TARGETED NANOMEDICINE FOR TREATING VASCULAR DISORDERS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH:

[0001] This invention was made with government support under grant numbers R1H HL13S223 an EM DK !00505 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

Field of Invention

[0002] This disclosure relates to compositions and methods for treatin vascular disorders including, for example, arteriovenous fistula (AVF) failure, stenosis, restenosis, and atherosclerosi s .

Technical Background

[0003] Chronic kidney disease (CK.D) affects approximately 37 million Americans and is defined as foe gradual loss of kidney function. CKD can result front diabetes, high blood pressure, glomerulonephritis _* polycystic kidne disease (PKD), an other disorders that lead to kidney failure over time. CKD increases the risk of cardio vascular disease and mortality as kidney function decreases. End-stage renal failure, also known as end-stage renal disease (ESRP; also known as end-stage kidney disease or ESKD), is foe final, permanent stage of chronic kidney disease, where kidney function has declined to the point that the kidneys can no longer function on their own. There are >700,000 patients in he ITS with ESRD. Most are treated with hemodialysis via the vascular access of arteriovenous fistula (AVF), a direct anastomosis between a peripheral artery and vein, which allows for higher blood flow (J ?. _S via a larger diameter) into and out of the dialyzer, However, AVF failure rate cast be up to 60%, with costs for maintenance of vascular access averaging nearly S3 billion a year in the US, Moreover, there are currently ao marketed drugs for treating AVF failure; anti-platelet agents (aspirins, dipyridamole, ..etc ), anti-coagulant drugs (Warfarin, heparin... etc.) and amihyperteosive drugs (angiotensin receptor blockers, calcium channel blockers . etc.) have no clinically meaningful benefit in reducing AVF failure,

(00«4| Atherosclerosis is a chronic inflammatory disease of foe arterial wall that arises from an imbalanced lipid metabolism an a maladaptive inflammatory response, potentiated by the fluid mechanical stresses imposed on the endothelium. Atherosclerotic lesions are known to originate and develop preferentially at arterial sites of curvature, branches, and bifurcations, where complex hemodynamic conditions of disturbed blood flow are associated with chronically endoplasmic reticulum-stressed endothelial phenotypes expressing pro- inflammatory and pro-coagulant molecules. For example, the sines of the carotid bifurcation with Us recirculation is athero-susceptible whereas the nearby distal carotid artery, with straight streamlines, is athero-protected (Figure 1). Treatment methodologies for atherosclerosis include, .for example, percutaneous coronary intervention or coronary angioplasty when atherosclerotic plaques affect the coronary arteries. In the case of carotid blockage angioplasty and stenting can also be used.

[0005] The success of percutaneous coronary intervention has bee dramatically improved by placement of coronary artery stents. In percutaneous coronary intervention, the stenose coronary artery is dilated, and in stenting the mechanical support provided by the metal stent prevents elastic recoil Still, hyperplasia forming a neomtima within the stent leads to slow r egrowth of the stenosis, referred to as in-stent restenosis. This has been addressed in many patients by using drug-eluting stents, for example releasing the roTOR inhibitor everolimuS _j which is now widely used. However, for patients who are particularly susceptible, such as diabetics, current therapy remains unsatisfactory. Moreover, usage of drug-eluting stent is associated with very late stent throbosis (VLST), a potentially catastrophic complication.

[0006] MicreRN s and Nmomedicim »?l MicroRNAs (miRNAs) are small son-coding RNA molecules, approximately 1 - 26 nucleotides in length, that regulate biological gene expression in diverse biological processes, miRN As reconserved across organisms and regulate gene tar gets via hybridization to the 3 ^! t iran slated region (UTR) of messenger RNA, thereby blocking the translation of degradation of m ^: RN A targets. Altered miRNA expression i associated with multiple diseases an targeting of miRNA can be a treatment strategy in these eases,

10 001 Micelles are nanopaitieles formed, for example , by self-assembly of amphiphilic block copolymers with a hydrophobic core that can serve as a reservoir for drug delivery. Micelles are small in size, allowing for penetration into tissues. They exhibit m vivo stability and are efficient in solubilizing water insoluble drugs, making them potentially useful therapeutic deliver} ¹ tools. In this context, polyelectrolyte complexes forming micelles are the association complexes formed between oppositely charged particles ,{e.g , polymer-polymer, po lymer-dmg an polymer-drug-polymer) . |qqΐ>9| Vascular disease (including complications with A VP and athersc l.eros.is interventions, as discussed above) although a leading cause of death worldwide, are significantly underserved by the nano-material community, especially relative to cancer nanomedicine which receives vastly more attention. One unique feature of vascular diseases is that pathological vascular remodeling, such as: stenosis, typically occurs in specific sites of curvature, branching, and bifurcation where disturbed blood flows cause constitutive activation of vascular endothelium. For instance, disturbed flow-induced endothelial acti vation and vascular remodeling contribute to AVF failure in patients with ESRX> undergoing AVF creation lor hemodialysis. Targeted nanomedic ine can augment future treatment of vascular diseases b suppressing endothelial activation and inhibiting stenosis“regionally” in diseased blood vessels.

SUMMARY OF THE DISCLOSURE

(«0010] This disclosure describes compositions and methods for treatin vascular disorders, including, for example, arteriovenous fistula (AVF) failure, stenosis, restenosis, and atherosclerosis,

(OOOli I In a first aspect, the present disclosure provides a targeted naaopartiele, comprising an inhibitor of nncroRN A~ 2 miR~92a). In some embodiments of the first aspect, the targeted nanoparticle comprises a polyeleetfolyte micelle and a targeting molecule. In some embodiments, the polyeleetrolyte micelle comprises a polyethylene glycol (PEG) domain and a domain of positively charged amino acids. In some embodiments, tire PEG domain comprises P EG having an averag molecular weight of about 1 ,000 to about 100,000 Daltons in some embodiments, the domain of positively charged amino acids comprises repeats of lysine (K), arginine (R), and/or histidine (H). In some embodiments, ihe donutin of positively charged amino acids comprises repeats comprising about 2 to about 100 residues. In some embodiments, the the domain of positively charged amino acids comprises 30 repeats of lysine (K30). In some embodiments, the targeting molecule comprises a peptide comprising the amino acid sequence REKA (SEQ ID NO: 1 ), VHPKQHR (SEQ ID NO; 2), NNQ iVNIJ EK VAQLEA (SEQ ID NO: 3), DITWDQLWDLMK (SEQ ID NO; 4), CREICA (SEQ ID NO; 5), CGVHPKQHR (SEQ ID NO: 6). or GGSPGWVRCG (SEQ ID NO: 7). In some embodiments, the targeted nanoparticle comprises VHPKQHR-PEG-K30, CGVHPKQIIR-PEG-K30, NNQRJVNLKEICVAQLEA-PEG-KSO, DITWDQLWDLMIC- PEG-K30, REKA-PEG- O, CREKA-PEG- 0, or CGSPGWVRCG-PEG-K30. In some embodiments, the mlR-92a inhibitor comprises hsa-mIR-92a~3p. in some embodiments, the miR-92a inhibitor comprises a concentration of about 2 mM.

[00012] In a second aspect, tire present: disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a targeted naaoparticle comprising an raiR- 92a nhibitor and a pharmaceutically acceptable carrier, solvent, adjuvant and/or diluent. In some embodiments of the second aspect, the pharmaceutical composition further includes a secondary therapeutic agent in some embodiments, the secondary therapeutic agent comprises one or more of an anticoagulant, an irntiplatele agent, an angiotensin-eonvertiftg enzyme inhibitor, an angiotensin II receptor blocker, an angiotensin-receptor neprilysin inhibitor, a beta blocker, a calcium channel blocker, a cholesterol-lowering medication, a digitalis preparation, a diuretic, a vasodilator, an anti-niilannnatory medication, an IL~lb blocker, an ini!ammasome blocker, dehydroepiandrosterone sulfate, a myeloperoxidase inhibitor, a dipeptidyl peptidase-4 (DPP-4) inhibitor, a nitric oxide synthase activator, and/or a small GTPAse RhoA inhibitor. In some embodiments, the pharmaceutical composition is formnhued for oral, intravenous, topical, ocular, buccal, systemic, nasal, injection, transdermal, rectal, or vaginal administration in some embodiments, tito pharmaceutical composition is formulated for inhalation or insufflation

{00013] In a third aspect, the present disclosure provides a method of treating a vascular disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaeeuticaS composition comprisin a targeted nanoparticle comprising an miR-92a inhibitor. In some embodiments of the third aspect, the targeted nanopartfcle comprises a polyelectrolyte micelle and a targeti ng molecule . In some embodiments of the third aspect, the polyelectroiyte micelle comprises a polyethylene glycol (PEG) domain an a domain of positively charged amino acids. In some embodiments of the thi r aspect, the PEG domain comprises PEG havin an average molecular weight of about 1,000 to about 100,000 Daltons In some embodiments of the third aspect, the domain of positively charged amino acids comprises repeats of lysine ( ), arginine (R), and/or histidine CH). In some embodiments of the third aspect, the domain of positively charged amino acids comprises repeats comprising about 2 to about 100 residues. In some embodiments of the third aspect, the domain of posi tively charged amino acids comprises 30 repeats of lysine (K30). In some embodiments of the third aspect, the targeting molecule comprises a peptide comprising the amino acid sequence REKA (SEQ ID NO: i), VHPKQHR (SEQ ID NO; 2), NNQXiVNLKE VAQLEA (SEQ ID NO: 3), D!TWDQLWDLMK. (SEQ ID NO: 4), CREKA. (SEQ ID NO: 5), CGVHPKQHR (SEQ ID NO: 6), or CGSPGWVRGG (SEQ ID NO : 7). in some embodiments of the (bird aspect, the targeted nanopartieie comprises VHPKQHR-PEG-K30, CGVHPKQHR-PEG- O, NNQKIVNLK.EICVAQLEA-PEG-K30, DiTWDQLWDLMK-PEG- O, REKA-PEG-RJO, GREK A-PEG-K3i), or CGSRGWVRGG- PBG-K30. In some embodiments of the third aspect, the miR-¾a inhibitor comprises hsa~ miR-92a-3p. In some embodiments of the third aspect, th miR-92a inhibitor comprises a concentration o f about 2 mM.

{00814] In a fourth aspect, the presentdisclosure provides a method of treating a vascular disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a targeted nanopartieie comprising an niiR _^ 92a inhibitor, wherein the targeted nanopartieie is preferentially targeted to inflamed endothelial cells associated with the vascular disorder an reducing inflammation at the site of the inflamed endothelial cells. In some embodiments of she fourth aspect, the vascular disorder comprises one or more of arteriovenous .fistula (AVF) iMIure, stenosis, restenosis, and atherosclerosis . In some embodiments of the fourth aspect, the method results in one or more of greater lumen cross-sectional area, greater lumen diameter, or increased flow rate compared to a control at the site of the inflamed endothelial cells f 00015] In a fifth aspect, the present disclosure provides a method of promoting endothelial wound healing in asubject comprising administering to fob subject a therapeutically effective amount of a pharmaceutical composition comprising a targeted nanopartieie comprising an miR-92a inhibitor, wherein the targeted nanopartieie is preferentially targeted to inflamed endothelial cells associated with the endothelial wound and reducing inflammation at the site of the inflamed endothelial ceils. In some embodiments of the fifth aspect, the method further includes stimulating endothelial growth at the site of the wound.

1000161 These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompany ing claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in tire present description. BRIEF DESCRIPTION OF THE BRA WINGS [00017] The accompanying drawings are included to provide a further understanding of the methods and cornpositions of the disclosure _* and are incorporated in and constitute a part of this specification The drawings illustrate one or more embodiments) of the disclosure, and together wi th the descripti o serve to explain the principles and operation of the disclosure.

100018] Figure 1, Human carotid bifurcations with carotid sinus with recirculation (arrow)., which is aihero-susceptible, Other portions of the bifurcatio experience linear laminar flow, which is athero-proteeti ve.

[QQ0I9] Figure 2. Brachiocephalic fistula for hemodialysis. The cephalic arch and arteriovenous anastomosis wham endothelial cells are constantly activated by locally disturbed flow are sites most susceptible to stenosis and thrombosis, leading to arteriovenous fistula (AVF) failure.

100020] Figure 3, AVF maturatio requires limited inward remodelin and sufficient outward remodeling,

[60021 ] Figures 4A-4B. Flow at atheroselerosis-proRe carotid artery bifurcation (4 A) Aberrant blood flow; (4B) unregulated raicroRNA-92a (raiR-92a) in cultured endothelial ceils (ECs) as shown by the ratio of miR~92a/U6, miRN A RT~PCR (W u et ai Circulation 2011). PS; pulsatile shear flow (linear laminar flow); OS: oscillatory shear flow (dishibed flow)

100022] Figures 5A-5B. Disturbed flow in human (5A) brachiocephalic arteriovenous anastomosis an (5B) cephalic arch alter creation of arteriovenous fistula (AVF), which is similar to disturbed flow at aiherosclexosis-proue carotid artery bifurcation (Figure 4 A) [60023] Figure 0, miR-92a down-mgn fes Sirtuin 1 (SlRTl), KfUp eFlike factor 2 (KLF2), KruppeMilie factor 4 (KLF4), and endothelial nitric oxide synthase (eNOS), which leads to decreased flow-mediated dilation (FMD),

[00024] Figure 7. Association of preoperati ve FMD with 6-week AVF diameter and blood flow Preoperative FMD was positively associated with AVF maturation (Alien et al 1ASN, 2016),

[00023] Figure 8, Serum miR~92a levels were negatively associated with FMD in patients with coronary artery disease (Chen et al. Circulation 2015). [00026] Figures 9A~98. Relative serum miR-92a levels were increased in chronic kidney disease (CKD) patients (9A) and negatively associated with FMD (9B, Shang & gl. JASM, 2017).

[00027] Figures 10A-1QB. Relative CKD tnjR~92a serum levels. ( tOA) CKO npregulated relative iR~92a serum levels compared to control; (10B) normalised niRNA expression of endothelial protective genes in CKD serum-treated cultured ECs was observed with decreased miR-92-a levels by miR-92a inhibitors (arsti-miR92a) (Shaog etal JASN, 2017). {001)28] Figure 11. Hypothetical role for miR~92a in AVF maturation; miR-92a leads to decreased expression of endothelial protective genes which causes increased inward AVF remodeling and decreased outward AVF remodeling, which contribute t AVF failure. [00029] Figure 12, Workin hypothesis a combination of CKD and AVF blood flow increases miR-92a expression causing AVF maturation failure.

[0003Q] Figu e 13. Study design for rat study,

[00031] Figure 1 Modified, low-dose dietary adenine- induced CKD and AVF creation timeline, as described in Longer et at Kidney International 2010.

[Q0032] Figures 15. CKD rats have elevate serum mIR-92a levels (Shang et «/., JASN 2017), as seen in CKD patients.

[00033] Figure 16. Study design for mouse study.

[00034] Figure 17. Schematic of proposed effect of miR-92a inhibition on AVF de velopment using whole- body gene kn ock out.

[00035] Figures 18A-18B. Mouse carotid-jugular AVFs were created in iR-92a knockout (KO) and wild-type (WT) C57BL/6 mice, as described in Chun etal. J Vis Ex 2016. MiR-92a KO decrease NH in A VF veins, (18 A) Percent area of stenosis by NH (top) and Percent area of open lumen (bottom); (18B) Q«iward-to-inwatd remodeling ratio.

[00036] Figure 19. Schematic of proposed effect of iR-92a inhibition of AVF development using targeted nanomedicine,

[00037] Figures 20A-2QB. Dual-function nanoparticies (MP$J encapsulate miR inhibitor and target inflamed endothelial ceils. Vascular cell adhesion molecule (VCAM-i) presence in CKD rat tissue (20 A) Non-surgieal femoral artery; (20B) AVF artery. Dark color ^~

VC AMI narrow indicate endothelial cells.

[00038] Figure 1, Dual-function NPs encapsulate miR inhibitor and target inflamed endothelial cells. Assembly and structure of VC AM- 1 targeting nanoparticies wit miR inhibitors in the core. The peptide targeting VCAMI has the sequence VHPK.QHR (SEQ ID NO: 2).

(00 3$} Figure 22. Negatively stained TEM image of micelles formed via eooipiexatien ofREKA-PEG- O and miR-92a inhibitor. PEG is polyethylene glycol.

(00040] Figure 23. Preferential accumulation of VCAM- 1 -targeted mR-92a inhibitors to aorta in vivo.

100041] Figures 24A-24C, Comparison of control, naked and NP-eneapsuiated miR-92a inhibitor in treating neointhnai hyperplasia. Nsnoparticle-eneapsulated miR inhibitors enhance AYF remodeling when compared to controls and naked inhibitors via a reduction i venous neoinuma! hyperplasia and promoted venous lumen expansion. (24A) morphometric analysis of Open lumen area; (24B) Percent area of Open lumen; and (24C) percent area of stenosis by neoin timal hyperplasia (bottom) PBS (Filled circle), naked inhibi ors (ope square) and nasopnrtteie-encapsulafed inhibitors (shaded triangle).

100042] Figure 2 S. Study design for Bioinforraatics study

(00043] Figures 20A-26C, Bioinformaiics study. (26A) Enrichment analysis suggested pathway differences between AWs in miR 92a KQ vs WT mice; (26Bj Network analysis suggested interactions among gene group 1 and other genes; (26C) Gene group 1 is associated with the extracellular matrix and protease activity.

|00044] Figures 27A-27C. A o B ^" mice (27A) were led a high fat diet at week sixteen.

At eighteen weeks, a tail vein injection was performed of either naked miR-92a inhibitor or the raiR-92a inhibitor encapsulated in a VCAM i -targeted micellar nanoparticle, In both cases, a dose of 8 mg/kg or 4 mg/kg of body weight was administered. At twent weeks, the mice were euthanized and the size (-area) ofatherosclerotic lesions in the aortic root was measured. Data from injections of a dose of 8 mg/kg are shown in 27B. Data from injections of a dose of 4 mgik ate shown in 27C. 1 ,0 on the normalized vertical axis is the average size of the lesions in the mice that received injections of phosphate buffered saline (PBS ) shownin column 1. Column 2 shows results for injection of the naked inhibitor. Column 3 shows th results for injection of the micellar nimopartieie-deiivered control oligonucleotide of the same nucleic acid compositio in a scrambled sequence targeted to tissue displaying VCAM1, an indication of local inBammation. Colum 4 shows the results for the injection of the micellar natioparlicle-deilvere mIR-92a i nhibitor targeted to tissue di splay i ng VCAM I .

(0004S] Figures 28A-28C. (28A) 16-week old Apo E mice were fed a high fat diet and subjected to carotid partial ligation in the left carotid. This surgical procedure (Nam et al. A. 1. Phys Heart, 2009) introduces disturbed blood flow and induces stenosis in left carotid artery in 14 days. (28B) Three days after the partial carotid ligation, tail vein injection was conducted of either naked miR 92a; inhibitor or the raiR-i)2a inhibitor encapsulated in a VCAMI -targeted micellar nanopar kle. In both cases, a dose of 2 mg/kg of body weight wasadministered. Two more injections were conducted on days 6 and 9 after the partial carotid ligation. Mice were sacrificed 14 days after the partial carotid ligation to measure the stenosis in the partially-ligated left carotid arteries. (28C) Vertical axis is the average size of the stenosis in the partially-ligated carotid artery from the mice that received injections of phosphate buffered saline (PBS) shown in column 1 Column 2 shows the results for injection of the naked rnsR-92a inhibitor. Column 3 shows the results for injection of the micellar nanoparticle-delivere control oligonucleoii.de of the same nucleic acid composition in a scrambled sequence targeted to tissue display ing VCAMI _< an indication of local inflammation. Column 4 shows the results lor the injection of the micellar nanoparticle- delivered miR-92a inhibitor targeted to tissue displaying VCAMI. Representational cross- sections of each treatment group are shown below the data chart

DETAILED DESCRIPTION

[00046] Provided herein are compositions and methods for treating vascular disorders, including, for example, arteriovenous fistula (A VT) failure, stenosis, restenosis, an atherosclerosis. As used herein, the term “vascular disorder” refers to disorders, diseases, and/or damage to the vascular s stem of an individual The vascular system, also known as the ciculatory system, Includes the vessels (e.g., arteries, veins, capillaries, and lymph vessels) that cany blood and lymphatic fluid throughout the body.

[00047] It is to be understood that the particular aspects of the specification are described herein are not limited to specific embodiments presented, and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, withou t limitation

[00048] Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “Including”} will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of my other component;, feature, element, or step or group of components, features, elements, or steps. Any of the ten»s: ^s ‘comprising, ^M “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms, while retaining their ordin l meanings. 0O«49] As used herein, the singular forms “a ” “an;' and “the” include plural referents unless the context early indictates otherwise.

|0b05b] In some embodiments, percentages disclosed herein can vary in amount by AH),20 or 30% from values disclosed and remain wi thin the scope of the contemplated disclosure.

[QQ05I I Unless o therwis indicated or otherwise evident from the contex t and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unitof the lower limit of the range, unless the context clearly dictates otherwise.

{00052] As used herein, ranges and amounts cm be expressed as “about” a particular value or range. About also includes the exact amount For example, “about 5% ’ means “about 5%” and also “5% A The term “aboit” can also refer to 10% of a given value or range of values. Therefore, about 5% also means 4,5% - 5.5%, for example

|00iS3{ As used herein, the terms “or” and “and/or” are milked to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or x” can refer to *V alone, “y” alone, V alone, “x, y, and x,” “( and y) or ” “x or (y and x},” or “x or y or z ^"

[00054] “Pharmaceutically acceptable ⁵ ' refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio or which have otherwise been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animats. [00055] “Therapeutically effective amount” or “effective amount” refers to that amount of a therapeutic agent, such as an raiR-92A inhibitor, which when administered to a subject, is sufficient to effect treatment (e.g., improve symptoms) for a disease or disorder described herein, such as, for example, A VP failure, stenosis, restenosis, or atherosclerosis. The amount of it compound which constitutes a “therapeutically effecti ve amount” or “effective amount” ea» vary depending on the compound, the disorder and its severity, and the age, weight, sex, and genetic background of the subject to he treated, but can be determined by one of ordinary skill in the art.

[00856] "Treating” or "treatment” as used herein refers to the treatment o f a di sease or disorder describe herein, in a subject, preferably a human, and includes: inhibiting, relieving, ame liorating, or slowing progression of the di sease or disorder or one or more symptoms of the disease or disorder

{00857] “Subject” refers to a warm blooded animal such as a mammal, preferably a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and disorders described herein.

[00058] "Pharmaceutical composition” as used herein refers to composition that includes one or more therapeutic agents disclosed herein, such as an mlR-92A inhibitor, a pharmaceutically acceptable carrier, a solvent» an adjuvant, and/or a diluent, or any combination thereof

{08859] As used herein, the term “vascular disease” can also be described as a "vascular disorder.”

(00060] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinaryskill in the art to meet the desired need. In general, the disclosed materials and methods provide improvements in treating vascular disorders as described herein.

OVERVIEW

{00061] Cardiovascular diseases un desired vascular remodeling, and obstruction of blood vessels are potentiated by disturbed blood flow such as vortex formation and other manifestations of local turbulence and flow complexity, interacting with other factors such as imbalanced lipid metabolism and uremia. Disturbed blood flow opregulates the production of a n croRNA, ro¾-92a, 'which increases local inflammation, and romotes local,pathological vascular remodeling and stenosis. Disclosed herein are engineered nanoparticles that can locally deliver an inhibitor against miR-92a that retards the development of localized atherosclerotic lesions in ApoE knockout mice. The nanoparticles also inhibit pathological vascular remodeling in arteriovenous fistuke (A VP) in mice. It is further contemplated that the inflammation targeting nanopariicles carrying an mi _: R~92a inhibitor are also effectiveat

-i l treafing and/or preventing restenosis, which often occurs after insertion of a stent to relieve coronary artery blockage, for example, and afterangioplasty.

(00062] Moreover, the present disclosure demonstrates that this targeted nanopartiele significan tly reduced vascular stenosis, which is a narrowing of the arterial lumen that disrupts local bloo flosv and leads to a wide range of vascular diseases in the brain, heart, and legs.

100063] The preferred vascular access for hemodialysi is the arteriovenous fistula (AVF) that is surgically created by a direct anastomosis between a peripheral artery' and vei (Figure 2). Many AVFs fail to mature suffteiendy for adequate dialysis due to vascular stenosis (Figure 3 ). The failure rate of newly create AVF varies from 25-60%, with the most common causes of A VF fail ore being pathological vascular remodeling,, insult to the endothelial layer, stenosis, and thrombosis. The cephalic arch where endothelial cells are constantly activated by locally disturbed flow is one of the sites most susceptible to stenosis slid thrombosis, leading to AVF fail ure. The annual cost of treating vascular access dysfunction totals over one billion dollars in the Unite States of America, largely due to new AVF creation, interventional procedure with angioplasty aid stent placement, as well as complications from prolonged catheter use. There is currently no pharmacological treatment fcr AVF failure, representing a significant unmet clinical need.

{ 0064] MicroR As are small ncn-eoding RNA molecules known to regulate pathological processes related to endothelial ceil function and cardiovascular health. MicroRN As (miRs), includin raiE-92a, are thought to play a role in arteriovenous fistula maturation. (Figures % 3, 6 and 12). Studies have shown that serum miR-92a levels were negatively associated with FMD in patients with coronary artery disease (Chen et aL Circulation 2015). Studies have also shown that preoperative FMD was positively associated with AVF maturation (Allon et aL JAS ,2016),

{00065] Studies have shown that die CKD milieu increases serum miR-92a levels in patients and that serum miR--92a was likely derived front endothelial cells (EC’s) (S mg ei el. JASN, 2017) ECs cultured with CKD serum increased their miR-92a expression and had reduced levels of several molecules critical for maintaining endothelial homeostasis, inclu ing smuio-l , Krappel-Kke factors 2 and 4, and endothelial nitric oxide synthase (Figure 10), which can lead to a decreased flow mediated dilation (Figure 6). Inhibiting raiR-92a rescued those vasoprotective molecules in cultured ECs in the CKD milieu (Figure 10). [00066] To date, there a three clinically relevant delivery platforms for modulating gene and miRNA expression «sing therapeutic nucleotides: (i) naked therapeutic nucleotides, (ii) lipid nanoparticles, and (Hi) conjugate nucleotide systems. Yet they suiter from moderate to serious disadvantages, including rapid degradation, immunogenicity, poor circulation half- life, high cytotoxicity, and poor cellular uptake.

[00067] The significance of the present disclosure includes at least two aspects. First, it provides novel nanomedicine approaches to treat vascular disorders with unmet medical need. Second, it integrates targeted nanomedicine and RNA therapeutics to creat a new avenue ^" for the treatment of various vascular diseases includin stabilization oi ^' AFV or vein grads, restenosis, and atherosclerosis. This disclosure further provides, in pari, peptide- ^: targeted polyelectrolyle complex rising micelles to deliver therapeutic nucleotides to vascular cells, promoting AVF maturation and reducing AVF failure, reducing restenosis, and reducing atherosclerotic lesions.

[00068] Compositions

[QQ06.9] In some embodiments, pharmaceutical compositions contemplated herein include therapeutically effective amount of a targeted nanoparticle including one or more inhibitors of endothelial inflammation, such as, for example, an miR-92a inhibitor. Such compositions may further include an appropriatepharmaceutically acceptable carrier, solvent, adjuvant, diluent, or any combination thereof The exact nature of the carrier, solvent, adjuvant, or diluent will depend upon the desired use (e.g , route of administration) for the composition, and may range from bemg sultable or acceptable for veterinary uses to being sui table or acceptable for human use,

[00070] In some embodiments, pharmaceutical compositions contemplated herein include one or more nanoparticles that cam- the one or more miR-92a inhibitors, for example, inside the nanopartkle, atached to an external surface of the nanoparticle, or both. In some: embodiments, the nanoparticles include one or more targeting moeities attached thereto to en able targeted del iver of the nauopartiele to a desired location. For example, the targeting moeity can target the nanoparticle to a site of endothelial inflammation associated with a vascular disease or disorder or wound.

[00071] Any miR-92a inhibitor is contemplate herein. For example, contemplated ttiR- 92a inhibitors include those available from Dharmacon. Other contemplated m:iR-92a inhibitors include custom niiRIDIAM Hairpin, Inhibitor (hsamlR-92a~3p. MIMAT0000Q92; RefiffH-300510-06), (00072] Such compositions optionally include secondary therapeutic agents (possibly also carried on or in contemplated nanoparticles).

(00073] miR-92a inhibitors of the present disclosure can be administered through a variety of routes and in various compositions. For example, pharmaceutical compositions containing miR~92a inhibitors can he formulated for oral, intravenous, topical, ocular, buccal, systemic _* nasal, injection, transdemial, rectal, or vaginal administration, or formulated in a form suitable for administration by Inhalation or insufflation. In some embodiments of the present disclosure, administration is oral or intravenous.

(00074) A variety of dosage schedules is contemplated by foe present disclosure. For example, a subject cart be dosed monthly, every other week, weekly, daily, or multiple times per day. Dosage amounts and dosing frequency can vary based on the dosage form and/or route of administration, an foe age, weight, sex, and/or severity of the subject ’s disease. In some embodiments of the present disclosure, _. One or more miR- 2a inhibitors is administered orally, and the subject is dosed on a daily basis.

|0UO75j The therapeutic agents (also referred to a “compounds” herein) described herein (e.g., mlR-92a inhibitors and secondary therapeutic agents), or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example, in an amount effective to provide a therapeutic benefit to subject having the particular disease being treated. As used herein, therapeutic benefit refers to the eradication: or amelioration of the underlying disease being treated and/or eradication or ameli oration of one or more of th symptoms associated with the underlying disease such that asubject being treated with the therapeutic agent reports an improvement in feeling or condition, notwithstanding that the subject may still be afflicted with the underlying disease.

(00076) Non-limiting examples of contemplated secondary therapeutic agents include those fo can promote vascular health, inhibit vascular inflammation, promote endothelial health, suppress smooth muscle proliferation/restenosis _* reduce thrombosis, reduce oxidate stress, suppress smooth muscle pfoiiieratiots/restenosis, reduce excessive, abnormal, or imbalanced degradation and synthesis of extracellular matrix, an promote foe health and functional phenotype of endothelial cells and vascular smooth muscles. Examples of agents to promote vascular health can include anticoagulants (blood thinhers), antiplatelet agents, angiotensin-converting enxyme (ACE) inhibitors, angiotensin II receptor blockers, angiotensin-receptor neprliysin inhibitors, beta blockers, calcium channel blockers, cholesterol-lowering medications, digitalis preparations, diuretics, and vasodilators. Further therapeutic agents contemplated for use herein include atrtMnflanuitatoiy medications. Still further therapeutic agents contemplated for use herein include IL~Ib blockers an infla masorae blockers. Other example of inhibitors of vascular inflammation include dehydroepiandrosierorie sulfate, inhibitors of myeloperox idase, inhibitors of dipeptidylpeptidase-4 (DFP-4}, inhibitors of inflammasorae, ac tivators of nitric oxide synthase, and inhibitor of small GTPAse RhoA.

|000?7] Determinat ion of an effective dosage of compound^} for a particular disease and/or mode of administration is well known. Effecti ve dosages can be estimated initially from in vitro activity and metabolism assays. For example, a initial dosage of compound for use in a subject can be formulated to achieve a circulating blood or serum concentration of tire metabolite active compoun that is at or above an IC Of the particular compound as measured in an tin vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via a given route of administration is well within the capabilities of a skilled artisan. Initial dosages of compound can also be estimated from in vivo data, such as from an appropriate animal model.

(06078] Dosage amounts of miR-92a inhibitors and secondary therapeutic agents can be in the range of from about 0.0001 mg kg/day about 0,001 mg/kg/day, or about 0.01 mg kg/day to about ! 00 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavailability of the compound, i ts metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, including particular condition being treated, the severity of existing or anticipated physiological dysfunction, the genetic profile, age, health, sex, diet, and/or weight of the subject. Dosage amounts and dosing intervals can he adjusted individually to maintain a desired therapeutic effect over time. For example, the compounds may be administered once, or once per week, several ti es per week (e.g„ every other day), once per day or multiple times per day, depending upon, among other tilings, the mode of administration, the specific indication being treate and the judgment of the prescribing physician. In cases of local administration or select! ve uptake, such as local topical admhii strati on, the effective local concentration of compound(s) and/or active metaboli e co potmd(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation. QQ079] For example, a dosage contemplated herein can include a single volume of about 0.1, 0.2, 03, 0.4. 0.5, 0.6, 0.7, 0.8, 0.9, 1 0, 1.5, 2.0, 2.5, or 3.0 mL of a pharmaceutics] composition having a concentration of a tniR-92a inhibitor at about OiffiOOL 0001 , 0.001, 0.01, 0,05, 0,1 , 0.2, 0.3, 0,4, 0,5, 0.6, 0.7, 0.8, 0.9, 1.0, 1 5, 2.0, 2.5, 3,0, 3.5, 4.0, 4.5, 5.0, 10, 15, 20, 50, 100, 200, 500, or 1000 mM in a pharmaceutically acceptable carrier. OO080| anoparticles

100081] The present disclosure contemplates use of polyeleetrolyie micelles (also referred to as nanoparticles herein) to deliver therapeutic agents. Polymers that b ar charge in an aqueous environment are called polyelectroiytes When oppositely charged polymers are mixed under the right conditions, _: they form complexes. Folyelectrolytes, including at least one attached to a non-charged water soluble block, can be mixed at a stoichiometric charge ratio with an oppositely charged homopolymer to form panicles of a relatively compact core surrounded by a dilute corona of neutral water soluble block. These nanometer-sized particles are calle polyelecirolyte complex micelles, polyion comple micelles, interpoiyeieclro!yle complex micelles, complex eoacervais core micelles, or polyelecirolyte micelles.

Polyeleci rolyte complexes composed of nucleic acid and positively charged polymers have been explored as a possibility to neutralize the charge on the molecule and protect it from enzymatic degradation, Polyelecirolyte complex micelles have great potential as gene delivery vehicles because of their ability to encapsulate charged nucleic acids, forming a core by neutralizing their charge, while simultaneously protecting the nucleic acids from nonspecific interactions and enzymatic degradation. Furthermore, to enhance specificity and transfection efficiency, polyelecirolyte comple micelles can he modified to include targeting capabilities.

|O0082] The contemplated polyelectrolyte micelles can comprise polyethylene glycol (PEG) domains as well as domains of positivel charged amino acids (e,g , repeated positively charged amino acids) PEG domains prevent macrophase separation, stabilizing the micelles. The domains further protect the nanoparticles from recognition by the reticuloendothelial system in the body . The PEG domain can be comprised of PEG having an average molecular weight of about 1,000 to about 100,000 Daltons (Da).

|00i83] The micelles can comprise a domain of positively charged amin acids, such as lysine, arginine, and/or histidine. This domain can complex with negatively charged therapeutic agents, such as miRNA inhibitors. The domain of repeated positively charged amino acids can include about 2 to about 100 residues QQ884] Contemplated nauopartides for use herein include, for example, polyelectrolyte complex micelles thatcan effectively incorporate negatively-charged nucleotides in the core and functionally display tissue-targeting peptides on the surface. These sell-assembled nanoscale carriers ( -20 ran in diameter) are formed by electrostatic interaction between two oppositely-charged polymers. Polyethylene glycol (FEGJ-20ftft was conjugated with poly- lysine on one side to form -positively-charged building blocks and conjugated with targeting peptides on the other side to functionalize foe nanoparticles to bind specific- eel! membrane molecules , Negatively-charged nucleotides are neutralized bypoly-lysine and encapsulated in the cores of the nanoparticles. This approach offers multiple advantages, including; (i) the nano-scale of micelles significantly increases the surface area ; volume ratio that can enhance specific targeting, and (ii) the self assembling feature of the polyelectrolyte micelles eliminates the use of chemical cross-linking agents, thereby re ucing possible toxicides ftiTOSSl Additional micelles are contemplated for use herein, such as those disclose in International Application No, PCT/U$2006/920760, Vieregg et al (I. Am, C!iem. Soc. 2018, 14ft, 1632-1638}, Lueekheide et al. (Nano Lett 2018, I S, 7111-7117), and Marras et al. (Polymers 2019, 11, 83), each of whic is incorporated by reference.

|00086) Targeting Molecules

{00087 j The present disclosure contemplate use of targeting molecules (or targetin moieties) with foe nanoparticles disclosed herein for targeted delivery of therapeutic compositions, such as nriR-92a inhibitors, or for incorporation into pharmaceuticalcomposition® as described herein. Targeting molecules can include peptides such as CGVHP QHR CSEQ ID NO; 6} or VHPKQHR (SEQ ID NO: 2), which were identified via phage display and allows for targeting of vascular endothelial cells through VCAM-L Peptide targeting molecules further include the amino acid sequence -NNQKiVN.LKEKVAQL.EA (SEQ IB NO: 3), which allows for the targeting of intercellular adhesion molecule 1 (ICAM-I). 1C AM- 1 is a cell surface glycoprotein typically expressed on endothelial cells. Peptide targeting molecules further includ foe amino aci _: sequence DITWDQL WDLMK (SEQ ID NO: 4), which allows for foe targeting E-selectis. E-selectin is a cell adhesion molecule expressed only on cytokine-activated endothelial cells. In another embodiment, foe targeting molecule is a peptide comprising the amino acid sequence REftA (SEQ ID NO: I) or the peptide comprising the amino acid sequence CREKA (SEQ ID NO: 5), both of which bin fibrin. CGSPGWVRCG (SEQ ID NO: 7) is a peptide identified by phage display to bind specifically to lung endothelial cells and can be displayed on this nanoparticie.

[00988] The expression of vascular cell adhesion molecule i (VCAMi) is low in healthy endothelium but increases i inflamed endothelial cells (ECs). To achieve effective targeting to VCAMI -expressing ECs, the micelles are fbnetlonahzed with a: VC AM I binding: peptide that has been shown to facilitate VCAMI -mediated intracellular internalization of nano- materials in endothelium in viiro and in vivo (Figure 21). in some embodiments, contemplated targeting peptides are posi tione at the periphery of the corona of thenanoparticles

[00089] In one embodiment, a contemplated targeted nanoparticie containing an mIR-92a inhibitor (2 mM) is VHPKQHR-PEG-K3Q In one embodiment, a contemplated targeted nanoparticie containing an miR-92a inhibitor (2 mM) is CGVHPKQHR-PEG-K30 in one embodiment, a contemplated targeted nanoparde!e containing an miR-92a inhibitor (2 mM) is NNQKIVNLKEKVAQLEA-PEG-K30. in one embodiment, a contempiated targeted nanoparticie containing an raiR.~92a inhibitor (2 mM) is DiTWDQL WDLMK~PEG-K30 In one embodiment, a contemplated targeted nanoparticie containing a miR-92a inhibitor (2 mM) is REKA-FEG-K30. In one embodi ment, a contemplated targeted nanoparticie containing an mi R~92a inhibitor (2 mM) is CREKA-PEG-K30. In one embodiment a contemplated targeted nanoparticie containing an miR-92a inhibitor (2 mM) is CGSPGWVRCG -PEG-K30.

(000991 lu some embodiments, contemplated nanoparticles containing an mi.R-92a inhibi tor exhibit a polydispersity of about 0.1 to about 0,3,

[00091] in some embodiments, contempiated nanoparticles containing an nhR-92a inhibitor contained with the core exhibit a spherical shape and have a diameter (in nanometers, n ) of about 10, 11, 12, 13 _» 14, 13, 16, 17 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, or 50 nm.

[00092] The nanoparticie elivery system of the present disclosure possesses multiple advantages compare with other nanoparirde-hased platforms. The first advantage is higher stability the therapeutically active components (e.g., mrR-92a inhibitors or other nucleic acid based therapeutics) can be encapsulated in the inner core of the polyeleetrolyte complex micelles and can therefor be protected by the outer layer of bioconipatihle polymers. Through this approach, first, the degradation of rntcleotkles of an miR-92a inhibitor by serum nucleases is prevented; second, the micelles ar capable of escaping renal clearance; and third, the immunogenic responses are avoided. The second advantage is higher safety: cell- targeting peptides:(e,g., (hose targeting to Vascular Cell Adhesion Molecule I (VCAM-l)) are covalen tly conjugated on the periphery of the polyeleetrolyte complex micelles, which significantly reduces cytotoxicity and increases circulation time by circumventing nonspecific interaction with serum components. The third advantage is higher specificity: with the defined chemical structures of the targeting peptides, the polyeleetrolyte complex micelles are able to bind specific receptors and penetrate targeted eells.The final advantage is higher scalability, ^' This approach does nor require chemical modifications on nucleotides for conjugation, nor does if need to engineer hard-to-reprodttce lipid nanoparticles. The synthesis of the core components in the micelles is highly automated. In addition, the targeting peptides are easily changeable to target different receptors.

|M993] Further, as shown herein, the use of targeted nanoparticies permits use of a loweramount of a therapeutic agent for the treatment: of a vasculardisorder or wound due to tire specific targeting of the therapeutic agent to the si te of the vascular disorder or wound. In ibis way, use of targeted nanoparticies can significantly lower the dosage of a therapeutic agent required to treat a vascular disorder or wound, which can significantly reduce costs associated with the treatment. For example, a therapeutically effective amount of a therapeutic agent to he delivered by a targeted nanoparticle can he at least about 10, 20, 30, 40. or 50% lower than the therapeutically effective amount of the naked (ήόh-targeted) therapeutic agent.

100094] Methods

I0009S] In some embodiments, methods of treating and/orpreventing a vascular disorder in subject in need thereof include administering to the subjec a therapeutically effective amount of one or more miR-92a inhibitors and optionally a secondary therapeutic agent. Treatable and/or preventable vascular disorders can include arteriovenous fistula (AVF) failure, stenosis , restenosis, and atherosclerosis .

{00096] In some embodiments, therapeutic methods contemplated herein can also treat and/or prevent complications associated with or promote endothelial wound healing (e.g., caused by trauma or surgery) by administering to the subject a therapeutically effective amount of one or more miR-92a inhibitors and. Optionally a second therapy and/or secondary therapeutic-agent. For example, treatment and/or prevention of complications associated with endothelial wound healing is associated with the reduction of inflammation at the site of the wound and the stimulation of endothelial growth. |qqΐ>97] I» some embodiments, therapeutic methods contemplated herein can also accelerate endothelial growth to treat wound healing (e.g., caused by trauma or surgery) by administering to the subject a therapeutically effecti ve amount of one or more miR-92a inhibitors and optionally a second therapy and or secondary therapeutic agent,

AVF failure addressed by the presen t disclosure can be of multiple types. These types can include maturation failure, in which a newly created AVF does not mature sufficiently (such as does not have adequate open lumen size or blood flow rate) to be used for dialysis. The failure can also be one of durability , whic means that an AVF is able to h used for dialysis, hut then later on develops problems (such as stenosis) and cannot be used. These: failures can result from pathological vascular remodeling, insult to the endothelial layer, stenosis, and thrombosis,

[90099] The AVFs contemplated by the present disclosure can be of a variety of subtypes. The subtypes can include forearm AVF (e.g,, snuff-box* distal ra focephalie or transposed radiobasilie), proximal forear AVF (e.g., proximal diocephalic, perfortor-combinations), brachiocephalic A VF, the brachial attery Ao-transpose basilic vein fistula, and lower extremity A VF.

[0Q01Q0I The present disclosure contemplates a variety of methods of administering the therapeutic agents, targeting molecules, and micelles disclosed herein, including local, oral, nasal, rectal, infra vaginal, topical, subcutaneous, intradermai, intramuscular (1M), intravenous (IV), intrathecal (IT), intracerebral, epidural, or intracranial administration. Local, in siiu administration of these compositi ons is contemplated.

[000101] The present disclosur contemplates methods that result in a variety of indications of improvement for the AVF. These indications can include a greater lumen cross-sectional area, a greater lumen diameter, and/or increased flow rate.

[0001.921 The present disc l osure contemplates use of the disclosed methods in conjunction with other treatments for failure of AVF. These other treatments can include percutaneous transluminal angioplast or endovascular dec-lotting techniques. Other inventions are contemplated, such as out-patient interventional procedur with angioplasty and/or s tent placement. EXAMPLES

[000103 The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.

Example 1: Formation and characterization of micelles.

[ 0 1041 introduction

[00 1051 Engineered polyeleetrolyte complex micelles were produced to inhibit dys regulated vascular miRNAs that promote AVF The approach is to encapsulate miRNA inhibitors in the core of {be micelle and use peptides on the periphery of the nanopariide corona to target markers. One strategy targets vascular ceil adhesion molecule 1 ( VCAM-1) oh the surface of endothelial cells using micelles conjugated with VCAM-1 -binding peptides that have the sequence valine-histidi e-pr ol me-Iysine-gly ne-histMme-argMne, or VHPK.QHR (SEQ IB NO: 2). The formation of these miRNA inhibitor-containing, peptide- targeted micelles is achieved via the electrostatic eomplexaiion between the negatively charged miRNA inhibitors and polycations containing targetin peptides that has been designed and synthesized (Figure 21), Both pol cation molecules contain three functional domains consisting of targeting peptides lor cellular or plaque localization, a polyethylene glycol (PEG) domain to prevent macrophase separation, and a poly lysine domain to complex with the negatively charged miRNA inhibitors. Combining: the miENA inhibitors with these targeting pepu de~PE G2000-po !y 1 v sine molecules should result in the formation of electrostatically driven, self-assembled micelles with a poly!ysme-m RNA inhibitor core, protected by a PEG corona that is decorated with the targeting peptide. By taking advantage of the benefit of self-assembly:, the micelle corona can be tailored to enable the targeting of diverse cell types and load the micelles with specific miRNA inhibitors, thereby providing the means to target various pathological mechanisms in a wide range of cells and contex ts [008100! Methods

[00010?| Material Synthesis and Purification

[OOi 1081 Targeting peptide-PEG(2000)~poly-L~ lysine with a degree of polymerization of 30 (Peptide-PEG-K30) was synthesized using standard Ihmrenylmethyioxycarbony! (FMOC) solid phase synthesis methods on m automated PS3 peptide synthesizer from Protein Technologies, Inc (Tucson, A 2, USA). FMOC protected amino acids were also purchased from Protein Technologies Inc. The heterobifonetionai molecule, FMOC-PEQ2000-COOH, was purchased from Jen em Technology USA (Allen, TX, USA). A peptide targeting VC AM-1 which has the sequence VHPKQBR (SEQ I© NO: 2> was used in this study. The targeting pepttde-FEG-JOO molecule was subsequently purified using reverse phase high performance liquid chiomatogmphy (HPL€, Shlmadzu Corporation, Japan) and confirmed using a Broker UiteaiieXiteme (Fremont, CA, USA) matrix-assiste laser desorption/ionization time of flight mass spectrometr (MALDI-TOF), The miRNA inhibitor molecules (nriRIDIAN nucroRNA Hairpin Inhibitors, Dharmaeon, USA) are single-stranded, chemically enhanced oligonucleotides diluted to a working stock solution of 100 mM. [mm\ Micelle Farm km fOOOll 0| Micelles of varying conceutrations were iormed by completing the targeting- pepfide-PEG-K30 and miRNA inhibitors at an equal charge molar ratio. First, the appropriate amount of deionized water was added, followed by the miRN A inhibitor, and finally the targetmg-pept:ide~PEG~K30, The sample was vortexed after each polymer addition. The completing of targeting-peptide-PEG-K30 with ralR inhibitors was performed under charge neutral conditions in which the number of charged monomer units torn the polylysine equaled the number of charged units in the miR inhibitors. Micelles were made by compiexing WiFKQliE-PEG-K30 with mlR~92a inhibitors and miR. inhibitor controls; DL$ was used to confirm the formation and measure the hydrodynamic diameter of all micelles. The size of polyelecirotyie complex micelles is determined by the length of the charged block covalently I hiked to the neutral non-charged polymer, as well as the ratio between them. fOOOlllJ Results: Characterization ø/ micelles |QQ0112| DynamicLight Scattering ( IS)

10001131 Micelle solutions containing 5 mM miRNA inhibitors were measured at 90° using a BI-200SM goniometer containing a red lase diode with a wavelength of 637 nm and TurboCorr digital correlator, ail from Brookhaven Instruments (Holtsville, NY, USA). Brookhaven Instruments Dynamic Light Scattering software was used to analyze the inverse Laplace transforms of the intensity autocorrelation functions using the non-negatively constrained least-squares (NNLS) algorithmic obtain multimodal size distribution data. |000I14| Transmission Ekarm Microscopy ffEMj

1000115) Negatively stained TEM samples were prepared by placing micelles on 400 mesh lacey carbon grids (Ted Pella, Redding, CA, USA) and then staining with a I wi% oran t acetate solution. Images were obtained using a FBI Tecnai P30 electron microscope operated at 300 kV.

[0601161 Zeta Potential

[000117J Zeta potential was determin d by measuring the pofyauions: miR.92a inhibitor, miR 33a inhibitor, and miR inhibitor control at a concentration 4 mM and the poly cations ^" VHPKQHR-PBG _‘ I 30 at a concentration of 9 pM (equal in molar charge to the miRNA inhibitors). Micellar solutions of the combined polyanion and polyeation were measured at the combined concentration of 13 mM (equal molar charge). All samples were dissolved in MQ water and measured at 25°C (Zetasi er Nano ZS, Matvera, Worcestershire, United Kingdom, N=3).

[0001181 Treatment before a mmtiimtioR

[0001191 To separate unbound, free polymers from the micelle preparation, the micelle solution was separated into filtered and concentrated fractions by a 50-kDa cut-off membrafte (Amicon Ukra-0.5 centrifugal filter devices, Millipom). The concentrated fraction was further washed with nuclease-free water and the volume of concentrate was evaluated after centrifugation and concentration of concentrated if action calculated.

[0001201 Only slight variations in the size of all the micelle constructs were expected, and the results shown In Table 1 confirms this result.

[0001211 Table I. Dynamic light scattering of compositions.

Dh - particle diameter.

[0001221 The average size o f all the micelles was 21.1*4 nm. Negatively s tained TEM confirmed the DLS observations. A characteristic TEM image is shown in Figure 22. Micelles between 15-20 nm in diameter were observed using TEM, which is in agreement with the DLS results. As another characterization method, the zeta-potential of the individualpolymers prior to eoraplexatlon and the micelle solutions was measured. The results, shown in Table 2, indicate that the iargeting~peptide-BEG-K30 molecules are highly positivel charged and the raiR inhibitors are highly negatively charged.

[00 123[ T able 2. Zeta potential of compositions.

|QQ9I24| These results were expected since targethig-pepiide-FEG-K30 contains posi lively charged poly-.L-ly sine and miR. inhibitors contain the negatively charged phosphate backbone. The micellar solutions all have values in between the miR inhibitors and the peptide-PEG-K30 molecules indicative of a mixture of the two components.

Example 2: Inhibition of i»kroRNA-92a and effects on pathological vascular remodeling under disturbed flow - AVF maturation failure {008125} Ititmihicthm

|QQM26>1 The most common causes of AVF failure are acti vation of endothelium, neoi imal hyperplasia, stenosis, and thrombosis at the arteriovenous anastomosis and cephalic arch, both sites are exposed to complex hemodynamics after AVF creation. (See Figure 2). Blood flow in veins is typicall steady, slow, and non-pulsatiie under normal physiological conditions but local vein geometry and flow parameters are drastically remodeled after access creation. At the sites of arteriovenous anastomosis and cephalic arch, increased flow rate and enhanced pulsatility due to AVF creation impose complicated patterns of mult idi rectional hemodynamics at variable frequencies leading to fluid disturbance featuring oscillation, flow reversal or recirculation. This complex hemodynamics, or disturbed flow, has been linked to the endothelial activation and consequent pathological vessel remodeling leading to a wide range of vascular diseases, comprising atherosclerosis and AVF failure. Figure 5A depicts the complex hemodynamics at the arteriovenous anastomosis (juxta-anastoniotic: venous segment in humans) after brachiocephalic AVF creation in a hemodialysis patient (Figure SB). Here, the vivo potency of the proposed targeted uanohiedicme approach in alleviating neoiutimal hyperplasia was determined in the carotid aneiy-jugnlar vein AVF mouse model.

[ 001271 Further, a previous study showed that endothelial health was associated with tire development of arteriovenous fistulas (A VPs) (Alton ei el JASN, 2016). MieroRNA (miRj- 92a is a major contributor to vascular endothelial dysfunction. It has been previously reported that patients with CKD have increased serum miR-92a levels when compared to non-GKD control subjects, and that scrum miR-92a is likely derived from the endothelium (Shang ei al. .1ASN, 2017). Thus, the relationship between miR-92a and AVF development in animal models was investigated (Figures 11-26). 0O«28| Methods

1009129) In young male Wisiar rats with norma! kidney function or with adenine diet- induced CKD, femotai AVFs were created and then AVF lumen diameter was assessed by ultrasound and AVF tissue miR-92a levels by RT-PCR at 4 weeks after creation, as described in Laager etal. Kinde International 2010. 1» a mouse carotid-jugular AVF model as described in Chnn ei al J Vis Exp 201 ( mift-92a inhibition was achieved using genetic and ph rmacological approaches; (1) whole-body knockout ( _: miR-92a---r- ^· ) with CS7BL/6 mice used as wild-type (WT) controls; or (2) nanoparticles (NPs) that encapsulate miR-92a inhibitors and target inflamed endothelium as described in Example 1 (unencapstdated (naked) niiR-92a inhibitors and saline were used as no- P and no-treatment controls, respectively).

1600130 j The anastomosis angles of mouse carotid-jugular A VFs (approximately 70-90°) are similar to human brachiocephalic AVFs in the literature, and this mouse AVF model is well established for the study of human AVF maturation failure and drug targets. Ten-week old male C57BL/6 mice received the inhibitor treatment (8 mg/kg body weight) intravenously, through the ail vein, at 1 day after AVF creation and were sacrificed I week later. Mouse AVF cross-sectional lumen area and the area of neoinfiraal hyperplasia were quantified by histology using Image J.

10001311 Results

{600132} When compared to AVFs in non-CKD rats, AVFs in CKD rats had increased niiR~92a in serum (3 fold, p<0.05) (Figure 15). In the knockout study, the percent open lumen area of A VF veins was larger in miR-92a-/ ^~ mice (72% of total area) than in WT mice (12%) (Figures I8A an 18B) In the Inhibitor study, both NP-encapsulaied (41%) and un-encapstiated (23%) mift-92a inhibitors resulted in larger open lumen area than saline control (5%), and the: effect of encapsulated inhibitors was more pronounced (Figure 24B). It was determined _^ that while both encapsulated an naked miR~92 a inhibitors resulted in greater open lumen area an smaller H area when compared to the PBS control, the effect of encapsulated inhibitors was more pronounced (Figure 24A). |000133| I» the mouse model, inhibition of miR~92a improved AVF development (Figure 24), Thys, this nanomedidne approach may offer a novel and effective therapy to enhance AVF maturation In C D patients

[0001341 Conclusion

[0901351 These experiments confirm that VC AM- 1 -targeted polyeleciroiyte complex micelles effectively deliver mlR-92a inhibitors preferentially to inflamed endothelial cells.

The experiments sought to determine the therapeutic effectiveness of VCAM-1 -targeted polyelectrolyie complex micelles in inhibiting endothelial miR~92a and suppressing pathological AVF vascular remodeling in vivo. These studies should further preclimcal development, and perhaps clinical testing, of a new therapeutic strategy to treat AVF failure, a still critically important disease with unmet medical need.

[0001361 Results item the experiments disclosed herein indicate that combination of mlRNA therapies and targeted nanomedidne is an attractive new strategy to provide preventative maintenance to AVF placements. First, prior data employing cultured cells, animal models (mouse and swine), and human genetics identified that increased expression of endothelial mlR-92a Is a major molecular signature of endothelial activation anti pathological vascular remodeling under disturbed flow. Second, genetic deletion of raiR-92a in experiments described herein significantly lessened AVF failure in a mouse model. Third, the novel uanopartiefes described here preferentially target inflamed endothelial cells and moreover, mtraeelfukr!y deliver miR-92a. inhibitors to the targeted cells. This is achieved by encapsulating mift-92a inhihiiors in the core of the po!yeleetroiyte complex micelle white displaying targeting peptides against Vascular Cell Adhesion Molecule 1 (VCAM-l) on the surface. The expression of VCAM-l increases in activated endothelium but remains low in healthy endothelium and VCAM- l -targeiing dri ves active binding of nano-materials to inflamed endothelial cells.

Example 3: Inhibitio of miR-92a and effects on pathological vascular remodeling under disturbed flow - atherosclerosis

1000137} Imtmduci n

[0001381 A cohort of endothelial miRs have been shown to be differentially expressed between athero-suscepiible arterial sites and at hero-proteeted regions In both mouse and swine models as well as in humans. In particular, a pro-mtlammatory micro RNA, miR-92a, is elevated in areas of athero-suscepribiiity and studies using cultured ceils, animal models(mouse and swine), and human genetics. (genome-wide association studies) have identified that increased presence of endothelial miRAQa is a major molecular signature of endothelial activation and pathological vascular remodeling under disturbed flow,

1090139j In the present study, the role of mi R ~92a as an underlying cause of the development Of atherosclerotic lesions which also present disturbed flow conditions, was examined. The ability of the . nanomedidne platform described in Example I to address this condition through, the targeted delivery of oligonucleotides that inhibi t miR-92a was examined.

100 1401 M&tetiafo mdMethmh

|000l4l 1 Atherosclerosis-prone apoli oprotein E-deficient (Apo E ) mice display poor lipoprotein clearance with subsequent accumulation of cholesterol ester-enriched panicles in the blood, which promote the development of atherosclerotic plaques. Therefore, the Apo E-/- mouse model is well established for the study of human atherosclerosis and drug targets. Moreover, like humna lesions, atherosclerotic lesions in Apo E ^“ ' ^“ develops in arterial regions exposed to distubed flow increased endothleial m£R-92a by disturbed flow has been linked to atherosclerosis in humans and in Apo E ^'' % The study design of treating atherosclerosis in Apo E ^" " ^" by targeted oaooparticles as described in Example I is is shown in figure 27 A.

|&0hl421 Results

1 001431 figure 27B shows a comparison between treatments administerin the naked miR-92a inhibitor (8 mg/kg) and the micelle-encapsulated and -targeted niiR~92a inhibitor (8 mg/kg), along with some controls. The statistically significant result in Figure 27B is that naked miR-92 inhibitor produces a 55% reduction in lesion size, while targeting the niiR~ 92a inhibitor to inflamed endothelium with a nanoparticle produces an 80% reduction, A follow-up series of experiments with the same protocol sho wed that, at a lower dosage of 4 mg/kg, the advantage of targeted nanoparticle delivery increased (80% reduction for targeted delivery ys 42% reduction for naked deli vert' ((data not shown) f QQ01441 Moreover, recent results demonstrated that when lower dosage of miR-92a inhibitor (4 mg/kg) was used, no reduction of the atherosclerosis was detected in Apo E ^; mice treated with naked miR-92a inhibitor (4 mg/kg) but the atherosclerosis lesion was significantly reduced by 70% in mice treated with 4 mg/kg miR-92 inhibitor encapsulated: in the VC AMI -targeting nanoparticles (Figure 27C).

(000145] These results collectively demonstrate dial the targeted nanqparii e approach significantly treat atherosclerotic lesions in viv in a well-established mouse model of atherosclerosis. Therefore, such approaches may have clinically meaningful therapeutic effects in humans.

Example 4; Inhibition of miR-92a and effects on pathological vascular remodeling under disturbed flow - Restenosis

100014&1 Introduction

1000147] The biology of restenosis and in-stent restenosis is similar to that of A VF failure both involving endothelial dysfunction, smooth muscle cell proliferation and matrix protein elaboration, and ultimately intimal hyperplasia. This similarity, and the preliminary success in the mouse model of AW reported herein, suggests that the therapeutic nanoparticles described in Example 1 can also inhibit the restenosis that often occurs after angioplasty and insertion of a stent to relieve coronary artery blockage, which is a potentially larger indication than AVF„ Over 1.8 million stents were implanted in the CIS in 2018, including drug eluting stents, and restenosis is anticipated to be in the range of 10-25% depending on the type of stent used. Thus, there exists a large unmet medical need far susceptible patients even when interventio with drug-eluting stents is used.

|0i)01481 Materials and Methods

[000149] Carotid arterial stenosis can be induced in mice by a surgical procedure in the carotid arteries which introduces disturbed- flow and increases miR~92a in endothelial cells. As shown in representati ve Figure 28A, the left external carotid, internal carotid, and occipital artery were ligated with 6-0 silk suture while the superior thyroid artery remained intact; this partial carotid ligation results in flow disturbance In left carotid artery, leading to increased endothelial miR-92a expression an causing stenosis in two weeks in vivo. This mouse model has been wkfely-osed to mimic the arterial stenosis in humans such as the in- stent restenosis. The treatment protocol Is show i Figure 2SB Specifically, three days after lire partial carotid ligation, a tail vein injection was conducted of either naked h«R-92a inhibitor or the mlR-92a inhibitor encapsulated in a ACAMT -targeted micellar nauoparticle, Nanoparticles described in Example i were used. In both cases, a dose of 2 mg/kg of body weight was administered. Two more injections were conducted in day 6 and 9 after the partial carotid ligation. Mice were sacrificed 14 days after the partial carotid ligation to measure the stenosis In the partially -ligated left carotid arteries.

[000150] Results

[090151] The results (Figure 28C) demonstrate that disturbed flow-induced stenosis in the partially-ligated carotid artery is significantly reduced by &7% by three injections of VCAMl-iargetin aiioparticle encapsulating 2 mg/fcg n«R-92a inhibitors. However, injections of miR~92a Inhibitors in the naked form had much less effect (30% reduction) on the disturbed flow-induced stenosis in mice.

[000152] Conclusion

[000153| The resitlts demonstrated tha the targeted uanopartici platform significantl treats arterial stenosis in vivo

[900154] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding an equi valents of the features shown and described or portions thereof, but i t is recognized that various modifications are possible within: the scope of the embodiments claimed. Thus, it shoul be understood that al though the present description has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may he resorted to by those skilled In the art, and that such modifications and variations are considered to be within the scope of these embodiments as defined fay the description and the appended claims. Although some aspects of the present disclosure can be identified herein as particularly advantageous, it is contemplated that the present disclosure is no t limited to these particularaspects of the disclosure,

[900155] Claims or descriptions that include “or” between one or more members of a group are considere satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactl one me er of the group is present in, employe in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employe in, o otherwise relevant to $ gi ven produc t; or process. |QQ0156| Furthermore, the disclosure encompasses ail variations, combinations, and permutations in which: one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any elernenti s} can he removed from the group.

It should It be understood that, in general, where the disclosure, or aspec ts of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. Fo purposes of simplicit , those embodimen ts have not been specifically set forth in haec verba herein.

Sequences.