNI SNLLVLHLSH

[0069] 310 320 330 340 350

[0070] NRISSVPAIN NRLEHLYLNN NS IEKINGTQ ICPNDLVAFH DFSSDLENVP

[0071] 360 370 380

[0072] HLRYLRLDGN YLKPPI PLDL MMCFRLLQSV VI

[0073] Detailed description of the invention

[0074] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0075] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0076] The terms “comprises,” “comprising,” “includes,” and / or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0077] A “subject” as used herein includes all members of the animal kingdom, including a non-human mammal and a human subject. Non-human mammals include, but are not limited to, non-human primates, rodents (including mice and rats) dogs, cats, domesticated animals and farm animals. In preferred embodiments, the subject is a human subject.

[0078] The terms “treatment”, “treat” or “treating” as used herein refers to obtaining a beneficial or desired clinical outcome. This includes a reduction in the number, extent, or duration of symptoms related to a disease, condition, or disorder; the prevention of further progression of symptoms related to a disease, condition, or disorder; a change in one or more disease associated biomarkers correlating with or predictive of a beneficial or desired clinical outcome of symptoms related to a disease, condition, or disorder; a reduction in inflammation; a reduction in pain; a reduction in damage from a disease, condition, or disorder; or a reduction in functional impairment related to a disease, condition, or disorder.

[0079] The terms “treatment”, “treat” or “treating” also includes the administration of the compounds, agents, drugs or pharmaceutical compositions of the present disclosure to prevent or delay the onset of one or more symptoms, complications, or biomarkers of a disease or condition; lessening or improving one or more symptoms; shortening or reduction in duration of a symptom; or arresting or inhibiting further development of a disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of a disease, condition, or disorder, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease, condition, or disorder.

[0080] The term “neurovascular leakage” as used herein refers to a condition where at least a part of the neurovascular system, such as blood vessel (e.g. an artery, vein or capillary), allows for passage of fluids, macromolecules, or cells through the layer of endothelial cells forming the wall of the blood vessel.

[0081] Neurovascular leakage can result from various physiological or pathological processes and may manifest as the breach of vascular integrity. This phenomenon can occur in various tissues and organs and is associated with conditions such as inflammation, injury, or diseases, including but not limited to vascular disorders, oedema, and pathological angiogenesis. Vascular leakage may encompass, for example, vascular permeability, microvascular permeability, or capillary permeability. Vascular leakage may be associated with a number of diseases or disorders, as known to one of ordinary skill in the art.

[0082] The term “vascular leakage-associated disease or disorder” as used herein refers to a disease, disorder, or condition which results in or is exacerbated by vascular leakage.

[0083] In a vascular leakage-associated disease or disorder, the cause of the vascular leakage may be inflammation. Examples of inflammation that may lead to vascular leakage include, but are not limited to, inflammation due to infection, physical trauma or injury, immune dysfunction or neurodegeneration. A vascular leakage- associated disease or disorder may be an infection, for example a viral, bacterial, fungal, or parasitic infection.

[0084] “Increased neurovascular permeability” as used herein refers to a condition in which the endothelial cell barriers separating the central nervous system from the systemic circulation (e.g. the blood-brain barrier) become more permeable than they are under normal physiological conditions, allowing foreign substances to pass through the barrier into the central nervous system.

[0085] Under normal physiological conditions, the neurovascular unit forming these barriers tightly regulate permeability, which prevents transport of foreign substances such as bacteria, fluid, blood cells, immune cells, large molecules, metabolic waste products and drugs into the brain. The leakage of such potentially harmful substances into the brain, which may disrupt normal brain function and cause or contribute to the progression of disease.

[0086] Unless indicated otherwise, the terms “increase” and “decrease” as used herein in the context of neurovascular permeability, neurovascular leakage or other physiological functions refer to increases and decreases relative to normal physiological conditions. For example, the increase or decrease may be relative to the level of function in a subject prior to the onset of a pathological state, or relative to the expected level of function in a healthy subject. Such information would be well within the common general knowledge of qualified medical professionals.

[0087] References to “increased” or “abnormal” permeability should be understood as referring to conditions under which one or more substances which would typically be excluded from a part of the central nervous system (e.g. the brain) by an endothelial cell barrier (e.g. the blood-brain barrier) under normal physiological conditions are able to pass through the barrier and access the part of the central nervous system.

[0088] References to an “increase” in endothelial cell to mesenchymal cell transition events at the blood-brain barrier should similarly be understood as referring to an increase relative to the number and / or frequency of events in the subject prior to the onset of a pathological state, or relative to the expected number and / or frequency of events in a healthy subject.

[0089] “Neurovascular unit dysfunction” as used herein refers to a condition in which the relationship between neurons, blood vessels and supporting cells such as glia is disrupted or impaired. This disruption or impairment may allow foreign substances such as bacteria, fluid, blood cells, immune cells, large molecules, metabolic waste products and drugs to enter the central nervous system. Neurovascular unit dysfunction may also result in impairments to blood supply and the removal of metabolic waste.

[0090] The term “effective amount” as used herein is an amount sufficient to affect any one or more beneficial or desired results. An effective amount may prevent, alleviate or ameliorate symptoms of a disease, condition, disorder, or adverse reaction. For prophylactic use, beneficial or desired results may include preventing or reducing the risk of developing the disease, condition, disorder, or adverse reaction, reducing the severity or delaying the onset of a disease, condition, or disorder or adverse reaction, or a complication or comorbidity associated with the disorder or adverse reaction. For therapeutic use, beneficial or desired results may include clinical results such as reducing one or more symptoms of a vascular leakage-associated disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, reducing or eliminating unwanted side effects or another medication, and / or delaying the progression of the disease in a subject.

[0091] Beneficial or desired results may also include reducing vascular leakage in a subject, reducing reactive oxygen species production in a subject, inducing apoptosis of neutrophils in a subject, or increasing survival of a subject having a vascular leakage-associated disease. An effective dosage can be administered in one or more administrations.

[0092] A therapeutically effective dosage of an agent, a drug, a compound, or a pharmaceutical composition as referred to herein is an amount sufficient to accomplish therapeutic or prophylactic treatment either directly or indirectly. The amount may vary from one subject to another and may depend upon one or more factors, such as, for example, subject gender, age, body weight, subject’s health history, and / or the underlying cause of the disease, condition, or disorder to be prevented, inhibited and / or treated.

[0093] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to a non-medicinal substance or compound that is suitable for use in the formulation of pharmaceutical compositions, drugs, or therapeutic agents. Pharmaceutically acceptable carriers or excipients are typically inert, non-toxic, and free from contaminants. This includes substances which enhance the solubility, bioavailability, and absorption of the active pharmaceutical ingredient, improve the stability of the active pharmaceutical ingredient, facilitate administration of the active pharmaceutical ingredient, or control the rate of release of the active pharmaceutical ingredient. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. In some embodiments, diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline.

[0094] Proline And Arginine Rich End Leucine Rich Repeat Protein (PRELP) in a proteoglycan present in connective tissue extracellular matrix. It is encoded by the PRELP gene. The human PRELP protein has a sequence of SEQ ID NO: I (UniProtKB: P51888)

[0095] Any reference to “PRELP” should be interpreted to encompass any functional variants, fragments, fusions or conjugates PRELP which substantially retain the activity of wildtype PRELP (SEQ ID NO: 1). “Functional variants” as used herein should be understood to include variants comprising one or more amino acids substitutions, insertions or deletions, or one or more post translational modifications, provided that such variants substantially retain the activity of wildtype PRELP.

[0096] In particular, PRELP variants comprising substitution as positions 33, 157, 334 and / or 348 relative to SEQ ID NO: 1 are within the scope of the claimed invention.

[0097] Post-translational modifications include phosphorylation, acetylation, methylation, glycosylation, ubiquitination, sumoylation, neddylation, sulfation, hydroxylation, phospholipidation, s-nitrosylation, carboxylation and deamidation.

[0098] In particular, PRELP variants comprising N-linked glycosylation of the asparagine residue at positions 124, 289, 230 and / or 327 of SEQ ID NO: 1 are within the scope of the claimed invention.

[0099] For example, the variants of PRELP having at least 75% identity to SEQ ID NO: 1, at least 80% identity to SEQ ID NO: 1, at least 95% identity to SEQ ID NO: 1, at least 96% identity to SEQ ID NO: 1, at least 97% identity to SEQ ID NO: 1, at least 98% identity to SEQ ID NO: 1, or at least 99% identity to SEQ ID NO: 1 are within the scope of the present invention.

[0100] Similarly, functional variants, fragments, fusions or conjugates of PRELP which retain at least 75%, at least 80%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% of the activity of wildtype PRELP, are within the scope of the present invention. “Activity” as used herein refers to any biologically relevant activity, including binding affinity for any relevant ligand or binding partner, and performance on any in vivo or in vitro assay. In certain embodiments, the ligand is TGFpRI, II, or III. “Activity” in this context specifically encompasses protective activity against TGF-P mediated vascular leakage and binding to TGF-p. Binding or other biologically relevant activity can be measured by any suitable assay known in the art. Selection of a suitable assay would be well within the common general knowledge of a person skilled in the art. In certain embodiments, activity is measured using an adhesion assay. Suitable adhesion assays to use in this context would be known to a person skilled in the art.

[0101] The present inventors surprisingly discovered that application of PRELP protein (SEQ ID NO: 1; UniProtKB: P51888) is protective against TGF-P mediated vascular leakage and promotes endothelial barrier integrity.

[0102] Without wishing to be bound by theory, the inventors have shown that PRELP application reduces abnormal vascular permeability, and protects endothelial cell barriers (such as the blood-brain barrier) through inhibition of Endothelial cell to mesenchymal cell transition events (EndMT), by direct binding with an EndMT factor, TGF-p.

[0103] Endothelial cell barriers include blood-brain barrier, blood-spinal cord barrier, blood-ocular barrier, and the blood-retinal barrier. Endothelial cell damage caused by activation of TGF-P is a feature of many neurological diseases, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, stroke, meningitis, traumatic brain injury, spinal cord injury, epilepsy, vascular dementia, stoke, haemorrhage, aneurysm and other cerebrovascular diseases, and cancer-associated angiogenesis.

[0104] TGF-P (Transforming Growth Factor beta) is a multifunctional cytokine and a member of the Transforming Growth Factor family. It exists in several isoforms, including TGF-P 1, TGF-P2, and TGF-P3. TGF-P 1 in particular promotes EndMT by up-regulating mesenchymal characteristics via a SMAD dependent pathway. PRELP also binds to TGF-P 1.

[0105] Accordingly, the present invention provides PRELP for use in a method of treating a disease characterized by abnormal neurovascular permeability. Preferably, the abnormal permeability is increased permeability.

[0106] The present invention also provides PRELP for use in a method of treating neurovascular unit dysfunction. Further provided is PRELP for use in a method of treating neurovascular leakage. In certain embodiments, the present invention provides PRELP for use in a method of preventing blood-brain barrier breakdown and / or reducing blood-brain barrier leakage. In some embodiments, the blood-brain barrier breakdown or blood-brain barrier leakage may be mediated by TGF-p.

[0107] In some embodiments, the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction. In some embodiments, the disease may be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

[0108] In some embodiments, the abnormal neurovascular permeability is caused by an increase in endothelial cell to mesenchymal cell transition events at the blood-brain barrier and / or the blood-CSF barrier. In some embodiments, the increased endothelial cell to mesenchymal cell transition events are caused by TGF-P mediated damage to cell-cell adhesion.

[0109] Neurovascular leakage, specifically in the brain, plays a significant role in the development and progression of dementia. In particular embodiments, the present invention provides PRELP for use in the treatment of dementia.

[0110] Potential mechanisms of neurovascular leakage which cause dementia include the following.

[0111] Blood-Brain Barrier (BBB) Disruption: The BBB is a barrier between the brain's blood vessels and the cells and other components that make up brain tissue. In healthy individuals, the BBB prevents potentially harmful substances in the blood from entering the brain. However, in people with dementia, the BBB often becomes leaky or disrupted, allowing harmful substances like toxins and inflammatory cells to enter the brain.

[0112] Increased Inflammation: Vascular leakage contributes to increased inflammation in the brain. Inflammatory cells and molecules that are normally restricted to the bloodstream can enter the brain tissue, leading to chronic inflammation, which is a key feature of many neurodegenerative diseases, including dementia.

[0113] Amyloid-P Accumulation: Amyloid-P is a protein that accumulates in the brains of people with Alzheimer's disease, a common form of dementia. Disruption of the BBB and vascular leakage can contribute to the accumulation of amyloid-beta in the brain.

[0114] Microvascular Dysfunction: The brain's micro vasculature, the network of tiny blood vessels that supply blood to the brain, often becomes dysfunctional in people with dementia. This can lead to reduced blood flow to the brain, hypoxia (lack of oxygen), and the accumulation of harmful substances in the brain tissue, all of which contribute to the development and progression of dementia.

[0115] Neurovascular Unit Dysfunction: The neurovascular unit is a functional unit comprising neurons, blood vessels, and glial cells (supporting cells in the nervous system). Dysfunction of the neurovascular unit, including vascular leakage, is associated with the development and progression of dementia.

[0116] Although vascular leakage is associated with dementia, it's not the only factor. Dementia is a multifactorial disease with a complex aetiology involving genetic, environmental, and lifestyle factors. Additionally, there are multiple types of dementia, each with its own unique characteristics and contributing factors. Nonetheless, vascular leakage and BBB disruption are considered key contributors to the pathogenesis of many forms of dementia.

[0117] In some embodiments, the increased neurovascular permeability is associated with administration of an immunotherapeutic agent. Accordingly, the inventions provide methods of preventing or minimising the increase in neurovascular permeability associated with or resulting from administration of an immunotherapeutic agent. Impairment of the blood-brain barrier allows blood-borne molecules (e.g., albumin and plasmin) and cells (e.g., leukocytes) to enter the brain, where they may have detrimental effects, including activating neuroinflammatory, innate and adaptive immune responses. These may trigger or exacerbate pathological mechanisms, leading to neurodegeneration and cognitive decline.

[0118] In particular, increased vascular permeability is known to be a side-effect of anti-Amyloid P immunotherapy, although treatment of increased vascular permeability associated with other conditions is also envisaged.

[0119] In some embodiments, PRELP is in combination with an anti-Amyloid- immunotherapeutic agent for treating abnormal vascular permeability caused by or associated with administration of the anti-Amyloid- immunotherapeutic agent. The anti-Amyloid-P immunotherapeutic agent may be administered before, simultaneously with, or subsequently to the anti-Amyloid-P immunotherapeutic agent.

[0120] In some embodiments, the anti-Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

[0121] In a fifth aspect, the present invention provides a method of treating a subject suffering from a disease characterized by abnormal neurovascular permeability or neurovascular unit dysfunction, comprising administering a therapeutically effective amount of PRELP to the subject. In some embodiments, the abnormal neurovascular permeability is increased permeability of the blood-brain barrier and / or the blood-CSF barrier.

[0122] In some embodiments, the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction. The disease may be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

[0123] In some embodiments, the disease is caused by increased endothelial cell to mesenchymal cell transition events at the blood-brain barrier and / or the blood-CSF barrier. In some embodiment, the increased endothelial cell to mesenchymal cell transition events are caused by TGF-P mediated damage to cell-cell adhesion. In some embodiments, wherein the abnormal vascular permeability is caused by or associated with administration of an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an anti- Amyloid-P immunotherapeutic agent. In some embodiments, the anti-amyloid beta agent is lecanemab or donanemab.

[0124] In a sixth aspect, the invention provides a method of preventing or reducing increased neurovascular permeability or neurovascular unit dysfunction caused by or associated with anti-Amyloid- immunotherapy, comprising administering an effective amount of PRELP to a subject before, simultaneously with, or subsequently to the anti-Amyloid-P immunotherapeutic agent.

[0125] In one embodiment, PRELP is formulated as a composition, for example a pharmaceutical composition.

[0126] A pharmacal composition of the invention comprises an PRELP and one or more of a pharmaceutically acceptable carrier, buffer, excipient, adjuvant, vehicle, diluent or stabiliser or any combination thereof.

[0127] Examples of carriers suitable for use with the present invention include water, saline, buffered saline, dextrose, ethanol, liposomes, glycerol, polyethylene glycol and combinations thereof.

[0128] In some embodiments, the formulation of the active drug concentrate can comprise a pharmaceutically acceptable tonicity agent, a buffering agent, and a pharmaceutically acceptable surfactant.

[0129] The pharmaceutical compositions of the invention may also contain one or more further therapeutically active agents in addition to the molecule or molecules of the present invention. For example, the pharmacal compositions of the invention may comprise one or more further active agents suitable for treating or managing a disease or disorder associated with neurovascular leakage.

[0130] Alternatively, or in addition, the pharmaceutical compositions of the invention may contain one or more of an anti-viral agent, an anti-inflammatory agent, an antibiotic agent, an anti-fungal agent, a chemotherapeutic agent, an analgesic agent, an immunotherapeutic agent, a neurotransmitter or neuromodulator, or another therapeutically active agent.

[0131] PRELP may be introduced, delivered, or applied as pharmaceutical composition or drug substance into the body of a human or animal subject by any suitable method for therapeutic or prophylactic purposes.

[0132] In some embodiments, PRELP is administered in the form of a pharmaceutical composition or formulation comprising PRELP protein. In other embodiments, PRELP is administered in the form of a nucleic acid, plasmid or vector, or cell comprising a nucleic acid, plasmid or vector. PRELP protein may be administered by any suitable method known in the art, including but not limited to oral administration, intravenous (IV) administration, intramuscular (IM) administration, subcutaneous (SC) administration, inhalation administration, topical administration, transdermal administration, rectal administration, nasal administration, intrathecal or intraventricular administration, intra-articular administration, intradermal administration, intraperitoneal administration, intranasal administration, intraocular administration, intradermal patch administration, buccal administration, sublingual administration. In preferred embodiments, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule, or foam.

[0133] Also within the scope of the present invention are methods of inducing the production of PRELP protein in the subject by gene therapy or cell therapy. Gene therapy includes the introduction, modification, or replacement of one or more genes expressing PRELP within the subject’s cells. Gene therapy can be executed using various methods, including the insertion of functional genes into a patient's cells, the repair of mutated genes, or the regulation of gene expression. Cell therapy may involve the introduction of living cells expressing PRELP into a subject. The cells may be derived from a subject’s own body or from a donor.

[0134] The present invention also provides a nucleic acid molecule comprising a polynucleotide sequence that encodes PRELP, or a functional fragment, fusion or conjugate thereof. The nucleic acid molecule may be suitable for use in therapy, such as gene therapy.

[0135] There is also provided a vector comprising a nucleic acid sequence of the invention and a host cell comprising such a nucleic acid.

[0136] In some embodiments, the host cell is a cell suitable for use in cell therapy. In other embodiments, the host cell is a cell suitable for use in protein manufacturing.

[0137] Also provided herein is a kit comprising PRELP and an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an anti-Amyloid-P immunotherapeutic agent. In some embodiments, the antiamyloid beta agent is lecanemab or donanemab. The kit may further comprise instructions for use. The instructions may be instructions for administration of the components of the kit for the purposes of treating one of the diseases or disorders mentioned herein. The components of the kit may be disposed separately in separate containers. The components of the kit may be provided in the form of pharmaceutically acceptable compositions.

[0138] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. The scope of the present invention will be limited only by the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

[0139] Examples

[0140] Example 1: Materials and Methods

[0141] For Antibodies, reagents, data accession number, software, and primes, all antibodies, reagents, accession number, software and algorithms, and primes used in this study were listed in Table 1 and Table 2.

[0142] 1.1. Omd- / - and Prelp- / - mice

[0143] Mouse lines were generated by Takeda Pharmaceutical Company and wild type and heterozygote founders were imported to our animal facility. All animal procedures were performed in accordance to the Animals (Scientific procedures) Act 1986 of the UK Government and housed in compliance with the Home Office Code of Practice. Mice were kept in individually ventilated cages (IVCs), in a 12 h 98 lightdark cycle and were fed a complete pelleted mouse diet and with constant access to water.

[0144] Briefly, Omdflox or Prelpflox ES cells were generated from C57BL / 6J ES cells by homologous recombination. Targeting vectors were constructed by insertion of the first LoxP sequence upstream of exon two, containing the initiation codon on the Omd or Prelp locus. A second LoxP sequence, neomycin resistant unit, and LacZ unit was inserted downstream of exon three. Cre expression plasmid was electroporated into the recombinant flox ES cells to generate ES cells harboring the knockout 104 allele. The resulting cells were injected into ICR tetrapioid blastocysts to generate chimeric male mice 105 which were backcrossed to C57BL / 6J females. Single knockout mice (OmdLacZ / LacZ and PrelpLacZ / LcicZ) were generated by cross breeding within the colony.

[0145] 1.2 Genotyping

[0146] Genotyping PCR reactions were performed as follows. Mouse ear punches were mixed with 180 pl DirectPCR Lysis Reagent (Viagen Biotech) and 0.4 mg / ml Proteinase K (Sigma) solution before rocking at 55 °C overnight. The samples were then incubated at 85 °C for 45 min before centrifugation and collection of the resulting lysate. OMD samples were genotyped using an Invitrogen kit. A master mix was prepared according to the manufacturers protocol using the primers described in Table 2 and added to 5 pl genomic DNA. Samples were incubated at 95oC for three min prior to 35 cycles consisting of 30 sec at 95 °C, 90 sec at 61 °C and 90 sec at 72 °C. The final extension was 10 min at 72 °C. This produced amplicons of different lengths: 298 bp in wild type mice with OMD-A and 0MD-B2 primers; 541 bp in knockout mice with 0MD-B2 and LacZ- 5756 primers; or both amplicons present in heterozygotic mice. PRELP samples were genotyped using a Multiplex PCR kit (Qiagen). A master mix was prepared according to the manufacturers protocol using the primers described in Table 2 and added to 2.5 pl genomic DNA. Samples were incubated at 95oC for 15 min prior to 40 cycles consisting of 30 sec at 94 °C, 90 sec at 63 °C and 90 sec at 72 °C. The final extension was 10 min at 72 °C. Amplicons were 846 bp for wild type mice with PRELP-A and PRELP-B primers, 634 bp for knockout mice with PRELP-C and LacZ-B primers, or both amplicons present in heterozygotic mice.

[0147] 1.3 Tissue processing and staining

[0148] Mouse brains were isolated and fixed in 4% PFA for 24 h before paraffin embedding and sectioning. Tissue for paraffin sectioning was processed in the Institute’s Pathology department using an automated machine (Leica ASP300S). Samples were sectioned into 5 pm slices on superfrost slides treated with poly-L-lysine, dried and stored at room temperature. Histological staining was performed in an automated system in the Pathology department. H&E, von Kossa, alcian blue, congo red and MSB, were performed in the Pathology department following department’s specific protocol for each stain.

[0149] Methylene blue and basic fuchsin staining was performed on semi -thin sections of xenografted tumors. For immunostaining, slides were dewaxed for 10 min in Histoclear and rehydrated in an ethanol-water graded series. Antigen retrieval was performed by boiling the samples for 15 min in citrate (pH 6.0) or Tris-EDTA buffer (pH 9.0) depending on the antibody. Sections were blocked for 1 h in 10% goat serum in PBS and were incubated overnight with primary antibodies at 4 °C. Detection was performed by incubation with anti-rabbit or anti-mouse Alexa Fluor 488 secondary antibodies, for 1 h at room temperature (1:500 dilution, Life technologies).

[0150] 1.4 -Galactosidase analysis

[0151] Mouse brains were isolated from adult mice and were fixed in 4% PFA at 4 °C briefly for 2 h with gentle agitation. Afterwards they were washed in PBS and left at 30% sucrose at 4oC overnight 142 before subsequently frozen in OCT. Cryosections 10 pm thick were washed twice in PBS + 2 mM MgC12 for 20 min and were stained overnight in X-gal at 37 °C. Sections were washed in PBS and then 144 either counterstained and mounted in Nuclear Fast Red (Vector Laboratories) or followed by IHC. For 145 IHC, briefly, samples were immediately blocked for 1 h with 10% goat serum and were incubated with 146 the primary antibodies overnight. Secondary staining was completed with anti-rabbit or anti mouse Alexa Fluo-488 antibodies, counterstained in DAPI and mounted.

[0152] 1.5 Immunocytochemisty

[0153] Coverslips with cell monolayers were washed in PBS and fixed with either 4% PFA or ice-cold methanol for 10 min. Samples were then washed and incubated for 1 h in blocking buffer. After blocking and incubation with primary antibodies overnight at 4 °C, the samples were incubated with secondary Alexa Fluor 488 (1 :500, Life Technologies) in blocking buffer for 1 h at room temperature, counterstained with Hoechst solution (Invitrogen) and mounted. Slides were imaged using a Zeiss LSM710 at 10X and 40X magnification. After laser intensity settings were optimized, images were processed on ImageJ and a standard threshold to remove background noise across all samples. 1.6 Microscope settings and image analysis

[0154] A preliminary analysis determined the thickness of samples, and the microscopic settings were adjusted accordingly to enable the detection of structures. In this setting, Hamamatsu ORCA-ER Digital Camera (Hamamatsu, Japan) and ^Manager software (Edelstein et al. 2010) were used to obtain fluorescent images on an Axioskop 2 Plus (Zeiss, Germany) from sections. A 40X differential interference contrast objective with an aperture of 0.95 and 0.25 working distance and 10X ocular lens were used to obtain each region of interest. Imaging parameters of laser intensity and exposure time were optimized and uniformly set in the same experiments. Images were then processed, despeckled, background subtracted and applied with a median filter at 2px in order to remove all background noise. Huang auto threshold was then applied, and the threshold was saved as an ROE This ROI was then used as the “outline” of immunostaining to measure, and regions of interest measured and quantified with FIJI software.

[0155] 1.7 Vascular capillaries leakage studies

[0156] 25 mg / ml 70 kDa Dextran-Texas Red was injected into mice by intravenously. After circulation for 3 h, mice were culled and brain tissues harvested. Perfusion was performed through the heart before 174 collecting tissues. Dye excess was washed out through fixation and washing in PBS before 175 cryoprotection. Sample preparation was done as described above. Vascular permeability was then 176 visualized with fluorescence microscopy. Vessels were traced on ImageJ and interior staining removed. The resulting external staining was then quantified by methods mentioned above.

[0157] 1.8 Expression profiling of meningeal vessels in RNA-seq analysis

[0158] 180 Sample preparation - Four wild-type mice, three Omd- / - and three Prelp- / - knockout mice meningeal samples were used for RNA-seq analysis. RNA was extracted via ARCTURUS PicoPure RNA Isolation kit for mouse samples or PureLinkTM RNA Mini Kit for HUVECs. In brief, meningeal vessels were excised and homogenized using a rotor-stator homogenizer. After centrifugation at 3,000 184 x g for 2 min and extracted in accordance with manufacturer’s instructions RNA was quantified and qualified by Agilent’s 2200 Tape Station, measuring RNA concentration and agarose gel electrophoresis. Library preparation - Samples were processed using the KAPA mRNA HyperPrep Kit (Roche KK8580) according to manufacturer’s instructions.

[0159] Briefly, mRNA was isolated from total RNA using Oligo dT beads to pull down poly-adenylated transcripts. The purified mRNA was fragmented using chemical hydrolysis (heat and divalent metal cation) and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase in the presence of Actinomycin D. This allows for RNA dependent synthesis while preventing spurious DNA- dependent synthesis. The second cDNA strand was synthesized using dUTP in place of dTTP, to mark the second strand. The resultant cDNA is then “A-tailed” at the 3’ end to prevent self-ligation and adapter dimerization. Full length xGen adaptors (IDT), containing two unique 8bp sample specific indexes, a unique molecular identifier (N8) and a T overhang are ligated to the A-Tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle 198 PCR (50 ng of starting material, 15 PCR cycles).

[0160] The high fidelity polymerase employed in the PCR is unable to extend through uracil. This means only the first strand cDNA is amplified for sequencing, 200 making the library strand specific (first-strand).

[0161] 1.9 Sequencing

[0162] Libraries to be multiplexed in the same run are pooled in equimolar quantities, calculated from Qubit and Bioanalyser fragment analysis. Samples were sequenced on the NextSeq 500 instrument (Illumina, San Diego, US) using a 75 bp single read run with a corresponding 16 bp UMI read.

[0163] 1.10 Data Analysis

[0164] Run data were demultiplexed and converted to fastq files using Illumina’s bcl2fastq Conversion Software v2.19. Fastq files were then aligned to the Mus musculus genome GRCm38 or Homo sapiens genome GRCh38 using RNA-STAR 2.5.2b then UMI deduplicated using Je-suite (1.2.1). Reads per transcript were counted using FeatureCounts and differential expression was estimated using Galaxy. Log2 fold change and p values of pairwise differential expression between wild type samples and knockout samples or PRELP treated and untreated samples were then analysed using Qiagen’s Ingenuity Pathway Analysis (version 48207413).

[0165] 1.11 Measurement of microglial morphological change

[0166] Microglial morphological change was measured using the previously reported protocol with minor modifications (Morrison and Dilosa, 2013). Briefly, images of Iba-1 staining were processed to remove noise and any background staining. A threshold was applied to produce a binary image and skeletonized which was analysed using Analyse Skeleton plugin. The image was then analysed, and the sum of all branch lengths were extracted and used for further quantification. All images were processed using the same parameters. Branch lengths were normalized by number of microglia per area and the resulting value was expressed as branch length per microglial density.

[0167] 1.12 Brain integrity studies

[0168] Transepithelial / transendothelial electrical resistance (TEER) were used to assess the brain integrity using an EVOM Volt-Ohmeter (World Precision Instruments). STX2 probes were disinfected and air dried before being inserted into upper and bottom chambers. Resistance was measured for empty wells containing media before determined across monolayers. Values were calculated by multiplying raw values by transwell growth area once the base value for empty wells had been subtracted. 70,000 cells per 24-well transwell insert were seed and allow them to grow for 4 days in control and PRELP conditioned media. TEER measurements were completed 96 h after PRELP treatment. For the membrane permeability, permeability assays were completed using an in vitro vascular permeability assay kit (Merck). In brief, inserts were hydrated and seeded with 0.5 x 105 cells / insert in media and incubated until a monolayer formed. PRELP conditioned media was applied and incubated for 24 h. FITC-Dextran was added to each insert and incubated for 20 mins in the dark before the insert was removed from the well. Media in the well was mixed and transferred to black 96-well opaque plate to measure fluorescence intensity at 485 nm and 535 nm excitation and emission respectively.

[0169] 1.13 Experimental design and statistical analysis

[0170] Experiments were performed at least in three independent replicates. All data shown as the mean ±239 SEM. All data was tested for normal distribution before a Student's T-test was used to calculate significance. A two- tailed student-t test was used for statistical analysis. *p < 0.05, **p < 0.01 and 241 ***p < 0.001. NS, not significant. Imaging data were analysed by NIH software ImageJ (Ljosa et al. 2012). N shown in the figure legends indicates the number of animals used in the experiments. For image analyses, five fields were randomly imaged per animal.

[0171] Example 2: PRELP is selectively expressed in vascular smooth muscle cells (vSMCs) and pericytes around brain vasculature

[0172] PRELP expression in the CNS was examined by X-gal staining of Prelp+ / LacZ mouse brain. The mouse expresses the lac-Z gene under the control of endogenous PRELP transcription elements (Papadaki et al. 2020). At embryonic stages, we observed expression in the cortical hem of the hippocampal allocortex (Figure 1A, arrowheads) and at sites of bone formation around the CNS (Figure IB). However, we did not observe any other strong PRELP expression in the head (Figure 1C).

[0173] At the adult stages, a unique X-gal staining was observed in mural cells around the CNS vessels, covering both the dorsal and ventral part of the brain, as well as around the optic nerves, the space between the two hemispheres (superior sagittal sinus) and the central canal of the spinal cord (Figure D-G). Enlarged images revealed X-gal staining (Figure 1H,I), reminiscent of pericytes which encircle capillaries and vascular smooth muscle cells (vSMCs) around large arterial and venous vessels (Chen et al. 2017, Vanlandewijck et al. 2018). Staining with PRELP antibodies (a-PRELP #15) identified secreted PRELP protein largely localized around large arterioles / venules (Figure 1J) and capillaries (Figure IK). In addition to vascular mural cells, we observed strong staining at ependymal layers of the ventricle walls (Figure IL), the choroid plexus (Figure IM), and non-pigmented layer of ciliary body of the retina (Figure IN).

[0174] The analysis of PRELP expression in pericytes and vSMCs, but not in endothelial cells has been confirmed by published single cell mRNA expression profiling data (He et al. 2016, Vanlandewijck et al. 2018, Zeisel et al. 2015) (Figure 2A). To confirm these previously published results in this study, we performed double staining to examine where PRELP was expressed. X-gal staining did not overlap with endothelial marker PECAM-1 positive cells (Figure 2B-D). We observed that X-gal staining was co-localised with a-SMA, marking vSMCs (Figure 2E-G). There was no detectable staining of X-gal with astrocyte marker GFAP on capillaries (Figure 2H-J) although single cell analysis indicated gene expression of PRELP in astrocyte subpopulations (Figure 2A). Double staining with pericyte marker, NG2, showed that co-staining was limited to the pericyte processes around the vasculature (arrowhead) and not the pericyte cell body (Figure 2K-M) (arrow in Figure 2M). This suggests that the receptor gene may be expressed by pericytes - localisation with the X-gal staining (Figure 2N-P), indicating PRELP was not expressed in the endothelial cells and expressed in vSMCs and in pericytes at specific locations, rather than astrocytes surrounding vascular capillaries.

[0175] In addition to the Prelp- / - mice, we examined the vasculature in Omd- / - mice. OMD is also a class II SLRP family, highly conserved with PRELP. We previously demonstrated that OMD and PRELP are both expressed in umbrella bladder epithelial cells and involved in bladder cancer initiation in a partially redundant manner (Papadaki et al. 2020). Omd expression was observed across the cerebrum, but not the cerebellum, optic nerves or optic chiasm (unpublished result). Omd was also strongly expressed in neurons and weakly expressed in cells around vasculature (unpublished result). Thus, besides wild-type controls, we also examined Omd- / - mice as the second control.

[0176] Examples 3: Cell-cell adhesion weakened in Prelp- / - mice results in leakage from vascular capillaries in mouse brain vSMCs and pericytes have important roles in controlling endothelial cell-cell integrity (Armulik et al. 2010, Hayes et al. 2022), therefore, we examined the effect of PRELP deletion on leakage from neural capillaries and the BBB. Firstly, the status of vascular integrity or BBB was assessed by immunoglobulin G (IgG) staining using one-year-old mice. IgG is a 160 kDa protein, which exclusively localizes in blood plasma. The intact BBB prevents IgG from passing through the vasculature and coming into contact with neural tissues. Using anti -mouse IgG- Alexa Fluor 594, we stained for plasma IgG in wild-type, Omd - / - and Prelp - / - mouse cerebellum (Figure 3A-C). In wild type, IgG staining was retained within the vasculature and along the walls of the blood vessels in a regular striated pattern perpendicular to the length of the vessel. In the Omd - / - mice brain, diffused staining outside the vasculature was not observed (Figure 3B). However, in the Prelp - / - brain, IgG was often found to be highly diffused outside vasculature. (Figure 3C,D). This suggests that PRELP, but not OMD, is responsible for the regulation of neurovasculature integrity. Disruption of the Prelp - / - BBB was most intense in the cerebellum, compared with the cortex (Figure 3E,G). This may reflect the higher levels of PRELP expression in the cerebellum. To confirm the BBB damage in Prelp- / - , we performed another leakage assay through injection of Texas Red conjugated 70 kDa-Dextran.

[0177] Consistent with the IgG staining result, Dextran was restricted to the vasculature in wild type animals (Figure 3H-J). In comparison, we detected areas of the posterior brain where the dye was detected outside of the vasculature in Prelp - / - (Figure 3K-M). One of the proposed mechanisms of vascular leakage is activation of EndMT of vascular endothelial cells (Sweeney et al. 2019), which is associated with a reduction in adherens and tight junctions.

[0178] Recently, we demonstrated that PRELP has the ability to activate mesenchymal -to-epithelial transition (MET), resulting in the enhancement in bladder epithelial cell-cell and retinoblastoma cells (Hopkins et al. 2022, Papadaki et al. 2020). As the EndMT / MEndT mechanism is largely conserved with EMT / MET, loss of PRELP may cause vascular leakage through activation of EndMT (Hong et al. 316 2018, Saito 2013).

[0179] We previously demonstrated the cell-cell junction dysfunction in response to the OMD and PRELP expression levels in bladder cancer cells using electron microscope analysis along with immunofluorescence analysis (Papadaki et al. 2020). Therefore, we performed immunostaining against an adherens junction marker (VE- cadherin) and tight junction markers (Claudin-5 and ZO-1). In Prelp- / - , we observed uneven, inconsistent VE- cadherin staining in contrast to the uniform staining in wild type and Omd- / - mice (Figure 4A-I) . Quantification of VE-cadherin signal revealed that there was significant reduction of VE-cadherin in Prelp - / - (Figure 4J). For claudin-5, we observed uniform membrane staining in the wild type and Omd- / - neuro vasculature, although in Prelp- / - mice presented significantly weaker expression (Figure 4K-T). ZO-1 formed a stripe-type staining pattern around the membrane in wild type mice, which was punctuated in Prelp- / - mice (Figure 4U- CC). ZO-1 staining intensity of Prelp- / - was also significantly reduced compared to controls (Figure 4DD). These observations indicate weakened cell-cell contacts between VSMCs in Prelp- / - mice.

[0180] Next, we examined neurovascular unit (NVU) components in the Prelp- / - mouse. The basement membrane (BM) is a relatively thick layer of secreted proteoglycans, laminins, collagens and perlecan which underlies endothelial cells. These components are organised by SLRP proteins, with most SLRPs able to bind collagen (Tashima et al., 2015) and PRELP specifically shown to interact with perlecan (Bengtsson et al. 2002). Laminin staining in wildtype and Omd - / - is intense, clearly surrounding blood vessels (Figure 8A-J). This intensity is lost in Prelp- / - mice, especially at sites with intense IgG leakage (Figure 8). Perlecan staining was also reduced in Prelp- / - Figure 8K-T). However, we did not observe a significant reduction of collagen IV staining in Prelp- / - compared with wild-type mice ( Figure 8U-DD), suggesting collagen IV expression was maintained independent of IgG leakage in the Prelp- / - vasculature (Figure 8AA-CC, arrows).

[0181] Next, we examined the effect of PRELP on the distribution of pericyte and astrocyte perivascular end-feet. Aquaporin 4 (AQP4) is a water channel found on astrocyte end-feet (Haj-Yasein et al. 2011). Double staining with AQP4 and IgG in mouse cerebellum revealed that there was a significant decrease in AQP4 signal around the vasculature in Prelp - / - , whereas no difference was observed in Omd -I- ( Figure 8EE-NN). We then examined the effect on pericytes. Pericytes were distinguished from endothelial cells by their nuclear morphology and the staining pattern of PDGFRp. The point of association between endothelial cells with pericytes was diminished ( Figure 800-XX), suggesting pericyte detachment from capillaries ( Figure 8UU, arrow). Example 4: mRNA expression profiling and the effect of PRELP on neuroinflammation in knockout mice

[0182] The meninges contain two sites highly expressing PRELP: the meningeal vessels and cells directly contacting with cerebrospinal fluid (Figure 5A), similar to ependymal cells and choroid plexus (Figure 1L,M). To elucidate PRELP mediated biological events and their molecular mechanisms, we performed mRNA expression profiling of meninges on wild-type and Prelp- / - mice as the meninges is easily dissected with less contamination of neural tissues. However, a major disadvantage of studying the meningeal vessels is a lack of astrocytes, although as PRELP is not expressed in astrocytes surrounding neurocapillaries, this may not be important (Figure 2H-J).

[0183] RNA for expression profiling was obtained from isolated meninges from wildtype (n = 3) and Prelp- / - (n = 3) mice. We identified 288 statistically differentially expressed genes (p < 0.01), of which 87 genes encode extracellular proteins. Ontological analysis was performed using the tools provided by GO consortium to produce four sets of analyses; GO Biological Processes (Figure 5B), GO Cellular components (Figure 5C), Gene set enrichment analysis (GSEA) of genes up-regulated in Prelp- / - (Figure 5D), and GSEA of genes down-regulated in Prelp- / - (Figure 5E).

[0184] The top 10 GO Biological Processes showed that two inflammation related processes of “Macrophage activation” and “Immune system process” were the most strongly affected followed by two adhesion related process; “Cell-cell adhesion” and “Cell-matrix adhesion”. “Blood circulation” was also significantly affected. In the case of GO Cellular components, the majority of affected cellular components were related to the extracellular matrix. This is probably a reflection of the extracellular localization of PRELP. In addition, eight categories were associated with change in cellular morphology, including “Cell projection part” and “Plasma membrane bounded cell projection”. As changes to cell-cell adhesion is a major biological event that induces alterations in cell morphology, these results suggest PRELP is a regulator of cell-cell adhesion.

[0185] To further elucidate PRELP function, we utilized GSEA. We analysed up-regulated and down regulated genes separately (Figure 5D,E). Interestingly, gene sets related to inflammation such as “Interferon y response”, “Interferon a response” and “Complement” were strongly affected in Prelp- / - meninges. Cell-cell adhesion related categories, “EMT” and “Apical Junction” were significantly affected (Figure 5E), which suggests that the functional role of PRELP as regulator of partial EMT may be conserved across tissues (Hopkins et al. 2022, Papadaki et al. 2020). In summary, ontological analysis proposes two main biological roles of PRELP within the meninges: cell-cell adhesion and inflammation.

[0186] Expression profiling data and vascular analyses suggest that, in the Prelp- / - brain, vascular leakage may trigger inflammation. We therefore examined the status of microglia and astrocytes in the mouse cerebellum by the labelling with Iba-1 and GFAP antibodies, respectively. Firstly, the number of microglia cell bodies was counted. Irrespective of morphological change (Figure 6A-C), quantification revealed that there was an increase in the number of Iba-1 positive microglia in Prelp - / - sections (Figure 6D). Microglial functional responses in accordance with the protocol established by Morrison (Morrison and Filosa 2013) was used with some minor modifications to examine inflammation and the sum of the branch lengths was used for quantification (Figure 6F). Quantification of branch length per microglial density revealed that there was a statistically significant decrease in Prelp - / - mice (Figure 6G), indicating increased microglial response and supporting the findings in our expression profiling (Figure 5B) as microglia are known as a neural type of macrophages (Masuda et al. 2020). Furthermore, we investigated the effect of PRELP on the morphology and the number of astrocytes using antibody to GFAP. However, there were no differences in astrocyte number, morphology, and staining intensity between wild-type, Omd- / - , and Prelp- / - mice ( Figure 99A-E).

[0187] While leakage of fluids from vasculature or ependymal layer can affect water content in the brain causing hydrocephalus (Karimy et al. 2017), we did not observe differences of water content between the wild type and Prelp- / - brain ( Figure 9F).

[0188] Example 5: Application of PRELP protein enhances endothelial cell-cell integrity by affecting EMT400 related events

[0189] To determine the role of PRELP protein in consolidating BBB integrity and elucidate its mechanism, we performed in vitro experiments using either PRELP conditional medium (PRELP CM) or purified recombinant PRELP protein, produced in Mimic Sf9 insect cells ( Figure 10) (Kosuge et al. 2021). All PRELP proteins showed a phenotypic effect in our assays as shown below.

[0190] To ensure that PRELP is not secreted from the vasculature, we examined the expression of PRELP in HUVECs and found extremely low expression. This is consistent with RNA-seq results in other papers that showed no or very low expression in mouse brain endothelial cells (Vanlandewijck et al. 2018). Thus, we examined the effect of PRELP on a transepithelial / transendothelial electrical resistance (TEER) using a simple Human umbilical vein endothelial cells (HUVECs) monolayer (Figure 7A). After confirming HUVECs formed a monolayer via PECAM1 immunostaining (Figure 7B), we found that application of PRELP CM significantly increased TEER (Figure 7C), indicating that PRELP can enhance endothelial cell-cell integrity. Furthermore, we also examined the effect of PRELP on permeability using fluorophore-tagged 70 kDa dextran. Under these conditions, PRELPCM did not reduce permeability compared to the control but was effective at preventing TGF-P mediated permeability, suggesting that PRELP can inhibit TGF-P signalling (Figure 7D).

[0191] EMT exists along a spectrum of different states. These partial EMT states, pEMT, are important for understanding human diseases such as cancer (Aiello et al. 2018, Brabletz et al. 2018, Lamouille et al. 2014, Nieto et al. 2016). Recently, we reported that PRELP regulates cell-cell adhesion of bladder umbrella epithelial cells and retinoblastoma cells through pEMT (Hopkins et al. 2022, Papadaki et al. 2020). Similar mechanisms, endothelial -mesenchymal transition (EndMT) has been demonstrated in vascular cells (Kovacic et al. 2019, Lamouille et al. 2014, Nieto et al. 2016). EndMT is important for regulating vascular leakage. Our expression profiling analysis of meninges showed that EMT was strongly affected by PRELP deletion (Figure 5E), suggesting that the vascular leakage in PRELP- / - mice might be caused by partial EndMT. TGF-P is a potent mediator of EMT and PRELP -mediated inhibition through the application of PRELP CM in TEER and permeability assay may increase cell adhesion via pMEndT (Figure 7C,D).

[0192] To elucidate the mechanism of PRELP action, we applied purified recombinant PRELP protein to HUVEC monolayers. HUVECs exhibit pEndMT states which contributes to cell-cell permeability (Guo et al. 2015). mRNA expression profiling was performed on HUVEC cultures incubated with PRELP for 48 h. Using Ingenuity Pathway Analysis (IP A) software, we performed ontological analysis to identify 1,903 significantly affected genes and 220 significantly affected canonical pathways. These pathways were largely classified into three categories; EndMT / cell adhesion ( Figure 11A), cancer (Figure 11B), inflammation (Figure 11C) and EMT related Signalling pathways. EndMT / cell adhesion events included “Regulation of the EMT pathway”, “Hepatic Fibrosis Signalling Pathway”, “Epithelial adherens junction signalling”, and “Integrin Signalling” (Figure 11A). As EMT is strongly implicated in cancer-related pathways, we found many associated pathways such as “Molecular Mechanism of Cancer”, and “Bladder Cancer Signalling” which were also affected (Figure 11B, (Brabletz et al. 2018). Furthermore, several interleukins related proinflammation pathways were negatively affected (IL-8, IL-3, IL-7, IL- 6, and IL-4) (Figure 11C), suggesting that PRELP may have an anti- inflammatory role as we discussed in the previous section (Effect of PRELP on neuroinflammation) and activated EMT through TGF-P Met and Wnt signalling was observed in the “Regulation of the EMT pathway” ( Figure 12).

[0193] Example 6: PRELP activates cell-cell adhesion of HUVEC cell culture and reverses TGF-b mediated pEndMT

[0194] The membrane localization of -catenin, an intracellular protein directly associated with cadherin molecules, was enhanced by PRELP (Figure 13A-F). These data indicate that PRELP enhances adherens junction formation and / or stability. We examined the effect of PRELP on tight junctions using ZO-1 (Figure 13G,H) and claudin-5 (Figure 13K,L) staining but could not detect tight junction formation in our conditions.

[0195] TGF-P is the strongest activator of EndMT. As observed in many other biological systems, TGF-P has complex and dual roles in vascular biology. This includes a dual role as an activator and an inhibitor of BBB function in context dependent manners (Diniz et al. 2019, Li et al. 2011). Application of TGF-P to HUVECs has been reported to cause damage to endothelial cell -cell adhesion through activation of pEndMT (Guo et al. 2015). Using this system, we examined the effect of PRELP on TGF-P mediated pEndMT. As shown in Figure 13, 20 ng / ml TGF-P resulted in the increase of P-catenin membrane staining (Figure 13C). PRELP application reversed all TGF-P mediated effects (Figure 13D,J,N) suggesting that PRELP can rescue TGF- p mediated vascular damage in association with inhibition of pEndMT and all of these may be associated with activation of pEndMT (Guo et al. 2015). Example 7: PRELP protein mediated inhibition of neuro-vascular leakage in mouse models

[0196] PRELP protein will be produced and administered to mice via a suitable administration route. Different administration options will be investigated to determine the most suitable method of administration.

[0197] Data showing PRELP protein inhibits neuro-vascular leakage in vivo will be generated using mouse models, including the Amyloid-Related Imaging Abnormalities (ARIA) model and a neuromuscular leakage model.

[0198] Table 1. List of antibodies, reagents, data accession number, and software used in this study

[0199]

[0200] Table 2. List of primers used in this study

[0201] References 1. Aiello NM, Maddipati R, Norgard RJ, Balli D, Li J, Yuan S, Yamazoe T, Black T, Sahmoud A, Furth

[0202] EE, et al. 2018. EMT Subtype Influences Epithelial Plasticity and Mode of Cell Migration. Dev Cell. Jun 18;45 :681 -695 e684.

[0203] 2. Alahmari A. 2021. Blood-Brain Barrier Overview: Structural and Functional Correlation. Neural Plast. 2021:6564585. 3. Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom

[0204] P, Strittmatter K, et al. 2010. Pericytes regulate the blood-brain barrier. Nature. Nov 25; 468:557-561. 4. Bengtsson E, Morgelin M, Sasaki T, Timpl R, Heinegard D, Aspberg A. 2002. The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor. J Biol Chem. Apr 26; 277: 15061-15068.

[0205] 5. Birke MT, Lipo E, Adhi M, Birke K, Kumar-Singh R. 2014. AAV-mediated expression of human PRELP inhibits complement activation, choroidal neovascularization and deposition of membrane attack complex in mice. Gene Ther. May; 21: 507-513.

[0206] 6. Bjamegard M, Enge M, Norlin J, Gustafsdottir S, Fredriksson S, Abramsson A, Takemoto M, Gustafsson E, Fassler R, Betsholtz C. 2004. Endothelium-specific ablation ofPDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities. Development. Apr; 131: 1847-1857.

[0207] 7. Brabletz T, Kalluri R, Nieto MA, Weinberg RA. 2018. EMT in cancer. Nat Rev Cancer. Feb; 18:128- 651 134.

[0208] 8. Chacon-Solano E, Leon C, Carretero M, Garcia M, Sanchez-Dominguez R, Quero F, Mendez-Jimenez E, Bonafont J, Ruiz-Mezcua B, Escamez MJ, et al. 2022. Mechanistic interrogation of mutation independent disease modulators of RDEB identifies the small leucine-rich proteoglycan PRELP as a TGF-beta antagonist and inhibitor of fibrosis. Matrix Biol. Aug; 111: 189-206.

[0209] 9. Chen J, Luo Y, Hui H, Cai T, Huang H, Yang F, Feng J, Zhang J, Yan X. 2017. CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development. Proc Natl Acad Sci U S A. Sep 5;114:E7622-E7631.

[0210] 10. Cho JG, Lee A, Chang W, Lee MS, Kim J. 2018. Endothelial to Mesenchymal Transition Represents a Key Link in the Interaction between Inflammation and Endothelial Dysfunction. Front Immunol.9:294.

[0211] 11. Dellett M, Hu W, Papadaki V, Ohnuma S. 2012. Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance. Dev Growth Differ. Apr;54:327-340.

[0212] 12. Diniz LP, Matias I, Siqueira M, Stipursky J, Gomes FCA. 2019. Astrocytes and the TGF-betal Pathway in the Healthy and Diseased Brain: a Double-Edged Sword. Mol Neurobiol. Jul;56:4653- 4679.

[0213] 13. Donahue JE, Berzin TM, Rafii MS, Glass DJ, Yancopoulos GD, Fallon JR, Stopa EG. 1999. Agrin in Alzheimer's disease: altered solubility and abnormal distribution within microvasculature and brain parenchyma. Proc Natl Acad Sci USA. May 25;96:6468-6472.

[0214] 14. Dozen A, Shozu K, Shinkai N, Ikawa N, Aoyama R, Machine H, Asada K, Yoshida H, Kato T, Hamamoto R, et al. 2022. Tumor Suppressive Role of the PRELP Gene in Ovarian Clear Cell Carcinoma. Journal of Personalized Medicine. 12: 1999.

[0215] 15. Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N. 2010. Computer control of microscopes using microManager. Curr Protoc Mol Biol. Oct; Chapter 14:Unitl4 20. 16. Gaengel K, Genove G, Armulik A, Betsholtz C. 2009. Endothelial -mural cell signaling in vascular development and angiogenesis. Arterioscler Thromb Vase Biol. May; 29: 630-638.

[0216] 17. Gaillard PJ, de Boer AB, Breimer DD. 2003. Pharmacological investigations on lipopolysaccharide induced permeability changes in the blood-brain barrier in vitro. Microvasc Res. Jan; 65:24-31. 18. Gautam J, Zhang X, Yao Y. 2016. The role of pericytic laminin in blood-brain barrier integrity maintenance. Sci Rep. Nov 3; 6:36450.

[0217] 19. Davaapil H, 2017: The role of the proteoglycans OMD and PRELP in the maintenance of brain vasculature; Thesis submitted to University College London for the degree of Doctor of Philosophy.

Claims

Claims1. Proline / arginine-rich end leucine-rich repeat protein (PRELP) for use in a method of treating a disease characterized by increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage.

2. PRELP for use according to claim 1, wherein the increased neurovascular permeability is increased permeability of the blood-brain barrier, the blood-spinal cord barrier, the blood-ocular barrier, and / or the blood-retinal barrier.

3. PRELP for use according to claim 3, wherein the increased neurovascular permeability is increased permeability of the blood-brain barrier.

4. PRELP for use according to any preceding claim, wherein the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction.

5. PRELP for use according to claim 4, wherein the disease is selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

6. PRELP for use according to any preceding claim, wherein the increased neurovascular permeability is caused by an increase in endothelial cell to mesenchymal cell transition events at the blood-brain barrier.

7. PRELP for use according to claim 6, wherein the increased endothelial cell to mesenchymal cell transition events are caused by TGF-P mediated damage to cell-cell adhesion.

8. PRELP for use according to any preceding claim, wherein the increased neurovascular permeability is caused by or associated with administration of an immunotherapeutic agent.

9. PRELP for use in combination with an immunotherapeutic agent for treating increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage.

10. PRELP for use in treating increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage, wherein PRELP is for administration simultaneously, separately or sequentially with an immunotherapeutic agent.

11. PRELP for use according any one of claims 8-10, wherein the increased neurovascular permeability is caused by or associated with administration of the immunotherapeutic agent.

12. PRELP for use according to any one of claims 8-11, wherein the immunotherapeutic agent is an anti- Amyloid-P immunotherapeutic agent.

13. PRELP for use according to claim 12, wherein the anti-Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

14. A combination comprising PRELP and an immunotherapeutic agent for use in treating increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage associated with the administration of the immunotherapeutic agent, wherein PRELP is for administration before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent.

15. The combination according to claim 14, wherein the immunotherapeutic agent is an anti-Amyloid-P immunotherapeutic agent.

16. The combination according to claim 15, wherein the anti-Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

17. PRELP for use in a method of preventing blood-brain barrier breakdown and / or reducing blood-brain barrier leakage.

18. PRELP for use according to claim 17 wherein the blood-brain barrier breakdown or blood-brain barrier leakage is mediated by TGF-p.

19. PRELP protein for use in a method of maintaining or enhancing blood-brain barrier integrity.

0. PRELP for use in a method of treating TGF-P mediated neurovascular permeability or blood-brain barrier leakage.

1. PRELP for use according to any preceding claim, wherein PRELP is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of PRELP and a pharmaceutically acceptable carrier.

22. PRELP for use according to any preceding claim, wherein the production of PRELP is induced in a subject to be treated by gene therapy or cell therapy.

23. A method of treating a subject suffering from a disease characterised by increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage, comprising administering a therapeutically effective amount of PRELP to the subject.

24. The method of claim 23, wherein the increased neurovascular permeability is increased permeability of the blood-brain barrier, blood-spinal cord barrier, the blood-ocular barrier, and / or the blood-retinal barrier.

25. The method of claim 24, wherein the increased neurovascular permeability is increased permeability of the blood-brain barrier.

26. The method of any one of claims 23-25, wherein the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction.

27. The method of claim 26, wherein the disease is selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

28. The method of any one of claims 23-27, wherein the disease is caused by increased endothelial cell to mesenchymal cell transition events at the blood-brain barrier and / or the blood-CSF barrier.

29. The method of claim 28, wherein the increased endothelial cell to mesenchymal cell transition events are caused by TGF-P mediated damage to cell-cell adhesion.

30. The method of any one of claims 23-29, wherein the subject to be treated is a human subject.

31. The method of any one of claims 23-30 wherein the increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage is caused by or associated with administration of an immunotherapeutic agent.

32. The method of claim 31, wherein the immunotherapeutic agent is an anti-Amyloid-P immunotherapeutic agent.

33. The method of claim 32, wherein the anti-amyloid P agent is lecanemab or donanemab.

34. A method of treating or preventing increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage, the method comprising administering a therapeutically effective amount of PRELP to a subject in need thereof.

35. The method of claim 34, wherein the increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage is caused by or associated with immunotherapy, the method comprising administering an effective amount of PRELP to a subject in need thereof before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent.

36. The method of claim 35, wherein the immunotherapeutic agent is an anti-Amyloid-P immunotherapeutic agent.

37. The method of claim 36, wherein the anti-Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

38. A method of treating or preventing increased neurovascular permeability, neurovascular unit dysfunction or neurovascular leakage caused by or associated with an the anti-Amyloid-P immunotherapeutic agent, wherein the method comprises administering PRELP in combination with the anti-Amyloid-P immunotherapeutic agent.

39. The method of claim 38, wherein PRELP is administered before, simultaneously with, separately to, or sequentially with the immunotherapeutic agent.

40. A method of treating a disease characterized by abnormal neurovascular permeability, wherein the method comprises administering PRELP in combination with an immunotherapeutic agent, optionally wherein the immunotherapeutic agent is an anti-Amyloid-P immunotherapeutic agent.

41. The method of claim 40, wherein the disease is a neurological disease, a psychiatric disease, a neurodegenerative disease, dementia, an infectious disease, a metabolic disease, an inflammatory disease, a traumatic brain injury, a cancer, a seizure disorder, an autoimmune disease, or an adverse drug reaction.

42. The method of claim 40, wherein the disease is be selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, vascular dementia, Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia, multiple sclerosis, traumatic brain injury, concussion, hypoxia, hypercapnia, epilepsy, non-epileptic seizure disorders, stroke, transient ischemic attack, depression, brain tumour, metastatic brain cancer, diabetic retinopathy, meningitis, or encephalitis.

43. The method of any one of claims 40 to 42, wherein the anti-Amyloid-P immunotherapeutic agent is lecanemab or donanemab.

44. The method of any one of claims 40 to 43, wherein PRELP and the immunotherapeutic agent are for separate, simultaneous or sequence administration.

45. The method of any one of claims 23 to 44, wherein the disease is Alzheimer’s disease.

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