WO2013058812A1

WO2013058812A1 - Targeted delivery to pancreatic islet endothelial cells

Info

Publication number: WO2013058812A1
Application number: PCT/US2012/023619
Authority: WO
Inventors: Donald E. Ingber; Kaustabh GHOSH; Mathumai Kanapathipillai
Original assignee: President And Fellows Of Harvard College; Children's Medical Center Corporation
Priority date: 2011-10-19
Filing date: 2012-02-02
Publication date: 2013-04-25

Abstract

The present invention provides, in part, compositions and methods of targeted delivery of an agent or cells to islet endothelial cells in the pancreas using a novel islet-targeting molecule. Accordingly, one aspect of the present invention provides a composition comprising a carrier particle attached to an islet targeting molecule, wherein an agent is associated with the carrier particle, and is therefore targeted to islet cells by the islet targeting molecule. In some embodiments, the islet targeting molecule is an islet-targeting peptide. In some embodiments, such a carrier particle is a nanoparticle or similar.

Description

TARGETED DELIVERY TO PANCREATIC ISLET ENDOTHELIAL CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Serial No: 61/549,042 filed on October 19, 2011 and U.S. Provisional Patent Application Serial No:

61/585,116 filed on January 10, 2012, the contents of which are each incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with U.S. Government Support under RL1 DE019023-01 and RL9 EB008539-01 awarded by the National Institutes of Health and the U.S. Government has certain rights this invention.

FIELD OF THE INVENTION

[0003] The present invention is directed to methods of pancreatic islet targeting and therapeutic delivery where nanomaterials e.g., polymeric nanoparticles, are developed to selectively home to islet endothelial cells and deliver therapeutic agents such as immunomodulatory drugs and regenerative cells locally to pancreatic islets for the treatment of Type 1 and Type 2 diabetes.

BACKGROUND OF THE INVENTION

[0004] Application of the principles of nanotechnology in medicine has led to the development of nanomaterials (e.g., liposomes and polymeric micelles) that can greatly improve the delivery and therapeutic efficacy of drugs by simultaneously increasing drug half -life, reducing toxic side -effects and controlling drug release kinetics (Shi, J et al, Nano Lett (2010) 10(9): 3223-30). Such therapeutic nanomaterials have mostly been used in cancer drug delivery where the leaky tumor vessels permit passive targeting of blood-borne nanoparticles to tumor tissue through the enhanced permeability and retention (EPR) effect (Peer, D. et al, Nat Nanotechnol (2007) 2(12):751-60). The ability of such therapeutic nanomaterials to treat diverse diseases will, however, depend on their ability to selectively target the desired tissue of interest and promote in situ tissue normalization (Shi, J et al., Nano Lett (2010) 10(9): 3223-30). Development of such smart nanomaterials will likely create new therapeutic

opportunities for the management of intractable diseases where systemic therapies result in a high risk/benefit ratio.

[0005] Type 1 diabetes is a debilitating and rapidly spreading autoimmune disease where the insulin- producing pancreatic islet β cells are progressively destroyed by the body's immune cells, leading to hyperglycemia at clinical diagnosis (Eizirik, D. L. et al., Nat Rev Endocrinol (2009) 5:(4): 219-26). One of the earliest events in the autoimmune destruction of islet β cells (insulitis) is the adhesion of blood leukocytes to inflamed islet vascular endothelium, following by extravasation of the immune cells into the islet parenchyma where they attack the islet β cells (Eizirik, D. L. et al, Nat Rev Endocrinol (2009) 5: (4): 219-26;Waldron-Lynch, F. et al., Nat Rev Drug Discov 10(6): 439-52). Given that the combination of metabolic and autoantibody tests can predict with 90% accuracy the 6-year risk of developing type 1 diabetes (Parving, H. H. et al, Diabetes Care (1999) 22:(3):478-83; Bingley, P. J. et al, Diabetes Care (2008) 31(l):146-50; Berchtold, P. et al, Schweiz Med Wochenschr (1996) 126(38): 1603-9), managing this debilitating disease at the early stage of leukocyte-islet vessel interaction has emerged as a viable therapeutic approach (Waldron-Lynch, F. et al, Nat Rev Drug Discov 10(6): 439-52). Anti-leukocyte proliferative drugs that reduce the number of circulating leukocytes, such as cyclosporine A and prednisone, have been reported to substantially extend endogenous insulin production while lowering the dependence on exogenous insulin treatment (Nikolova, G. et al, Dev Cell (2006) 10(3): 397-405; Cheng, J. et al, Biomaterials (2007) 28(5):869-76). Unfortunately, these systemically-administered drugs have been barred from clinical use due to severe side -effects, such as nephrotoxicity (Parving, H. H. et al, Diabetes Care (1999) 22:(3):478-83) and development of insulin resistance (Bingley, P. J. et al, Diabetes Care (2008) 31(l):146-50), as well as likely compromising the body's innate infection-fighting capacity (Berchtold, P. et al, Schweiz Med Wochenschr (1996) 126(38): 1603-9). Thus, the development of drug delivery approaches that can selectively inhibit leukocyte-islet vessel interactions without causing any adverse reaction would have important implications for the treatment of insulitis in subjects that are at high risk of developing type 1 diabetes.

[0006] Type 2 diabetes, on the other hand, is characterized by a progressive decline in islet mass and function resulting from insulin de-sensitization and subsequent chronic hyperglycemia. Previous research has reported that optimal pancreatic islet β cell growth and function requires soluble and physical cues from islet blood vessels (Lammert, E. et al, Science (2001) 294(5542):564-7; Nikolova, G. et al, Dev Cell (2006) 10(3):397-405). Therefore, by providing regenerative vascular cues to diabetic islets, it may be possible to stem the decline in islet mass and function and even restore them to their normal levels. Bone marrow represents an excellent source of vascular stem/progenitor cells that can be leveraged for pancreatic islet regeneration. One way to achieve high islet engraftment and therapeutic efficacy of these bone marrow-derived cells is to develop approaches that can selectively guide them to the microvessels of diseased islets following systemic delivery.

SUMMARY OF THE INVENTION

[0007] The present invention provides compositions and methods of targeted delivery of an agent or cells (e.g., stem cells or progenitor cells) to islet endothelial cells in the pancreas using an islet-targeting molecule. In some embodiments, an islet-targeting molecule is a peptide which specifically binds to islet endothelial cells. Accordingly, such compositions can be used for in situ diabetes therapy. [0008] Herein the inventors demonstrate the use of a pancreatic islet-targeting molecule, e.g., an islet- targeting peptide for the development of pancreatic islet-targeting nanoparticles for immunomodulatory therapy of autoimmune type 1 diabetes, and the use of this nanotechnological strategy to develop pancreatic islet-targeting cells, e.g., endothelial cells (EPCs), for treatment of type 2 diabetes. Using an islet-targeting molecule which is a unique islet-homing molecule, which in one instance is a peptide, the inventors demonstrate that polymeric nanomaterials exhibit 3-fold greater binding to islet endothelial cells and a 70-fold greater anti-inflammatory effect through targeted islet endothelial cell delivery of an immunosuppressant drug. The inventors also highlight the need to carefully tailor drug loading and nanoparticle dosage to achieve maximal vascular targeting and immunosuppression in the treatment of subjects that are at high risk of developing type 1 diabetes.

[0009] Herein, the inventors have developed islet-targeting polymeric nanomaterials for delivering therapeutic agents (e.g., pharmacologic drugs, stem/progenitor cells) selectively to pancreatic islets and stimulating in situ islet repair or regeneration. The inventors demonstrate using an amphiphilic poly(D,L- lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) polymer that spontaneously self-assembles in aqueous solution to form nanoparticles. PLGA and PEG are FDA-approved for use in a variety of clinical products (Cheng, J. et al., Biomaterials (2007) 28(5):869-76;Lu, J. M. Expert Rev Mol Diagn (2009) 9(4): 325-41); thus their block co-polymer is expected to be safe for use in humans. Using conventional carbodiimide chemistry, the inventors have covalently conjugated to this polymer an islet- targeting molecule, e.g., an islet-targeting peptide having a unique peptide sequence which homes specifically to the pancreatic islet vasculature (Yao, V. J. et al., Am J Pathol (2005) 166(2): 625 -36). The inventors islet-targeting peptide is modified from that previously reported to allow conjugation to block co-polymers and the like. The inventors demonstrate in vitro that these islet-targeting nanomaterials, which have an average size of 190 nm can bind preferentially to islet capillary endothelial (CE) cells. Additionally, the inventors have also demonstrated the release of an anti-inflammatory drug (genistein) from these nanoparticles, achieving -75% drug release over two days. Further, these genistein-loaded nanoparticles produce significant inhibition of islet endothelium inflammation in vitro. This

nanomaterials-based targeted delivery of anti-inflammatory agents to islets could be leveraged for the treatment of type 1 (juvenile) diabetes, which is an auto-immune disease marked by severe inflammation of the islet vasculature (insulitis).

[0010] Accordingly, one aspect of the present invention provides a composition comprising a carrier particle attached to an islet targeting molecule, e.g., an islet-targeting peptide, wherein an agent is associated with the carrier particle, and is therefore targeted to islet cells by the islet targeting molecule, e.g., an islet-targeting peptide. In some embodiments, such a carrier particle is a nanoparticle or similar carrier particle, and the island targeting molecule is a peptide, antibody, aptamer or other component that binds to pancreatic endothelial cells with high specificity. [0011] The inventors have also demonstrated use of the islet-targeting peptide to deliver stem cells or progenitors cells to islet cells for the treatment of type 2 diabetes. In contrast to type 1 diabetes, type 2 diabetes is characterized by a progressive decline in islet mass and function resulting from insulin de- sensitization and subsequent chronic hyperglycemia in the body. Here, the inventors demonstrate that endothelial progenitor cells (EPCs) can significantly enhance islet function in vitro. To selectively deliver EPCs to pancreatic islets for in situ islet normalization in type 2 diabetics, the inventors tethered the islet- targeting nanoparticles onto the surface of EPCs using biotin-streptavidin linkage. These EPC- nanoparticle conjugates exhibit significantly stronger binding to islet capillary endothelial cells compared to unmodified EPCs in vitro. Accordingly, the inventors have demonstrated an effective nanomaterials- based approach for targeted delivery of drugs or cells, e.g., EPCs, to pancreatic islet endothelial cells, which has direct therapeutic implications for both type 1 and type 2 diabetes. Further, this

nanoengineering approach can be adapted for the site-specific delivery of such therapeutic agents to other tissues and organs in the body.

[0012] Accordingly, another aspect of the present invention provides a composition comprising a islet-targeting molecule, e.g., an islet-targeting peptide which is attached to an affinity binding moiety, where the affinity binding moiety can attach to the cell surface of a cell, e.g., a stem cell or progenitor cells, such as but not limited an endothelial progenitor cell, thereby targeting the endothelial cell to islet cells by the islet-targeting peptide.

[0013] The advantages of the compositions as disclosed herein can be used for in situ diabetes therapy, reducing the risk of undesirable side effects from systematic administration of agents to treat diabetes

{e.g., anti-inflammatory agents and other agents used to treat diabetes) as well as significantly improving the engraftment of EPCs in pancreatic islets. In some embodiments, the carrier particles attached to the islet-targeting molecule, e.g., an islet-targeting peptide can incorporate imaging agents, e.g., contrast agents, bioluminescent agents, fluorescent dyes and the like, for simultaneous monitoring of the agent localization to pancreatic islet endothelial cells, and monitoring of the pancreatic islets.

[0014] In some embodiments, the islet-targeting molecule is an islet-targeting peptide which comprises the amino acid sequence CHVLWSTRKC (SEQ ID NO: 1) or a fragment thereof, e.g., at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9 contiguous amino acids of SEQ ID NO: 1.

[0015] In some embodiments, the carrier particle can be a nanoparticle, or other known carrier particle. In some embodiments, the carrier particle can be a liposome. In some embodiments, the islet-targeting peptide is covalently attached to a block polymer, where the block polymer or block co-polymer forms a carrier particle. In some embodiments, a block co-polymer comprises PLGA and PEG. In some embodiments, the block polymer is [PLGA-b-PEG-COOH]n.

[0016] In some embodiments, an islet targeting molecule is an antibody, aptamer or other component that binds to pancreatic endothelial cells with high specificity. [0017] In some embodiments, the islet-targeting peptide is an antibody or an antigen binding fragment thereof, for example but not limited to, a single chain antibody, a Fab portion of an antibody or a (Fab')₂ segment which binds to an antigen on the cell surface of the pancreatic CE cells. In some embodiments, where an islet targeting molecule is an antibody, the antibody can specifically bind to Ephrin A4 (Eph A4), as disclosed in Yao et al, Am. J. Path., 166(2); 625-636, which is incorporated herein in its entirety by reference.

[0018] In some embodiments, an islet-targeting molecule as disclosed herein is a polynucleic acid aptamer, or nucleic acid analogue or similar.

[0019] In some embodiments, the agent is associated on the inside or exterior of the carrier particle. In some embodiments, the agent is encapsulated inside the carrier particle.

[0020] In some embodiments, the agent is an agent used to treat diabetes, for example, an agent for the treatment of Type 1 diabetes. In some embodiments, the agent is an anti-inflammatory agent, for example, but not limited to, Genistein, cyclosporine A and prednisone and the like.

[0021] In some embodiments, an affinity binding moiety can associate with the islet-targeting molecule, e.g., an islet-targeting peptide, and the affinity binding moiety can be any moiety or molecule which attaches to a cell, e.g., stem cell or progenitor cells, e.g., EPC, and can be, for example, an antibody or antigen binding fragment thereof which targets cell surface antigen, for example targets the cell surface of a stem cell or progenitor cell, e.g., EPC, where the affinity binding moiety is associated with the islet- targeting molecule, e.g., an islet-targeting peptide as disclosed herein. In some embodiments, the affinity binding moiety can be part of a complex, e.g., where such a complex comprises an first affinity binding moiety: an affinity binding partner: a second affinity binding moiety, where the first affinity binding moiety associated with the islet-targeting molecule, e.g., an islet-targeting peptide, and the second affinity binding moiety associates with a specific cell, e.g., a stem cell or progenitor cell, e.g., a EPC, and where the first and second affinity binding moiety associate with a single affinity binding partner, thereby indirectly connecting the islet-targeting molecule, e.g., an islet-targeting peptide with the EPC cell.

[0022] In some embodiments, an affinity binding moiety which is bound to the islet-targeting molecule, e.g., an islet-targeting peptide is an antibody or an antigen binding fragment thereof, for example but not limited to, a single chain antibody, a Fab portion of an antibody or a (Fab')₂ segment which binds to an antigen on the cell surface of an EPC. In some embodiments, the affinity binding moiety and islet-targeting molecule, e.g., an islet-targeting peptide can be associated by a peptide bond, for instance the affinity binding moiety and islet-targeting molecule, e.g., an islet-targeting peptide can be a fusion protein, wherein the affinity binding moiety is fused to the carboxy portion or the N-terminal of the islet-targeting molecule, e.g., an islet-targeting peptide.

[0023] In some embodiments, the islet-targeting molecule, e.g., an islet-targeting peptide is associated with, or linked to an affinity binding moiety or carrier particle by any means commonly known by persons of ordinary skill in the art, for example, the islet-targeting molecule, e.g., an islet-targeting peptide and affinity binding moiety can be linked in the form of a fusion protein, for example, where the affinity binding moiety is fused to the carboxy or N-terminal portion of the islet-targeting molecule, e.g., an islet- targeting peptide. It is encompassed that alternative arrangements of a fusion protein of the islet-targeting molecule, e.g., an islet-targeting peptide and affinity binding moiety are useful in the methods and compositions of the present invention, for example where the affinity binding moiety is fused to the N- terminal portion of the islet-targeting molecule, e.g., an islet-targeting peptide.

[0024] In some embodiments, the islet-targeting molecule, e.g., an islet-targeting peptide is associated with, or linked to a carrier particle by any means commonly known by persons of ordinary skill in the art, for example, by chemical or covalent conjugation, van der Waals forces and the like, as discussed herein.

[0025] The methods and compositions as disclosed herein are useful in the delivery of agents, e.g., anti-inflammatory agents, RNAi molecules and the like, or cells, e.g., stem cells or progenitors, e.g., EPC to pancreatic islet endothelial cells. In some embodiments, the methods and compositions as disclosed herein are useful for the treatment and/or prevention (e.g., prophylactic treatment) of type 1 or type 2 diabetes and diabetes-related disorders.

[0026] In some embodiments, the methods to deliver an agent or cell, e.g., stem cell or progenitor cell, such as an EPC to pancreatic islets is a cultured pancreatic islet endothelial cell, e.g., in vitro, for example in embodiments where the cultured pancreatic islet endothelial cells are in an assay to test therapeutic agents and compounds which promote survival of islet cells and/or insulin production. In alternative embodiments, the methods to deliver an agent or cell, e.g., stem cell or progenitor cell, such as an EPC to pancreatic islet endothelial cells which are part of an organ, such as the pancreas, or are present in a subject (e.g., in vivo delivery) such as an animal or human subject. In some embodiments, the islet cell is a precursor islet cell, or present in a population of embryonic stem cells or islet progenitor cells.

[0027] Another aspect of the present invention relates to a method of delivering an agent or cell, e.g., stem cell or progenitor cell, such as an EPC to pancreatic islet endothelial cell in a subject, the method comprising administering a composition as disclosed herein to the subject. In some embodiments, the subject is human.

DESCRIPTION OF THE DRAWINGS

[0028] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] Figures 1A-1C show a schematic representation and physicochemical characterization of islet- targeting nanomaterials. Figure 1A shows carbodiimide chemistry was used to covalently conjugate islet targeting peptide (CHVLWSTRKC) to the amphiphilic PLGA-b-PEG-COOH block co-polymer, which undergoes spontaneous self-assembly in aqueous solutions to form nanoparticles. Figure IB shows 'H- NMR spectrum of polymer-peptide conjugate displaying the peaks characteristic of tryptophan (W) residue in the peptide (arrow), which is absent in the unmodified polymer. Figure 1C shows transmission electron microscopy (TEM) and dynamic light scattering analysis reveal that the islet-targeting nanoparticles have an average diameter of 190 ± 40nm. Scale bar on TEM image = 200 nm.

[0030] Figures 2A-2B show nanoparticle binding to islet capillary endothelial (CE) cells in static culture. Figure 2A shows that under static culture conditions, nanoparticles displaying islet-targeting peptide (CHVLWSTRKC; Pep I) exhibit a 3-fold increased binding (***p<0.001) to islet CE cells compared to skin CE cells, as shown in the representative fluorescent images of coumarin-loaded nanoparticles and quantified in the bar graph. This preferential binding to islet CE cells is specific to Pep I since the scrambled peptide (PepX) containing the same amino acids does not exhibit any binding preference. Scale bar = 20 μπι. Figure 2B shows that islet CE cells treated with coumarin-loaded nanoparticles were stained with lysotracker^™ red to label acidic organelles. Quantitative analysis of green (nanoparticle) and red (lysotracker) images reveals approx. 100% colocalization; Mx (green colocalizing with red) = 97.86%; My (Red colocalizing with green) = 99.99%.

[0031] Figure 3 shows nanoparticle binding to islet capillary endothelial (CE) cells under flow. To mimic nanoparticle binding to islet endothelium in vivo, islet and skin CE cells were cultured in parallel microfluidic channels and the NP-Pep I and NP-Pep X suspensions (10 ug/ml) were flown over them at a wall shear rate of 2 dyne/cm². Quantitative analyses of fluorescence images showed NP-Pep I binding specificity similar to that seen under static conditions (***p<0.001). Scale bar = 100 μπι.

[0032] Figures 4A-4C show immunosuppressive effect of islet-targeting nanomaterials. Figure 4A shows Genistein, a pharmacologic inhibitor of receptor tyrosine kinase, blocks leukocyte binding to TNF- a-stimulated islet CE cells in a dose dependent manner (***p<0.001). Figure 4B shows Genistein was encapsulated into NP-Pep I nanoparticles at 5% (w/w) and drug release measured over 48 hours. Figure 3C shows Islet CE cells treated with genistein-loaded islet-targeting nanoparticles (NP-Gen; 10 ug/ml) 18h prior to TNF-a stimulation exhibit significant inhibition in leukocyte binding (***p<0.001).

[0033] Figures 5A-5C show the regulation of therapeutic and physicochemical properties of NP-Gen as a function of drug and nanoparticle dosage. Figure 5A shows NP-Gen concentration of 10 μg/ml produced the most significant inhibition (***p<0.001) in leukocyte/islet CE cell binding while higher doses were progressively less effective. This trend is attributed to the pro-inflammatory effect of nanoparticles, as indicated by increased leukocyte binding to islet CE cells treated with higher doses of blank NP-Pep I nanoparticles. Figure 5B shows Increasing genistein loading into NP-Gen nanoparticles results in a significantly greater immunosuppressive effect, as indicated by a marked decrease in leukocyte binding to islet CE cells. Figure 5C shows NP-Gen nanoparticles loaded with increasing amounts of genistein exhibit a progressive increase in their average size.

[0034] Figures 6A-6C shows islet CE cells treated with blank or genistein-loaded (NP-Gen) nanoparticles. Figure 6A shows there are no apparent changes in cell morphology of islet CE cells when treated with blank or genistein-loaded (NP-Gen) nanoparticles. Figure 6B shows a histogram demonstrating no loss in cell viability of islet CE cells treated with blank or genistein-loaded (NP-Gen) nanoparticles when compared with untreated cells. Cell viability measurement is normalized with respect to untreated islet CE cells. Scale bar = 100 μπι. Figure 6C shows a histogram demonstrating that insulin- producing islet β cells (Min6) treated with unmodified or islet-targeting nanoparticles show no apparent loss in cell viability when compared with untreated cells. Cell viability measurement is normalized with respect to untreated islet β cells.

[0035] Figure 7A-7D shows endothelial progenitor cells (EPCs) enhance islet function in vitro. Figure 7A shows that in 'contact' co-culture, EPCs restore normal in vi^'vo-like functionality in whole mouse islets in vitro by significantly enhancing glucose-sensitivity of insulin secretion. Figure 7B shows that when cultured with a mouse insulinoma (Min6) islet beta cell line, EPCs significantly enhance islet functionality by significantly upregulating insulin secretion in response to high glucose. Figure 7C shows the enhancement of glucose stimulated insulin secretion is independent of increase in total insulin production by or proliferation of Min6 cells. Figure 7D shows the enhancement in insulin secretion and sensitivity by islet beta cells is a unique characteristic of immature or primitive (low passage) EPCs as this effect is not seen with mature (higher passage) EPCs in culture.

[0036] Figure 8 shows a schematic view of the steps involved in surface modification of EPCs with islet-targeting nanoparticles. Biotinylated islet-targeting nanoparticles were tethered onto the surface of biotinylated EPCs using a streptavidin linkage to obtain nanoengineered EPCs.

[0037] Figure 9A-9B shows the optimization of EPC surface modification. Figure 9A shows EPCs that were treated with varying concentrations of biotin and analyzed by flow cytometry. Figure 9B shows EPCs which were treated with varying concentrations of streptavidin and analyzed by flow cytometry. Biotin and streptavidin concentrations of 1 mM (0.9 mg/ml) and 50 μg/ml were found to be optimal (arrows).

[0038] Figures lOA-lOC show nanoparticle tethering to biotinylated EPC surface and its optimization using flow cytometry. Figure 10A shows that the high affinity biotin-streptavidin linkage was leveraged for specific tethering of biotinylated nanoparticles to biotinylated EPC surface. Unmodified EPCs exhibit very low levels of background NP binding. Figure 10B shows scanning electron micrograph where the conjugated NPs are clearly visible on the EPC surface (scale bars: left-10 μιη; right-1 μπι). Figure IOC shows 15 minute EPC/nanoparticle incubation was optimal for EPC surface tethering of biotinylated nanoparticles.

[0039] Figures 11A-11B show preferential binding of nanoengineered EPCs to islet CE cells in vitro. Figure 11A shows EPCs modified with islet-targeting nanoparticles exhibit significantly stronger adhesion to islet CE cells than unmodified EPCs or EPCs modified with control biotin or nanoparticles. Figure 11B shows EPCs modified directly with the islet targeting peptide exhibit significantly stronger adhesion to islet CE cells as compared to unmodified EPCs. [0040] Figure 12 shows the nanoengineered EPCs to exhibit robust vasculogenesis in vitro. Phase and fluorescent images show that, similar to unmodified EPCs, those modified with biotin alone or biotin-NP- Streptavidin complex undergo rapid capillary network formation when plated on Matrigel in vitro. Total tube length, an index of EPC vasculogenic property, was measured from the acquired phase images using Image J^® software.

[0041] Figure 13 depicts an 1H-NMR spectrum indicating that the islet-targeting peptide (Pep I; CHVLWSTRKC) displays peaks characteristic of tryptophan (W) residue at 7.2-7.6 ppm, which is used as a reference to confirm successful polymer-peptide conjugation.

[0042] Figure 14 shows data from dynamic light scattering analysis indicating coumarin incorporation into islet-targeting nanoparticles does not alter particle size, with the average particle diameter remaining -190 nm.

[0043] Figures 15A and 15B depict 1H-NMR spectra. Figure 15A shows that 1H-NMR spectrum of the scrambled peptide (Pep X; CVHWTLSRKC) is similar that of the islet-targeting peptide, with the tryptophan (W) peaks appearing at the expected 7.2-7.6 ppm. Figure 15B Carbodiimide chemistry was used to covalently conjugate the scrambled peptide to the PLGA-b-PEG-COOH block co-polymer. 1H-

NMR spectrum of the polymer-peptide conjugate displays peaks characteristic of the peptide's tryptophan (W) residue (arrow), thereby confirming successful peptide conjugation.

[0044] Figure 16 shows islet CE cells treated with coumarin-loaded nanoparticles stained with Mitotracker™ red to label mitochondria. Overlay of green (nanoparticle) and red (Mitotracker™) images reveals no detectable colocalization. Scale bar = 10 μιη.

DESCRIPTION OF THE INVENTION

[0045] Accordingly, the present invention provides compositions and methods of targeted delivery of an agent or cells to islet endothelial cells in the pancreas using an islet-targeting molecule. Accordingly, one aspect of the present invention provides a composition comprising a carrier particle attached to an islet targeting molecule, wherein an agent is associated with the carrier particle, and is therefore targeted to islet cells by the islet targeting molecule. In some embodiments, the islet targeting molecule is an islet- targeting peptide. In some embodiments, such a carrier particle is a nanoparticle or similar.

[0046] Another aspect of the present invention provides a composition comprising an islet-targeting molecule, e.g., an islet targeting peptide which is attached to an affinity binding moiety, where the affinity binding moiety can attach to the cell surface of a cell, e.g., an endothelial progenitor cell, thereby targeting the endothelial progenitor cell to islet endothelial cells by the islet-targeting molecule, e.g., an islet- targeting peptide.

[0047] Other aspects of the present invention relate to the use of the compositions as disclosed herein in a method to treat diabetes, e.g., Type 1 or Type 2 diabetes. For example, in one embodiment, a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide attached to the carrier particle can be used to deliver at least one agent, (e.g., an anti-inflammatory agent, or other agent used in diabetes treatment) to islet cells in a method to treat Type 1 diabetes. In an alternative embodiment, a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide attached to the affinity binding moiety, where the affinity binding moiety is attached to a cell, e.g., endothelial progenitor cell (EPC), can be used to deliver at least one EPC to islet endothelial cells in a method to treat Type 2 diabetes.

[0048] Accordingly, the present invention allows treatment of diabetes by specifically targeting agents or cells to islet endothelial cells, thus increasing the efficacy of treatment by allowing lower doses and a more targeted therapy approach, and also limiting any potential side effects of the agent and/or cells that may occur by non-specific targeting of agent or cell (e.g., EPC). Moreover, by specific targeting to islet endothelial cells in the pancreas, the amount of agent, in some instances, anti-inflammatory agent, RNA interference etc., administered into a subject in need of treatment can be minimized because the effect of the agent, e.g., anti-inflammatory agent, RNA interference, or amount of EPC is concentrated to specifically target islet endothelial cells.

[0049] In some embodiments, the islet β cells are cells expressing Neuropilin-2. Morphology of islet cells is an ovoid shape, about 75 μιη to 175 μιη in size (long axis). Islet cells tend to be located more towards the tail end of a pancreas (away from the duodenal cavity). Markers that can be used to detect islet cells include but are not limited to glucagon for islet-a cells, insulin for islet-β cells, somatostatin for islet-γ cells, and pancreatic polypeptide for islet-PP cells. Markers that can be used to detect ductal cells include, but are not limited to, cytokeratins (CK) 7, CK 8, CK18, CK 19, mucin MUC1, carbonic anhydrase II, and carbohydrate antigen 19.9 (sialyl-Lewis-a). Morphology of ductal cells is small, round, approximately 10 μιη across the cell, appears to be a tightly packed, cuboidal epithelium. Morphology of acinar cells include a larger size than ductal cells, shape, and zymogen granules present within acinar cells. Markers that can be used to identify acinar cells include but are not limited to carboxypeptidase A and amylase.

[0050] As disclosed herein, the inventors have demonstrated use of an islet-targeting molecule which is an islet-homing peptide to attach to nanoparticles or cells, to generate islet-targeted nanoparticles or islet-targeted stem cells, e.g., EPCs, respectively, to deliver the nanoparticles to pancreatic islet endothelial cells. Thus, the inventors have demonstrated a superior delivery drug vehicle to deliver agents, for example, which can be encapsulated in the nanoparticles or on the exterior of the nanoparticles, for the treatment of insulitis and Type 1 diabetes. Additionally, the inventors have demonstrated delivery of cells, e.g., progenitor cells, e.g., EPCs to islet endothelial cells, to treat Type 2 diabetes to prevent or decrease the rate of β-islet cell loss.

[0051] In some embodiments, an agent associated with (e.g., encapsulated or on the exterior of) the nanoparticle can be an agent used to treat Type 1 or Type 2 diabetes, and in some embodiments, for the treatment of Type 1 diabetes, the agent can be an anti-inflammatory agent, for example, but not limited to Genistein, cyclosporine A, prednisone, mesalamine (5-aminosalicylic acid), simvastatin (inhibitor of HMG-CoA reductase), Herbimycin A (a receptor tyrosine kinase inhibitor- similar to Genistein), or docosahexaenoic acid (DHA; n-3 fatty acid), or derivatives or analogues thereof. In some embodiments, an agent associated with (e.g., encapsulated or on the exterior of) the nanoparticles can be a short interfering RNA (RNAi agent) or micro interfering RNA (miRNA)-complex, or a modified RNA

(modRNA). Such RNAi agents, miRNA or modRNA which are suitable for use include any which inhibit inflammation or inhibit the auto-immune response in Type 1 diabetes. In some embodiments, suitable RNAi agents, miRNA or modRNA include any which can be used to treat Type 1 diabetes. In some embodiments, exemplary examples of siRNA or shRNA agents for inhibiting inflammation include, but are not limited to, GATA3-shRNA (Lee, C. C. et al.,Mol Ther (2008) 16(l):60-5) and siRNAs against IL- 13 (Lively, T. N. et al, J Allergy Clin Immunol (2008) 121(l):88-94) and IL-5 (Huang, H. Y. et al, Gene Ther (2008) 15(9):660-7) for reducing airway inflammation, and transdermal delivery of siRNA against the natriuretic peptide receptor A (NPRA) that exhibits immune modulating effects (Wang, X. et al., Genet Vaccines Ther (2008) 6:7).

Definitions

[0052] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0053] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0054] The term "target cell" as used herein refers to a cell which comprises cell surface antigens, such as for example but not limited to, cell surface receptors or glycoprotein or other cell surface markers which the islet-targeting molecule as disclosed herein can recognize and bind thereto.

[0055] The term "cell marker" refers to, without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes which are specifically expressed or present in pancreatic islet endothelial cells. In some embodiments, exemplary islet endothelial markers include, without limitation, von Willebrand factor, CD31, induction of endothelial cell leucocyte adhesion molecule- 1, uptake of acetylated low density lipoprotein, as well as express VEGF and angiostatic factors such as endostatin and pigment epithelial-derived factor.

[0056] The term "islet-targeting molecule" as used herein refers to any agent or molecule which can bind specificity and selectivity to target pancreatic islet endothelial cells, for example, a capillary endothelial (CE) cell as disclosed herein. An islet-targeting molecule can be for example, but not limited to, a peptide, antibody, aptamer, and or variants thereof, where the islet-targeting molecule functions as an agent that homes in on, or preferentially associates or binds to a pancreatic islet endothelial cell.

[0057] The terms "islet-targeting peptide" refers to a peptide sequence of SEQ ID NO: l or a fragment thereof which has affinity, or binds to a molecule on the surface of a target islet endothelial cell, for example a capillary endothelial (CE) cell, where the islet-targeting peptide functions as an agent that homes in on or preferentially associates or binds to a pancreatic islet endothelial cell.

[0058] The term "endothelial cell" as used herein refers to cells that line the inside surfaces of blood vessels, and lymph vessels and making up the endothelium. Endothelial cells are typically but not necessarily thin, flattened cells. An islet endothelial cell can be identified by expression of cell surface markers which include, without limitation, von Willebrand factor, CD31 , induction of endothelial cell leucocyte adhesion molecule- 1, uptake of acetylated low density lipoprotein, as well as express VEGF and angiostatic factors such as endostatin and pigment epithelial-derived factor.

[0059] The term "β-cell" or "islet β-cell" as used herein refers to an insulin producing cell of the pancreas. Pancreatic β-cells can be identified by one of ordinary skill in the art, and include, for example, but are not limited to, expression of one or more of the markers pancreatic and duodenal homeobox 1 (PDX-1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Ηηί3β, PCI/3, Beta2, Nkx2.2, Nkx6.1, GLUT2, PC2, ZnT-8, and those described in Zhang et al, Diabetes. 50(10):2231-6 (2001). In some embodiment, the β-cell marker is a nuclear β-cell marker. In some embodiments, the β-cell marker is PDX-1 or PH3.

[0060] The term "synergy" or "synergistic" as used herein refers to the interaction of two or more agents so that their combined effect is greater than each of their individual effects at the same dose alone.

[0061] The term "additive" as used herein in the context of one agent has an additive effect on a second agent, refers to an increase in effectiveness of a first agent in the presence of a second agent as compared to the use of the first agent alone. Stated in another way, the second agent can function as an agent which enhances the physiological response of an organ or organism to the presence of a first agent. Thus, a second agent will increase the effectiveness of the first agent by increasing an individual's response to the presence of the first agent.

[0062] The term "selectively target" as used herein refers to the ability of the islet-targeting molecule to home in on or bind to a pancreatic islet target cell with a greater affinity than to non-target cells {e.g., non-islet cells). For example, islet-targeting molecule can bind to a pancreatic islet targeting cell with about 10%, about 20%, about 30%, about 40%, preferably about 50%, more preferably about 60%, more preferably about 70%, still more preferably about 80%, still more preferably about 90%, still more preferably about 100% or greater affinity for the target pancreatic islet endothelial cell relative to non- target cells. [0063] The term "carrier particle" as used herein refers to any entity with the capacity to associate with and carry (or transport) an agent in the body. As discussed herein in some embodiments, a carrier particle can carry both an insoluble agent and a soluble agent simultaneously. In alternative embodiments, a carrier particle can carry an insoluble agent or a soluble agent. Carrier particles can be a lipid particle, such as but not limited to a liposome or a protein or peptide carrier particle. Carrier particles as disclosed herein include any carrier particle modifiable by attachment of an islet-targeting molecule known to the skilled artisan. Carrier particles include but are not limited to liposomal or polymeric nanoparticles such as liposomes, proteins, and non-protein polymers. Carrier particles can be selected according to (i) their ability to transport the agent of choice and (ii) the ability to associate with the islet-targeting molecule as disclosed herein.

[0064] The term "nanoparticle" as used herein refers to a microscopic particle whose size is measured in nanometers. A carrier particle here can be a nanoparticle.

[0065] The term "lipid particle" refers to lipid vesicles such as liposomes or micelles.

[0066] The term "micelle" as used herein refers to an arrangement of surfactant molecules (surfactants comprise a non-polar, lipophilic "tail" and a polar, hydrophilic "head"). As the term is used herein, a micelle has the arrangement in aqueous solution in which the non-polar tails face inward and the polar heads face outward. Micelles are typically colloid particles formed by an aggregation of small molecules and are usually microscopic particles suspended in some sort of liquid medium, e.g., water, and are between one nanometer and one micrometer in size. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center. This type of micelle is known as a normal phase micelle (oil-in- water micelle). Inverse micelles have the headgroups at the center with the tails extending out (water-in-oil micelle). Micelles are approximately spherical in shape. Other phases, including shapes such as ellipsoids, cylinders, and bilayers are also possible. The shape and size of a micelle is a function of the molecular geometry of its surfactant molecules and solution conditions such as surfactant concentration, temperature, pH, and ionic strength. The process of forming micellae is known as micellisation.

[0067] The term "polymer" as used herein, refers to a linear chain of two or more identical or non- identical subunits joined by covalent bonds. A peptide is an example of a polymer that can be composed of identical or non-identical amino acid subunits that are joined by peptide linkages.

[0068] The term "stabilized liposome" as used herein refers to a liposome that comprises a cryoprotectant and/or a long-circulating agent.

[0069] The terms "encapsulation" and "entrapped," as used herein, refer to the incorporation of an agent in a lipid particle. An agent can be present in the aqueous interior of the lipid particle, for example a hydrophilic agent. In one embodiment, a portion of the encapsulated agent takes the form of a precipitated salt in the interior of the liposome. The agent may also self -precipitate in the interior of the liposome. In alternative embodiments, an agent can be incorporated into the lipid phase of a carrier particle, for example a hydrophobic and/or lipophilic agent.

[0070] The term "affinity binding moiety" refers to an agent that homes in on or preferentially associates or binds to at least one of the following selected from; a particular tissue, cell type, cell surface marker, cell surface receptor, infecting agent or other area of interest, and the like. Examples of an affinity binding moiety includes, but is not limited to, an antibody, an oligonucleotide, an antigen, an antibody or functional fragment thereof, a ligand, a receptor, one member of a specific binding pair, a polyamide including a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a steroid or steroid derivative, a hormone, e.g., estradiol or histamine, a hormone-mimic, e.g., morphine, or other compound having binding specificity for a target. In the methods of the present invention, an affinity binding moiety promotes localization of the attached islet-targeting molecule to a cell to be delivered, for example e.g., a stem cell or progenitor cell, such as an endothelial progenitor cell (EPC).

[0071] The term "affinity moiety" refers to a molecule on the surface of a particular cell type, e.g., a stem cell or progenitor cell, such as an endothelial progenitor cell (EPC). Examples of an affinity moiety include, but are not limited to, an antibody, an antigen binding fragment of an antibody, an antigen, a ligand, a receptor, one member of a specific binding pair, a polyamide including a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a steroid or steroid derivative, a hormone, e.g., estradiol or histamine, a hormone-mimic, e.g., morphine, or other compound having binding specificity for a cellular target.

[0072] The term "marker" as used herein describes a characteristic and/or phenotype of a cell. Markers can be referred to as "cell-surface markers" and are often a cell-surface protein or glycoprotein expressed on the surface of a cell which can be used for binding of a targeting moiety to a target cell of interest. Markers will vary with specific cells. Markers are characteristics, whether morphological, functional or biochemical (enzymatic) characteristics particular to a cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a cell marker can also be any molecule found within a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art.

[0073] The term "progenitor" or "precursor" cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by

differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[0074] The term "stem cell" as used herein, refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term "stem cell" refers to a subset of progenitors that have the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one embodiment, the term stem cell refers generally to a naturally occurring mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also "multipotent" because they can produce progeny of more than one distinct cell type, but this is not required for "stem-ness." Self -renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self- renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as stem cells might proceed toward a differentiated phenotype, but then "reverse" and re-express the stem cell phenotype, a term often referred to as "dedifferentiation" or "reprogramming" or "retrodifferentiation" by persons of ordinary skill in the art.

[0075] In the context of cell ontogeny, the adjective "differentiated", or "differentiating" is a relative term meaning a "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, stem cells can differentiate to lineage-restricted precursor cells (such as a mesodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as an cardiomyocyte precursor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further. [0076] The term "pancreas" refers to a glandular organ that secretes digestive enzymes and hormones. In humans, the pancreas is a yellowish organ about 7 in. (17.8 cm) long and 1.5 in. (3.8 cm) wide. It lies beneath the stomach and is connected to the small intestine, muscular hose-like portion of the

gastrointestinal tract extending from the lower end of the stomach (pylorus) to the anal opening. Most of the pancreatic tissue consists of grapelike clusters of cells that produce a clear fluid (pancreatic juice) that flows into the duodenum through a common duct along with bile from the liver. Pancreatic juice contains three digestive enzymes: tryptase, amylase, and lipase that, along with intestinal enzymes, complete the digestion of proteins, carbohydrates, and fats, respectively. Scattered among the enzyme -producing cells of the pancreas are small groups of endocrine cells, called the islets of Langerhans that secrete two hormones, insulin and glucagon. The pancreatic islets contain several types of cells: alpha-2 cells, which produce the hormone glucagon; beta cells (also referred to herein as "pancreatic β-cells"), which manufacture the hormone insulin; and alpha-1 cells, which produce the regulatory agent somatostatin. These hormones are secreted directly into the bloodstream, and together, they regulate the level of glucose in the blood. Insulin lowers the blood sugar level and increases the amount of glycogen (stored carbohydrate) in the liver; glucagon has the opposite action. Failure of the insulin-secreting cells to function properly results in diabetes or diabetes mellitus.

[0077] As used herein, an "antibody" or "functional fragment" of an antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab')₂ fragments, F(ab) fragments, Fv fragments, single domain antibodies, dimeric and trimeric antibody fragment constructs, minibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule and/or which bind a cell surface antigen. The term "antibody" also encompasses antibodies and fragments thereof, for example monoclonal antibodies or monoclonal antibody fragments such as, for example, Fab and F(ab')2 receptor.

[0078] As used herein, the term "agent" refers to an agent that can be transported by the carrier particle and islet-targeting molecule to the target pancreatic islet endothelial cell, for example a CE cell. An agent can be a chemical molecule of synthetic or biological origin. In some embodiments, an agent is generally a molecule that can be used in a pharmaceutical composition, for example the agent is a therapeutic agent. An agent as used herein also refers to any chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition, and are herein referred to as "therapeutic agents". An agent for use in the invention as disclosed herein can affect the body therapeutically, or which can be used in vivo for diagnosis. Examples of therapeutic agents include agents used in the treatment of diabetes, including compounds, therapeutic nucleic acids including nucleic acid analogs, RNAi agents and modified synthetic RNAs (modRNA). An agent can be a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject for imaging purposes in the subject, for example to monitor the presence or progression of disease or condition, and are herein referred to as "imaging agents" or

"diagnostic agents".

[0079] The term "agent" also typically refers to any entity which is normally not present or not present at the levels being administered in the target cell. Agent can be selected from a group comprising:

chemicals; small molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; aptamers; antibodies; or fragments thereof. A nucleic acid sequence can be RNA or DNA, and can be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide or fragment thereof can be any protein of interest, for example, but are not limited to: mutated proteins; therapeutic proteins and truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins can also be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, minibodies, triabodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.

[0080] The term "hydrophilic" as used herein refers to a molecule or portion of a molecule that is typically charge -polarized and capable of hydrogen bonding, enabling it to dissolve more readily in water than in oil or other hydrophobic solvents. Hydrophilic molecules are also known as polar molecules and are molecules that readily absorb moisture, are hygroscopic, and have strong polar groups that readily interact with water. A "hydrophilic" polymer as the term is used herein, has a solubility in water of at least 100 mg/ml at 25 °C.

[0081] The term "soluble agent" or "hydrophilic agent" and "hydrophilic drug" are used interchangeably herein, refers to any organic or inorganic compound or substance having biological or pharmacological activity and adapted or used for a therapeutic purpose having a water solubility greater than 10 mg/ml.

[0082] The term "hydrophobic" as used herein refers to molecules that tend to be non-polar and prefer other neutral molecules and non-polar solvents. Hydrophobic molecules in water often cluster together. Water on hydrophobic surfaces will exhibit a high contact angle. Examples of hydrophobic molecules include the alkanes, oils, fats, and greasy substances in general. Hydrophobic materials are used for oil removal from water, the management of oil spills, and chemical separation processes to remove non-polar from polar compounds. Hydrophobic molecules are also known as non-polar molecules. Hydrophobic molecules do not readily absorb water or are adversely affected by water, e.g., as a hydrophobic colloid. A "hydrophobic" polymer as the term is used herein has a solubility in water less than 10 mg/ml at 25°C, preferably less than 5 mg/ml, less than 1 mg/ml or lower.

[0083] The term "lipophilic" as used herein is used to refer to a molecule having an affinity for lipid molecules or fat molecules, pertaining to or characterized by lipophilia. Lipophilic or fat-liking molecules refers to molecules with an ability to dissolve in fats, oils, lipids, and non-polar solvents, for example such as hexane or toluene. Lipophilic substances tend to dissolve in other lipophilic substances, while hydrophilic (water-loving) substances tend to dissolve in water and other hydrophilic substances.

Lipophilicity, hydrophobic and non-polarity (the latter as used to describe intermolecular interactions and not the separation of charge in dipoles) all essentially describe the same molecular attribute; the terms are often used interchangeably

[0084] The term "insoluble agent" or "hydrophobic agent" or "hydrophobic drug" are used

interchangeably herein, refers to any organic or inorganic compound or substance having biological or pharmacological activity and adapted or used for a therapeutic purpose having a water solubility of less than 10 mg/ml. Typically an insoluble agent is an agent which is water insoluble, poorly water soluble, or poorly soluble in such as those agents having poor solubility in water at or below normal physiological temperatures, that is having at least less than lOmg/ml, such as about <5 mg/ml at physiological pH (6.5- 7.4), or about <1 mg/ml, or about <0.1 mg/ml.

[0085] The term "aqueous solution" as used herein includes water without additives, or aqueous solutions containing additives or excipients such as pH buffers, components for tonicity adjustment, antioxidants, preservatives, drug stabilizers, etc., as commonly used in the preparation of pharmaceutical formulations.

[0086] The term "protein" as used herein, refers to a compound that is composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation.

Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids.

[0087] The incorporation of non-natural amino acids, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the peptides (or other components of the composition, with exception for protease recognition sequences) is desirable in certain situations. D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid- containing forms. Thus, the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane -permanent complexes (see below for further discussion), and prolonged intravascular and interstitial lifetimes when such properties are desirable. The use of D-isomer peptides can also enhance transdermal and oral trans- epithelial delivery of linked drugs and other cargo molecules. Additionally, D-peptides cannot be processed efficiently for major histocompatibility complex class II-restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism. Peptide conjugates can therefore be constructed using, for example, D-isomer forms of cell penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides.

[0088] The term "derivative" as used herein refers to polypeptides, peptides and antibodies which have been chemically modified, for example but not limited to by techniques such as ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or addition of other molecules.

[0089] As used herein, "variant" with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild- type polynucleotide or polypeptide). A "variant" of an islet-targeting peptide, for example the amino acid of SEQ ID NO:l, is meant to refer to a molecule substantially similar in structure and function to either the entire molecule of SEQ ID NO:l, or to a fragment thereof, where the function of the variant is substantially the same ability to bind to a pancreatic islet endothelial cell, such as a CE cell as compared to the wild type islet-targeting peptide of SEQ ID NO:l. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules not found in the other, or if the sequence of amino acid residues is not identical.

[0090] The term "functional derivative" or "functional fragment" or "mimetic" are used interchangeably herein, and refers to a molecule or compound which possess a biological activity (either functional or structural) that is substantially similar to a biological activity of the entity or molecule it is a functional derivative of. The term functional derivative is intended to include the fragments, variants, analogues or chemical derivatives of a molecule.

[0091] The term "fragment" of a polypeptide, protein or peptide or molecule as used herein refers to any contiguous polypeptide subset of the molecule. Fragments of an islet-targeting peptide, such as, for example a fragment of SEQ ID NO:l can have the same binding affinity for binding to a pancreatic islet endothelial cell as that of the full length islet-targeting peptide of SEQ ID NO: 1. Stated another way, a fragment of an islet-targeting peptide is a fragment of SEQ ID NO:l which can bind with the same, or lower or higher affinity to its ligand on the target pancreatic islet endothelial cell. Fragments as used herein typically are soluble (i.e., not membrane bound).

[0092] The term " functional fragments" as used herein is a polypeptide having an amino acid sequence which is smaller in size than, but substantially homologous to, the polypeptide it is a fragment of, and where the functional fragment polypeptide sequence is about at least 50%, or 60% or 70% or at 80% or 90% or 100% or greater than 100%, for example 1.5-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold effective biological action as the polypeptide from which it is a fragment of. Functional fragment polypeptides may have additional functions that can include decreased antigenicity, increased DNA binding (as in transcription factors), or altered RNA binding (as in regulating RNA stability or degradation).

[0093] Fragments of an islet-targeting peptide, for example functional fragments of SEQ ID NO: l useful in the methods as disclosed herein have at least 30% of the ability of SEQ ID NO: 1 to target pancreatic islet endothelial cells. Stated another way, a fragment or functional fragment of an islet-targeting peptide which result in at least 30% of the same activity as compared to full length peptide, for example functional fragments of SQE ID NO: l to bind to pancreatic islet endothelial cells. It can also include fragments that decrease the wild type activity of one property by at least 30%. Fragments as used herein are soluble (i.e. not membrane bound). A "fragment" can be at least about 6, at least about 9 or more nucleic or amino acids, and all integers in between. Exemplary fragments include C-terminal truncations, N-terminal truncations, or truncations of both C- and N-terminals (e.g., deletions of, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 8, at least 10, or more amino acids deleted from the N-termini, the C-termini, or both). One of ordinary skill in the art can create such fragments by simple deletion analysis. Such a fragment of an islet-targeting peptide of SEQ ID NO: l can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or more than 10 amino acids deleted from the N- terminal and/or C-terminal amino acids of an integrin or integrin ligand as those proteins are defined herein. Persons of ordinary skill in the art can easily identify the minimal peptide fragment of an integrin and/or integrin ligand useful as targeting agents and in the compositions and methods as disclosed herein, by sequentially deleting N- and/or C-terminal amino acids from the integrin and/or integrin ligand and assessing the function of the resulting peptide fragment to bind their respective binding partner, i.e., of a fragment integrin to bind its respective integrin ligand, and/or an integrin ligand fragment to bind its integrin. One can create functional fragments with multiple smaller fragments. These can be attached by bridging peptide linkers. One can readily select linkers to maintain wild type conformation. In some embodiments, a fragment of islet-targeting peptide can comprise fragments of SEQ ID NO: l joined together in series.

[0094] As used herein, "homologous" or "homologues" are used interchangeably, and when used to describe a polynucleotide or polypeptide, indicates that two polynucleotides or polypeptides, or designated sequences thereof, when optimally aligned and compared, for example using BLAST, version 2.2.14 with default parameters for an alignment (see below) are identical, with appropriate nucleotide insertions or deletions or amino-acid insertions or deletions, in at least 70% of the nucleotides, usually from about 75% to 99%, and more preferably at least about 98 to 99% of the nucleotides. The term "homolog" or

"homologous" as used herein also refers to homology with respect to structure and/or function. With respect to sequence homology, sequences are homologs if they are at least 50%, at least 60 at least 70%, at least 80%, at least 90%, at least 95% identical, at least 97% identical, or at least 99% identical. The term "substantially homologous" refers to sequences that are at least 90%, at least 95% identical, at least 97% identical or at least 99% identical. Homologous sequences can be the same functional gene in different species.

[0095] Determination of homologs of the genes or peptides of the present invention can be easily ascertained by the skilled artisan. The terms "homology", "identity" and "similarity" refer to the degree of sequence similarity between two optimally aligned peptides or between two optimally aligned nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by similar amino acid residues (e.g., similar in steric and/or electronic nature such as, for example conservative amino acid substitutions), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of similar or identical amino acids at positions shared by the compared sequences, respectively. A sequence which is "unrelated" or "non-homologous" shares less than 40% identity, though preferably less than 25% identity with a sequence of the present application.

[0096] The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties. Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

"Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Thus, a "conservative substitution" of a particular amino acid sequence refers to substitution of those amino acids that are not critical for polypeptide activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitution of even critical amino acids does not reduce the activity of the peptide, (i.e., the ability of the peptide to penetrate the BBB). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H.

Freeman and Company (1984).) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of the peptide (e.g., the ability of an binding moiety to bind or associate with a nucleic acid). Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents. Conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol (1999) 217: 721-739 and Taylor et al, J. Theor. Biol. 119(1986):205-218 and S. French and B. Robson, . Mol. Evol. 19(1983):171. Conservative amino acids encompassed for use in the methods as disclosed herein include conservative substitutions that are suitable for amino acids on the exterior of a protein or peptide {e.g., amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.

[0097] As used herein, the term "non-conservative" refers to substituting an amino acid residue for a different amino acid residue that has different chemical properties. The non-conservative substitutions include, but are not limited to aspartic acid (D) being replaced with glycine (G); asparagine (N) being replaced with lysine (K); or alanine (A) being replaced with arginine (R). "Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed can be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.

[0098] In one embodiment, the term "islet-targeting peptide homolog" refers to an amino acid sequence that has at least 40% homology or identity to the full length amino acid sequence of the islet-targeting peptide of SEQ ID NO: l and which binds or associates with pancreatic islet endothelial cells, e.g., CE cells. As a non-limiting example, an islet-targeting peptide fragment homologue is at least 40% homologous or identical to the full length amino acid sequence of SEQ ID NO: l, more preferably at least about 50% homologous or identical, or at least about 60% homologous or identical, or at least about 70% homologous or identical, or at least about 75% homologous or identical, or at least about 80%

homologous or identical, or at least about 85% homologous or identical, or at least about 90%

homologous or identical, or at least about 95% homologous or identical. As discussed above, the homology is at least about 40% to 99% and all integers in between (i.e., 45%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.).

[0099] In one embodiment, the term "islet-targeting peptide fragment" refers to an amino acid sequence that comprises at least 3, or at least 4, or at least 5, or at least 6 or at least 7 or at least 8, or at least 9 consecutive amino acids of the full length amino acid sequence of the islet-targeting peptide of SEQ ID NO: l and which binds or associates with pancreatic islet endothelial cells, e.g., CE cells. As a non- limiting example, an islet-targeting peptide fragment is at least 40% identical to the full length amino acid sequence of SEQ ID NO: l, more preferably at least about 50% identical, or at least about 60% identical, or at least about 70% identical, or at least about 75% identical, or at least about 80% identical, or at least about 85% identical, or at least about 90% identical, or at least about 95% identical or more. As discussed above, the identity is at least about 40% to 99% and all integers in between (e.g., 45%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.).

[00100] As used herein, the term "sequence identity" means that two polynucleotide or amino acid sequences are identical (e.g., on a nucleotide-by-nucleotide or residue -by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T. C, G. U. or 1) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[00101] The term "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which can include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence can be a subset of a larger sequence. The term "similarity," when used to describe a polypeptide, is determined by comparing the amino acid sequence and the conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

[00102] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[00103] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981), which is incorporated by reference herein), by the homology alignment algorithm of Needleman and Wunsch ( . Mol. Biol. 48:443- 53 (1970), which is incorporated by reference herein), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), which is incorporated by reference herein), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. (See generally Ausubel et al. (eds.), Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999)). [00104] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show the percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle ( . Mol. Evol. 25:351-60 (1987), which is incorporated by reference herein). The method used is similar to the method described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989), which is incorporated by reference herein). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

[00105] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described by Altschul et al. (J. Mol. Biol. 215:403- 410 (1990), which is incorporated by reference herein). (See also Zhang et al. , Nucleic Acid Res. 26:3986- 90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated by reference herein). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information internet web site. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990), supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992), which is incorporated by reference herein) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. [00106] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), which is incorporated by reference herein). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1 , more typically less than about 0.01, and most typically less than about 0.001.

[00107] As used herein, "gene silencing" induced by RNA interference refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, about 10%, about 20%, about 30%, about

40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without introduction of RNA interference. In one preferred

embodiment, the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.

[00108] The term "reduced" or "reduce" as used herein generally means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease, or any integer decrease between 10-100% as compared to a reference level.

[00109] The term "increased" or "increase" as used herein generally means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any integer increase between 10- 100% as compared to a reference level, or about a 2-fold, or about a 3-fold, or about a 4-fold, or about a 5- fold or about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[00110] As used herein, the term "RNAi" refers to any type of interfering RNA, including but are not limited to, siRNA, shRNAi, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of down-stream processing of the RNA (e.g., . although siRNAs are believed to have a specific method of in vivo processing resulting in the cleavage of mRNA, such sequences can be incorporated into the vectors in the context of the flanking sequences described herein). RNAi molecules as used herein are any interfering RNA, or RNA interference molecules, such as nucleic acid molecules or analogues thereof, for example RNA-based molecules that inhibit gene expression. RNAi refers to a means of selective post-transcriptional gene silencing. RNAi, for example use of a siRNA can result in the destruction of specific mRNA, or prevents the processing or translation of RNA, such as mRNA. [00111] The term "short interfering RNA" (siRNA), also referred to herein as "small interfering RNA" is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi. An siRNA can be chemically synthesized, it can be produced by in vitro transcription, or it can be produced within a host cell. siRNA molecules can also be generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated.

[00112] The term "therapeutically effective amount" refers to an amount that is sufficient to effect a therapeutically significant reduction in a symptom associated with diabetes when administered to a typical subject who has Type 1 or Type 2 diabetes. A therapeutically significant reduction in a symptom is, e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150% or more as compared to a control or non-treated subject. The amount can also cure or cause the cancer to go into remission, slow the course of cancer progression, slow or inhibit tumor growth, slow or inhibit tumor metastasis, slow or inhibit the establishment of secondary tumors at metastatic sites, or inhibit the formation of new tumor metastasis.

[00113] The term "treat" or "treatment" refer to the therapeutic treatment, wherein the object is to prevent or slow down the development or spread of a disease, inhibiting a disease, i.e., arresting or slowing down the development of a disease or a clinical symptom of the disease; or relieving a disease, i.e., causing regression of a disease or a clinical symptom of the disease. Beneficial or desired clinical results include, but are not limited to, alleviation of a symptoms, diminishment of extent of a disease, stabilized (i.e., not worsening) state of a disease, delay or slowing of the disease progression, amelioration or palliation of a disease state, and remission (whether partial or total), whether detectable or undetectable. In some embodiments, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. In some embodiments, "treating" or "treatment" of a diabetes can include preventing the disease, i.e. preventing a clinical symptom of the disease in a subject that can be exposed to, or predisposed to, a disease, but does not yet experience or display a symptom of the disease (e.g., do not display a symptom of diabetes) but where the subject is predisposition to develop diabetes, e.g., obesity, genetic mutation linked to developing diabetes (e.g., a mutation or polymorphism in a diabetes susceptibility gene) etc.

[00114] The term "effective amount" as used herein refers to the amount of therapeutic agent of pharmaceutical composition to alleviate at least some of the symptoms of the disease or disorder, e.g., diabetes. The term "effective amount" includes within its meaning a sufficient amount of

pharmacological composition to provide the desired effect. The exact amount required will vary depending on factors such as the type of diabetes (e.g., Type 1 or Type II), the severity of the diabetes, the age of the subject, the species being treated, the age and general condition of the subject, the mode of administration and so forth. Thus, it is not possible to specify the exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. [00115] The terms "composition" or "pharmaceutical composition" are used interchangeably herein and refer to compositions or formulations that usually comprise an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells. Such compositions can be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, parenteral, intravenous, intraarterial, subcutaneous, intranasal, sublingual, intraspinal, intracerebroventricular, and the like. An islet-targeting molecule: earner particle: agent complex, and/or islet-targeting molecule: affinity binding moiety: EPC cell complex. In another embodiment, another variation could include both carrier particle and affinity binding moiety for EPC targeting, i.e. islet-targeting molecule: affinity binding moiety: carrier particle: EPC cell complex can be administered a composition as disclosed herein can be part of a subject, for example for therapeutic, diagnostic, or optionally, prophylactic purposes to prevent the onset of diabetes. In some embodiments, an islet-targeting molecule: carrier particle: agent complex, and/or islet-targeting molecule: affinity binding moiety: EPC cell complex can also be added to cultured pancreatic islet endothelial cells, for example pancreatic islet endothelial cells as part of an assay for screening potential pharmaceutical compositions, and the pancreatic islet endothelial cells can be part of a transgenic animal for research purposes. In addition, compositions for topical (e.g., oral mucosa, respiratory mucosa) and/or oral administration can form solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral rinses, or powders, as known in the art are described herein. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, University of the Sciences in Philadelphia (2005) Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed.

[00116] The terms "composition" or "pharmaceutical composition" are used interchangeably herein and refer to compositions or formulations that usually comprise an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells. Such compositions can be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, ocular and nasal administration and the like.

[00117] The "pharmaceutically acceptable carrier" means any pharmaceutically acceptable means to mix and/or deliver the targeted delivery composition to a subject. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and is compatible with administration to a subject, for example a human. For the clinical use of the methods of the present invention, targeted delivery composition of the invention is formulated into pharmaceutical compositions or pharmaceutical formulations for parenteral administration, e.g., intravenous; mucosal, e.g., intranasal; enteral, e.g., oral; topical, e.g., transdermal; ocular, e.g., via corneal scarification or other mode of administration. The pharmaceutical composition contains a compound of the invention in combination with one or more pharmaceutically acceptable ingredients. The carrier can be in the form of a solid, semi-solid or liquid diluent, cream or a capsule. These pharmaceutical preparations are a further object of the invention. Usually the amount of active compounds is between 0.1- 95% by weight of the preparation, preferably between 0.2-20% by weight in preparations for parenteral use and preferably between 1 and 50% by weight in preparations for oral administration. The

"pharmaceutically acceptable carrier" means any pharmaceutically acceptable means to mix and/or deliver the targeted delivery composition to a subject. The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and is compatible with administration to a subject, for example a human. A diblock copolymer as described herein is a pharmaceutically acceptable carrier as the term is used herein. Other pharmaceutically acceptable carriers can be used in combination with the block copolymer carriers as described herein.

[00118] The term "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically, " "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[00119] As used herein, the terms "administering," and "introducing" are used interchangeably herein and refer to the placement of the pharmaceutical composition comprising an islet-targeting molecule: carrier particle: agent complex, and/or islet-targeting molecule: affinity binding moiety: EPC cell complex as disclosed herein into a subject by a method or route which results in at least partial localization of the complexes and agents (e.g., agents encapsulated or on the outside of the carrier particle, or EPC) at a desired site. The agents of the present invention can be administered by any appropriate route which results in an effective treatment in the subject.

[00120] The term "disease" or "disorder" is used interchangeably herein, and refers to any alteration in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder can also relate to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, inderdisposion or affectation.

[00121] The terms "diabetes" and "diabetes mellitus" are used interchangeably herein. The World Health Organization defines the diagnostic value of fasting plasma glucose concentration to 7.0 mmol/1 (126 mg/dl) and above for Diabetes Mellitus (whole blood 6.1 mmol/1 or 110 mg/dl), or 2-hour glucose level 11.1 mmol/L or higher (200 mg/dL or higher). Other values suggestive of or indicating high risk for Diabetes Mellitus include elevated arterial pressure 140/90 mm Hg or higher; elevated plasma triglycerides (1.7 mmol/L; 150 mg/dL) and/or low HDL-cholesterol (less than 0.9 mmol/L, 35 mg/dl for men; less thanl.O mmol/L, 39 mg/dL women); central obesity (males: waist to hip ratio higher than 0.90; females: waist to hip ratio higher than 0.85) and/or body mass index exceeding 30 kg/m²;

microalbuminuria, where the urinary albumin excretion rate 20 μg/min or higher, or albumin:creatinine ratio 30 mg/g or higher). The term diabetes encompasses all forms of diabetes, e.g., Type 1, Type 2 and Type 1.5.

[00122] As used herein, the term "treating" and "treatment" refers to administering to a subject an effective amount of a composition so that the subject as a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term "treatment" can include prophylaxis. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already diagnosed with a cardiac condition, as well as those likely to develop a cardiac condition due to genetic susceptibility or other factors such as weight, diet and health.

[00123] As used herein, the terms "administering," "introducing" and "transplanting" are used interchangeably in the context of the placement of a composition as disclosed herein, e.g., islet-targeting molecule: carrier particle: agent complex and/or an islet-targeting molecule: affinity binding moiety: EPC cell complex into a subject, by a method or route which results in at least partial localization of the introduced composition at a desired site, such that the islet-targeting molecule can transport the complex to pancreatic islet endothelial cells in the subject. The compositions as disclosed herein, e.g., islet- targeting molecule: carrier particle: agent complex and/or an islet-targeting molecule affinity binding moiety: EPC cell complex can be administered directly to the pancreas, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the islet-targeting molecule and associated complex remain functional, and where the attached cells (e.g., EPCs) remain viable. The period of viability of the attached cells (e.g., stem cell or progenitor cells, such as EPCs) after administration to a subject can be as short as a few hours, e. g. twenty-four hours, to a few days, to as long as several years. In some instances, the cells can also be administered at a non-pancreatic location, such as in the liver or subcutaneously, and allow migration of the implanted stem cells or progenitor cells to the pancreatic islet endothelial cells by way of the attached islet-targeting molecule.

[00124] The term "autoimmune disease" is used interchangeably herein with "immune response mediated disorder" as used herein refers to disorders in which the hosts' immune system contributes to the disease condition either directly or indirectly. Examples of disorders which are mediated by the immune response include diabetes.

[00125] As used herein, the term "medicament" refers to an agent that promotes the recovery from, and/or alleviates a symptom of a diabetes-mediated condition.

[00126] As used herein, the term "patient" refers to a human in need of the treatment to be administered.

[00127] The term "subject" and "individual" are used interchangeably herein, and refer to an animal, for example a human, to whom treatment, including prophylactic treatment, with a composition as described herein, is provided. The term "mammal" is intended to encompass a singular "mammal" and plural "mammals," and includes, but is not limited: to humans, primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and bears. Preferably, the mammal is a human subject. As used herein, a "subject" refers to a mammal, preferably a human. The term "individual", "subject", and "patient" are used interchangeably. Preferably, the mammal is a human subject.

[00128] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a composition for delivering "a drug" includes reference to two or more drugs. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[00129] The terms "decrease," "reduced," "reduction," "decrease," or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, "reduced", "reduction" or "decrease" or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level. [00130] The terms "increased" 'increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[00131] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[00132] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[00133] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00134] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00135] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[00136] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[00137] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%. The present invention is further explained in detail by the following examples, but the scope of the invention should not be limited thereto.

Islet-targeting molecules.

[00138] In some embodiments, an islet targeting molecule is a peptide or an antibody, aptamer or other component that binds to pancreatic endothelial cells with high specificity.

[00139] In some embodiments, the islet-targeting peptide is an antibody or an antigen binding fragment thereof, for example but not limited to, a single chain antibody, a Fab portion of an antibody or a (Fab')₂ segment which binds to an antigen on the cell surface of the pancreatic CE cells. In some embodiments, where an islet targeting molecule is an antibody, the antibody can specifically bind to Ephrin A4 (Eph

A4), as disclosed in Yao et al. , Am. J. Path., 166(2); 625-636, which is incorporated herein in its entirety by reference. Any Anti-Eph A4 antibody can be used as an islet-targeting molecule, including anti-Eph A4 antibodies which are commercially available, for example, from AbChem™, Acris™,

Sinobiological™, Sigma®, Santa Cruz Biotechnology™ and similar companies. In some embodiments, an islet-targeting molecule which is an antibody or a fragment thereof can bind with specific affinity to any cell surface marker expressed on the islet endothelial cell. For example, but without limitation, in some embodiments, an islet-targeting molecule can be an antibody with specific binding affinity for at least one or more of Willebrand factor, CD86 (B7-2), ICOS ligand (ICOS-L), endothelial surface phenotypic markers including CD 105, CD31, and CD 146, endothelial cell leucocyte adhesion molecule- 1, acetylated low density lipoprotein, VEGF and angiostatic factors such as endostatin and pigment epithelial-derived factor.

[00140] In some embodiments, an islet-targeting molecule as disclosed herein is a polynucleic acid aptamer, or nucleic acid analogue or similar. Without wishing to be bound by theory, aptamers are single- stranded DNA or RNA molecules that can fold into a 3D structure. With this 3D structure, they can bind with ultra-high affinity and specificity to their target molecules (e.g., cell-surface markers expressed on islet endothelial cells). Aptamers can be generated from a combinatorial library in a cascade process called systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have significant advantages compared to other islet-targeting molecules in that they are very small, bind to their target molecules with very high affinity and specificity and cause no immunogenic reactions, and are also non- toxic. In addition, they are easily synthesized in vitro. [00141] Aptamers have been generated for various cell populations, and can be covalently immobilized to a carrier particle as disclosed herein or to an EPC cell as disclosed herein. An aptamer for targeting porcine CD31 -positive cells have been developed to and covalently bound to starPEG-coated devices, which demonstrated that aptamers against CD31 -positive cells were able to attach the cells selectively to the synthetic devices (Hoffman et al, J. Biomed. Mat. Res. A. (2008) 84:614-621).

[00142] Accordingly, in some embodiments, an aptamer can be used as an islet-targeting molecule as disclosed herein. In some embodiments, an aptamer, or oligonucleotide islet-targeting molecule is specific for binding to Eph A4 expressing islet endothelial cells. In some embodiments, an islet-targeting molecule is an aptamer which binds to CD31 expressed on islet endothelial cells, such as an endothelial-cell-binding aptamer as disclosed in Strahm Y et al., J Invasive Cardiol., (2010) Oct;22(10):481-7, which is incorporated herein in its entirety by reference. In some embodiments, an islet-targeting molecule is an aptamer which binds to one or more cell-surface markers expressed on islet endothelial cells, selected from, but not limited to, Willebrand factor, CD86 (B7-2), ICOS ligand (ICOS-L), endothelial surface phenotypic markers including CD 105, CD31, and CD 146, endothelial cell leucocyte adhesion molecule- 1, acetylated low density lipoprotein, VEGF and angiostatic factors such as endostatin and pigment epithelial-derived factor. Methods to generate islet endothelial cell specific aptamers for use as islet- targeting molecules are disclosed in Meyer et al. , J. Nucleic Acids, (2011), Article ID 904750, 18 pages, entitled "Cell-Specific Aptamers as Emerging Therapeutics" which is incorporated herein in its entirety by reference.

Islet-targeting peptide

[00143] In some embodiments, an islet targeting molecule is a peptide. In some embodiments, an islet targeting molecule is a peptide which comprises SEQ ID NO: 1 of the amino acid sequence

CHVLWSTRKC (SEQ ID NO: 1) (also referred to herein as "Pep I") or a fragment or variant thereof, e.g., at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9 contiguous amino acids of SEQ ID NO: 1. In some embodiments, an islet targeting peptide fragment can comprise a deletion of one or more amino acids from either the C-terminal or N-terminal or both C- and N-terminal of the amino acid sequence of SEQ ID NO: 1. In some embodiments, an islet targeting peptide fragment has a similar biological activity of binding to pancreatic islet capillary endothelial (CE) cells as compared to the wild type islet targeting peptide of SEQ ID NO: 1. In some embodiments, an islet targeting peptide fragment has at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or 1.2-fold, or 1.5-fold, or 1.75 fold, or 2-fold, or 3 -fold, or 4-fold, or 5 -fold or greater than 5 -fold biological activity of binding to pancreatic islet CE cells as compared to the wild type islet targeting peptide of SEQ ID NO: 1.

[00144] In some embodiments, the islet targeting peptide can be a homologue of SEQ ID NO: 1 or any modification which does not decrease the biological activity of the amino acid sequence of SEQ ID NO: l to bind to and target islet CE cells. In some embodiments, a islet-targeting peptide homologue can have at least one variant amino acid (e.g., substituted amino acid, and/or deleted amino acid, or inserted amino acid) from any portion of the peptide of SEQ ID NO: 1. In some embodiments, where the homologue comprises at least one substitution, the substituted amino acid is a non-conservative or conservative amino acid, or can be a synthetic amino acid or amino acid analogue. In some embodiments, a islet-targeting peptide homologue can have at least one, or at least 2, or at least 3, or at least 4, or at least 5 or more variants (e.g., substituted amino acid, and/or deleted amino acid, or inserted amino acid) in the amino acid sequence as compared to the peptide of SEQ ID NO: 1, where the variation can be continuous or intermittent with the sequence of SEQ ID NO: 1. In some embodiments, an islet targeting peptide homologue has a similar biological activity of binding to pancreatic islet endothelial cells, e.g., islet capillary endothelial (CE) cells as compared to the wild type islet targeting peptide of SEQ ID NO: 1. In some embodiments, an islet targeting peptide homologue fragment has at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or 1.2-fold, or 1.5-fold, or 1.75 fold, or 2-fold, or 3-fold, or 4-fold, or 5-fold or greater than 5-fold biological activity of binding to pancreatic islet endothelial cells, e.g., islet capillary endothelial (CE) cells as compared to the wild type islet targeting peptide of SEQ ID NO: 1.

[00145] In some embodiments, the islet targeting molecule can be an islet targeting peptide which can comprise non-natural or synthetic amino acids, as disclosed herein. In some embodiments, an islet targeting molecule which is an antibody or aptamer can also comprise non-natural or synthetic amino acids which can be selected according to conventional approaches known by persons of ordinary skill in the art.

Carrier Particle

[00146] One aspect of the present invention relates to compositions and methods for the delivery of at least one islet-targeting molecule, e.g., an islet-targeting peptide which is associated with a carrier particle, wherein the carrier particle comprises an agent, for delivery of the agent to target pancreatic islet CE cells. As disclosed herein, the composition comprises a carrier particle comprising an insoluble agent and/or a soluble agent, wherein the carrier particle is attached to or conjugated to at least one islet-targeting molecule, where the islet-targeting molecule binds to (or has specific affinity for) to a cell surface marker expressed on pancreatic islet endothelial cells. As discussed previously, an islet-targeting molecule which binds to (e.g., has specific affinity for) a cell surface marker expressed on an islet endothelial cell can be, for example, but not limited to, a peptide, an antibody or aptamer, or modified versions thereof.

[00147] In some embodiments, the carrier particles are micro-lipid particles or nano-lipid particles, e.g., liposomes, spheres, micelles, or nanoparticles. In some embodiments the carrier particles are unilammar, (meaning the carrier particles comprise more than one layer or are multi-layered). In some embodiments, a first layer contains agents that facilitate cryoprotection, long half-life in circulation, or both (PEG, hyaluronan, others).

[00148] In some embodiments, as disclosed herein, the carrier particle can be a polymer, such as block co-polymer. In some embodiments, such a block co-polymer can be a PLGA-PEG co-polymer, for example, but not limited to [PLGA-b-PEG-COOH]n. In some embodiments, where a block co-polymer is [PLGA-b-PEG-COOH]n, there can be various blend composition of PLGA to PEG, for example different ratios such as (75:25, 50:50 etc. , and vice versa), and can in some embodiments, be or include other biodegradable polymers such as polycaprolactone, polylactic acid and polyglycolide.

[00149] In some embodiments, a co-polymer useful in the compositions and methods as disclosed herein is a synthetic biocompatible and biodegradable copolymer, for example, such as but not limited to any one or a combination of the following: polylactides, polyglycolides, polycaprolactones,

poly anhydrides, poly(glycerol sebacate), polyamides, polyure thanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxy valerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(acrylic acid), polyvinylpyrrolidone, polyhydroxycellulose, polymethyl methacrylate.

[00150] In some embodiments, a co-polymer useful in the compositions and methods as disclosed herein is a synthetic biocompatible and non-degradable copolymer, for example, such as but not limited to any one or a combination of the following: polyethylene glycol, polypropylene glycol, pluronic (Poloxamers 407, 188, 127, 68), poly(ethylenimine), polybutylene, polyethylene terephthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate, nitrocellulose.

[00151] In some embodiments, a co-polymer useful in the compositions and methods as disclosed herein is a Natural biodegradable polymer, for example, such as but not limited to any one or a combination of the following: chitin, chitosan, elastin, gelatin, collagen, silk, alginate, cellulose, poly- nucleic acids, poly( amino acids), hyaluronan, heparin, agarose, and/or pullulan.

[00152] In some embodiments, a copolymer useful in the compositions and methods as disclosed herein is can be a combination of biodegradable/biocompatible/natural polymers.

[00153] Carrier particles as disclosed herein include any carrier particle modifiable by attachment of at least one islet-targeting molecule known to the skilled artisan. Carrier particles include but are not limited to liposomal or polymeric nanoparticles such as liposomes, proteins, and non-protein polymers. Carrier particles can be selected according to (i) their ability to transport the agent of choice and (ii) the ability to associate with a targeting moiety as disclosed herein. In some embodiments, a carrier particle can comprise at least one, or at least about 2, or at least about 3, or between about 4-5, or between about 5-10, or between about 10-20, or between about 20-50, or between about 50-100, or between about 100-200, or between about 200-500 or more than 500, or any integer between 1-500 or more islet-targeting molecules per carrier particle. It is assumed that multiple islet-targeting molecules per carrier particle will increase the efficiency of targeting the carrier particle to target pancreatic islet endothelial cells, however, one of ordinary skill in the art should determine the maximum about of islet-targeting molecules without interfering with the ability of the effect of an agent attached on the outside of a carrier particle, or the ability of the carrier particle to release the agent at the site of the targeted pancreatic islet endothelial cell.

[00154] In some embodiments, carrier particles include colloidal dispersion systems, which include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. In some embodiments, a carrier particle is a liposome, a dendrimers, a nanocrystal, a quantum dot, a nanoshell or a nanorod, or similar structures.

[00155] In some embodiments, the carrier particle comprises a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al, Current Op. Biotech. (1995) 6:698-708). Other carrier particles are cellular uptake or membrane-disruption moieties, for example polyamines, e.g., spermidine or spermine groups, or poly lysines; lipids and lipophilic groups; polymyxin or polymyxin- derived peptides; octapeptin; membrane pore-forming peptides; ionophores; protamine; aminoglycosides; polyenes; and the like. Other potentially useful functional groups include intercalating agents; radical generators; alkylating agents; detectable labels; chelators; or the like.

[00156] One can use other carrier particles, for example lipid particle or vesicle, such as a liposome or microcrystal, which may be suitable for parenteral administration. The particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as the antisense oligonucleotide is contained therein. Positively charged lipids such as N-[I-(2,3dioleoyloxi)propyll-N,N,N-trimethyl- anunoniummethylsulfate, or "DOTAP," are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patents Nos. 4,880,635; 4,906,477;

4,911,928; 4,917,951; 4,920,016; and 4,921,757 which are incorporated herein by reference. Other non- toxic lipid based vehicle components may likewise be utilized to facilitate uptake of the agent carried {e.g., encapsulated or on the outside of the carrier particle) by the pancreatic islet endothelial cell.

[00157] In some embodiments, a carrier particle is a liposome. Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles possessing a single membrane bilayer or multilameller vesicles, onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer. In one preferred embodiment, the liposomes of the present invention are unilamellar vesicles. The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.

[00158] Liposomes useful in the methods and compositions as disclosed herein can be produced from combinations of lipid materials well known and routinely utilized in the art to produce liposomes. Lipids can include relatively rigid varieties, such as sphingomyelin, or fluid types, such as phospholipids having unsaturated acyl chains. "Phospholipid" refers to any one phospholipid or combination of phospholipids capable of forming liposomes. Phosphatidylcholines (PC), including those obtained from egg, soy beans or other plant sources or those that are partially or wholly synthetic, or of variable lipid chain length and unsaturation are suitable for use in the present invention.

[00159] Synthetic, semisynthetic and natural product phosphatidylcholines including, but not limited to, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy

phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholines for use in this invention. All of these phospholipids are commercially available. Further, phosphatidylglycerols (PG) and phosphatic acid (PA) are also suitable phospholipids for use in the present invention and include, but are not limited to, dimyristoylphosphatidylglycerol (DMPG), dilaurylphosphatidylglycerol (DLPG), dipalmitoylphosphatidylglycerol (DPPG),

distearoylphosphatidylglycerol (DSPG) dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dilaurylphosphatidic acid (DLPA), and dipalmitoylphosphatidic acid (DPP A).

Distearoylphosphatidylglycerol (DSPG) is the preferred negatively charged lipid when used in formulations. Other suitable phospholipids include phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, and phosphatidic acids containing lauric, myristic, stearoyl, and palmitic acid chains. For the purpose of stabilizing the lipid membrane, it is preferred to add an additional lipid component, such as cholesterol. Preferred lipids for producing liposomes according to the invention include

phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in further combination with cholesterol (CH). According to one embodiment of the invention, a combination of lipids and cholesterol for producing the liposomes of the invention comprise a PE:PC:Chol molar ratio of 3:1 : 1. Further, incorporation of polyethylene glycol (PEG) containing phospholipids is also contemplated by the present invention.

[00160] Liposomes useful in the methods and compositions as disclosed herein can be obtained by any method known to the skilled artisan. For example, the liposome preparation of the present invention can be produced by reverse phase evaporation (REV) method (see e.g., U.S. Pat. No. 4,235,871), infusion procedures, or detergent dilution. A review of these and other methods for producing liposomes can be found in the text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1. See also Szoka Jr. et al., (1980) Ann. Rev. Biophys. Bioeng. 9:467). A method for forming ULVs is described in Cullis et al , PCT Publication No. 87/00238, Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles". Multilamellar liposomes (MLV) can be prepared by the lipid-film method, wherein the lipids are dissolved in a chloroform-methanol solution (3: 1, vol/vol), evaporated to dryness under reduced pressure and hydrated by a swelling solution. Then, the solution is subjected to extensive agitation and incubation, e.g., 2 hour, e.g., at 37°C. After incubation, unilamellar liposomes (ULV) are obtained by extrusion. The extrusion step modifies liposomes by reducing the size of the liposomes to a preferred average diameter. Alternatively, liposomes of the desired size can be selected using techniques such as filtration or other size selection techniques. While the size-selected liposomes of the invention should have an average diameter of less than about 300 nm, it is preferred that they are selected to have an average diameter of less than about 200 nm with an average diameter of less than about 100 nm being particularly preferred. When the liposome of the present invention is a unilamellar liposome, it preferably is selected to have an average diameter of less than about 200 nm. The most preferred unilamellar liposomes of the invention have an average diameter of less than about 100 nm. It is understood, however, that multivesicular liposomes of the invention derived from smaller unilamellar liposomes will generally be larger and can have an average diameter of about less than 1000 nm. Preferred multivesicular liposomes of the invention have an average diameter of less than about 800 nm, and less than about 500 nm while most preferred multivesicular liposomes of the invention have an average diameter of less than about 300 nm.

[00161] In another embodiment, the carrier particle is a cyclodextrin-based nanoparticle. Polycation formulated nanoparticles have been used for drug delivery into the brain as well as for systemic delivery of siRNA. A unique cyclodextrin-based nanoparticle technology has been developed for targeted gene delivery in vivo. This delivery system consists of two components. The first component is a biologically non-toxic cyclodextrin-containing polycation (CDP). CDPs self-assemble with siRNA to form colloidal particles about 50 nm in diameter and protects si/shRNA against degradation in body fluids. Moreover, the CDP has been engineered to contain imidazole groups at their termini to assist in the intracellular trafficking and release of the nucleic acid. CDP also enables assembly with the second component. The second component is an adamantane-terminated polyethylene glycol (PEG) modifier for stabilizing the particles in order to minimize interactions with plasma and to increase the attachment to the cell surface targeting markers on the pancreatic islet endothelial cells). Thus, the advantages of this delivery system are: 1) since the CDP protects the siRNA from degradation, chemical modification of the nucleic acid is unnecessary, 2) the colloidal particles do not aggregate and have extended life in biological fluids because of the surface decoration with PEG that occurs via inclusion complex formation between the terminal adamantane and the cyclodextrins, 3) cell type-specific targeted delivery is possible because some of the PEG chains contain at least one or more islet-targeting molecule, e.g., an islet-targeting peptide, 4) it does not induce an immune response, and 5) in vivo delivery does not produce an interferon response even when a siRNA is used that contains a motif known to be immunostimulatory when delivered in vivo with lipids.

[00162] The glycosaminoglycan carrier particles disclosed in U.S. Patent Appl. No. 20040241248 and the glycoprotein carrier particles in WO 06/017195, which are incorporated herein in their entirety by reference, are useful in the methods of the present invention. Similar naturally occurring polymer-type carriers known to the skilled artisan are also useful in the methods of the present invention. [00163] Soluble non-protein polymers are also useful as carrier particles. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylrnethacrylamidephenol,

polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palitoyl residues. Furthermore, the therapeutic agents can be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels. The therapeutic agents can also be affixed to rigid polymers and other structures such as fullerenes or Buckeyballs.

Conjugation of islet-targeting molecule with the earner particle or Affinity binding moiety

[00164] In all aspects and embodiments of the present invention, an islet-targeting molecule can be associated with a carrier particle or an affinity binding moiety. In some embodiments, an islet-targeting molecule is for example, but not limited to, a peptide, antibody or aptamer and the like.

[00165] In some embodiments, a carrier particle as disclosed herein can be associated with the islet- targeting molecule. The association of a carrier particle with an islet-targeting molecule can be a non- covalent or covalent interaction, for example, by means of chemical cross-linkage or conjugation. In the composition and methods disclosed herein, an islet-targeting molecule is associated with a carrier particle, for example liposome.

[00166] As used herein, the term "associated with" means that one entity is in physical association or contact with another. Thus, a targeting moiety "associated with" a carrier particle can be either covalently or non-covalently joined to the carrier particle. The association can be mediated by a linker moiety, particularly where the association is covalent. The term "association" or "interaction" or "associated with" are used interchangeably herein and as used in reference to the association or interaction of a islet- targeting molecule, with a carrier particle for example, nanoparticle or liposome, refers to any association between the islet-targeting molecule with the carrier particle, for example a liposome comprising a hydrophilic agent and/or a hydrophobic agent, either by a direct linkage or an indirect linkage.

[00167] An indirect linkage includes an association between an islet-targeting molecule with a carrier particle for example liposome, wherein the islet-targeting molecule and the carrier particle are attached via a linker moiety, e.g., they are not directly linked. Linker moieties include, but are not limited to, chemical linker moieties. In some embodiments, a linker between a islet-targeting molecule and the carrier particle is formed by reacting the polymer and a linker selected e.g., from the group consisting of p-nitrophenyl chloroformate, carbonyldiimidazole(CDI), Ν,Ν'-disuccinimidyl carbonate(DSC), cis-aconitic anhydride, and a mixture of these compounds.

[00168] A direct linkage includes any linkage wherein a linker moiety is not required. In one embodiment, a direct linkage includes a chemical or a physical interaction wherein the two moieties, i.e. the islet-targeting molecule and carrier particle interact such that they are attracted to each other.

Examples of direct interactions include covalent interactions, non-covalent interactions,

hydrophobic/hydrophilic, ionic (e.g., electrostatic, coulombic attraction, ion-dipole, charge -transfer), van der Waals, or hydrogen bonding, and chemical bonding, including the formation of a covalent bond. Accordingly, in one embodiment, an islet-targeting molecule and the carrier particle are not linked via a linker, e.g., they are directly linked. In a further embodiment, a targeting moiety and the carrier particle are electrostatically associated with each other.

[00169] As used herein, the term "conjugate" or "conjugation" refers to the attachment of two or more entities to form one entity. For example, the methods of the present invention provide conjugation of an islet-targeting molecule of the present invention joined with another entity, for example a carrier particle, for example a liposome, or an affinity binding agent. The attachment can be by means of linkers, chemical modification, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion or by any means known to one skilled in the art. The joining can be permanent or reversible. In some embodiments, several linkers can be included in order to take advantage of desired properties of each linker and each protein in the conjugate. Flexible linkers and linkers that increase the solubility of the conjugates are contemplated for use alone or with other linkers as disclosed herein. Peptide linkers can be linked by expressing DNA encoding the linker to one or more proteins in the conjugate. Linkers can be acid cleavable, photocleavable and heat sensitive linkers. Methods for conjugation are well known by persons skilled in the art and are encompassed for use in the present invention.

[00170] According to the present invention, the islet-targeting molecule, e.g., an islet-targeting peptide can be linked to the carrier particle entity via any suitable means, as known in the art, see for example U.S. Patent Nos. 4,625,014, 5,057,301 and 5, 514,363, which are incorporated herein in their entirety by reference. For example, the agent to be transported by the carrier can be covalently conjugated to the carrier particle, where the carrier particle itself is conjugated to at least one islet-targeting molecule, either directly or through one or more linkers. In one embodiment, the carrier particle of the present invention is conjugated directly to an agent to be transported. In another embodiment, the carrier particle of the present invention is conjugated to an agent to be transported to pancreatic islet endothelial cells via a linker, e.g., a transport enhancing linker.

[00171] A large variety of methods for conjugation of at an islet-targeting molecule with a carrier particle are known in the art. Such methods are e.g., described by Hermanson (1996, Bioconjugate

Techniques, Academic Press), in U.S. 6,180,084 and U.S. 6,264,914 which are incorporated herein in their entirety by reference and include e.g., methods used to link haptens to carriers proteins as routinely used in applied immunology (see Harlow and Lane, 1988, "Antibodies: A laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). It is recognized that, in some cases, an islet-targeting molecule or carrier particle can lose efficacy or functionality upon conjugation depending, e.g., on the conjugation procedure or the chemical group utilized therein. However, given the large variety of methods for conjugation the skilled person is able to find a conjugation method that does not or least affects the efficacy or functionality of the entities to be conjugated.

[00172] In some embodiments, the outer surface of the liposomes can be modified with a long- circulating agent, e.g., PEG, e.g., hyaluronic acid (HA). The liposomes can be modified with a cryoprotectant, e.g., a sugar, such as trehalose, sucrose, mannose or glucose, e.g., HA. In some embodiments, a liposome is coated with HA. HA acts as both a long-circulating agent and a

cryoprotectant. The liposome is modified by attachment of the targeting moiety. In another embodiment, the islet-targeting molecule, e.g., an islet-targeting peptide is covalently attached to HA, which is bound to the liposome surface. Alternatively, a carrier particle is a micelle. Alternatively, the micelle is modified with a cryoprotectant, e.g., HA, PEG.

[00173] A method for coating the liposomes or other polymeric nanoparticles with an islet-targeting molecule, e.g., an islet-targeting peptide are disclosed in U.S. Provisional Application No. 60/794,361 filed April 24, 2006, and International Patent Application: PCT/US07/10075 filed April 24, 2007 with are incorporated in their entirety herein by reference.

[00174] In some embodiments, the outer surface of the liposomes can be further modified with a long- circulating agent. The modification of the liposomes with a hydrophilic polymer as the long-circulating agent is known to enable to prolong the half -life of the liposomes in the blood. Examples of the hydrophilic polymer include polyethylene glycol, polymethylethylene glycol, polyhydroxypropylene glycol, polypropylene glycol, polymethylpropylene glycol and polyhydroxypropylene oxide. In one embodiment, a hydrophilic polymer is polyethylene glycol (PEG). Glycosaminoglycans, e.g., hyaluronic acid, can also be used as long-circulating agents.

[00175] In some embodiments, an islet-targeting molecule, e.g., an islet-targeting peptide can be conjugated to a cryoprotectant present on the liposome, e.g., HA. Crosslinking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), N- hydroxysuccinimide (NHS), and a water soluble carbodiimide, preferably l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC). As is known to the skilled artisan, any crosslinking chemistry can be used, including, but not limited to, thioether, thioester, malimide and thiol, amine -carboxyl, amine- amine, and others listed in organic chemistry manuals, such as, Elements of Organic Chemistry, Isaak and Henry Zimmerman Macmillan Publishing Co., Inc. 866 Third Avenue, New York, N.Y. 10022. Through the complex chemistry of crosslinking, linkage of the amine residues of the recognizing substance and liposomes is established.

[00176] In some embodiments, after an islet-targeting molecule, e.g., an islet-targeting peptide is associated with (e.g., conjugated or covalently attached) to the carrier particle by way of covalent linkage to the cryoprotectant, or by way of covalent linkage to another islet-targeting molecule covalently linked to the cryoprotectant, the lipid particle may be lyophilized. The lipid particle may remain lyophilized prior to rehydration, or prior to rehydration and encapsulation of the agent of interest, for extended periods of time. In one embodiment, the lipid particle remains lyophilized for about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years or more prior to rehydration.

[00177] The term "cryoprotectant" as used herein refers to an agent that protects a lipid particle subjected to dehydration-rehydration, freeze -thawing, or lyophilization-rehydration from vesicle fusion and/or leakage of vesicle contents. Useful cryoprotectants in the methods of the present invention include hyaluronan/ hyaluronic acid (HA) or other glycosaminoglycans for use with liposomes or micelles or PEG for use with micelles. Other cryoprotectants, but are not limited to, include disaccharide and

monosaccharide sugars such as trehalose, maltose, sucrose, maltose, fructose, glucose, lactose, saccharose, galactose, mannose, xylit and sorbit, mannitol, dextran; polyols such as glycerol, glycerin, polyglycerin, ethylene glycol, prolylene glycol, polyethyleneglycol and branched polymers thereof; aminoglycosides; and dimethylsulfoxide.

[00178] In some embodiments, a liposome can be with a cryoprotectant. One preferred cryoprotectant of the present invention is hyaluronic acid or hyaluronan (HA). Hyaluronic acid, a type of

glycosaminoglycan, is a natural polymer with alternating units of N-acetyl glucosamine and glucoronic acid. Using a crosslinking reagent, hyaluronic acid offers carboxylic acid residues as functional groups for covalent binding. The N-acetyl-glucosamine contains hydroxyl units of the type -CH₂-OH which can be oxidized to aldehydes, thereby offering an additional method of crosslinking hyaluronic acid to the liposomal surface in the absence of a crosslinking reagent. Alternatively, other glycosaminoglycans, e.g., chondroitin sulfate, dermatan sulfate, keratin sulfate, or heparin, may be utilized in the methods of the present invention. Cryoprotectants are bound covalently to discrete sites on the liposome surfaces. The number and surface density of these sites will be dictated by the liposome formulation and the liposome type.

[00179] In one embodiment, the final ratio of cryoprotectant ^g) to lipid (μπιοΐε) is about 50 μg/μmole, about 55 μg/μmole, about 60 μg/μmole, about 65 μg/μmole, about 70 μg/μmole, about 75 μg/μmole, about 80 μg/μmole, about 85 μg/μmole, about 90 μg/μmole, about 95 μg/μmole, about 100 μg/μmole, about 105 μg/μmole, about 120 μg/mole, about 150 μg/mole, or about 200 μg/mole. In one embodiment, the ratio of cryoprotectant ^g) to lipid (μπιοΐε) is a range from 3-200 μg per mole lipid.

[00180] To form covalent conjugates of cryoprotectants and liposomes, crosslinking reagents have been studied for effectiveness and biocompatibility. Crosslinking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Through the chemistry of crosslinking, linkage of the amine residues of the recognizing substance and liposomes is established. Covalent attachment of the cryoprotectant HA is described in e.g., U.S. Pat. No. 5,846,561.

[00181] Subsequent to the covalent addition of the cryoprotectant, the lipid particles may be lyophilized. The lyophilized lipid particles may be rehydrated and the islet-targeting molecule (layer 2) covalently attached to the lipid particle. Alternatively, the islet-targeting molecule may be covalently attached to the lipid particle without prior lyophilization and rehydration.

[00182] In some embodiments, the carrier particles are coated with a second layer containing islet- targeting molecule, e.g., an islet-targeting peptide. In such embodiments virtually any agent or drug can be encapsulated in the carriers via lyophilization and reconstitution with an agent suspended in aqueous solution. For example, as disclosed herein, use of the amphiliphic poly (D,L-lactide-co-glycolide)-block- poly(ethylene glycol) (PLGA-b-PEG-COOH) co-polymer as disclosed in the Examples allows for spontaneous self-assembly into nanoparticles in aqueous solution. Accordingly, if the aqueous solution comprise an agent to be delivered to pancreatic islet endothelial cells by the islet-targeting

molecule:carrier particle complex, the agent will automatically be encapsulated in the carrier particle nanoparticle on spontaneous self-assembly. Such amphiliphic poly (D,L-lactide-co-glycolide)-block- poly(ethylene glycol) (PLGA-b-PEG-COOH) co-polymers which self-assemble are advantage as it simplifies optimization and large-scale production of carrier-particles encapsulating an agent of interest, e.g., diabetic treatment or anti-inflammatory agent.

[00183] In one embodiment, the invention provides a method of coating a lipid particle that is pre- conjugated with a cryoprotectant, wherein the cryoprotectant has a functional group attached. The attached functional group may be activated and a least one islet-targeting molecule, e.g., an islet-targeting peptide can be crosslinked to the activated functional group to form a two-layer coated lipid particle which can then be lyophilized for storage purposes prior to use for drug or agent encapsulation.

[00184] In one embodiment, the invention is directed to a method to generate immunoliposomes for targeting pancreatic islet endothelial cells, comprising a composition which comprises an islet-targeting molecule, e.g., an islet-targeting peptide for targeted delivery to pancreatic islet endothelial cells and a carrier particle associated with the islet-targeting molecule, wherein the carrier particle comprises at least one agent.

[00185] In one embodiment, the invention provides liposomes that may be stored in a lyophilized condition prior to encapsulation of drug or agent, or prior to the attachment of at least one islet-targeting molecule, e.g., an islet-targeting peptide.

[00186] Suitable methods for conjugation of an islet-targeting molecule, e.g., an islet-targeting peptide with carrier particle include e.g., carbodimide conjugation (Bauminger and Wilchek, (1980) Meth.

Enzymol. 70: 151-159). Alternatively, a molecule can be coupled to an islet-targeting molecule, e.g., an islet-targeting peptide as described by Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-7273 (1996), and Nagy et al, Proc. Natl. Acad. Sci. USA 95:1794-1799 (1998), each of which are incorporated herein by reference. Another method for conjugating one can use is, for example sodium periodate oxidation followed by reductive alkylation of appropriate reactants and glutaraldehyde crosslinking.

[00187] One can use a variety of different linkers to conjugate the islet-targeting molecule, e.g., an islet- targeting peptide to a carrier particle, for example but not limited to aminocaproic horse radish peroxidase (HRP) or a heterobiofunctional cross-linker, e.g., carbonyl reactive and sulfhydryl- reactive cross-linker. Heterobiofunctional cross linking reagents usually contain two reactive groups that can be coupled to two different function targets on proteins and other macromolecules in a two or three-step process, which can limit the degree of polymerization often associated with using homobiofunctional cross-linkers. Such multistep protocols can offer a great control of conjugate size and the molar ratio of components.

[00188] The term "linker" refers to any means to join two or more entities, for example a peptide with another peptide, or a liposome. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins to be linked. The linker can also be a non-covalent bond, e.g., an organometallic bond through a metal center such as platinum atom. For covalent linkages, various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like. To provide for linking, the effector molecule and/or the probe can be modified by oxidation, hydroxylation, substitution, reduction etc. to provide a site for coupling. It will be appreciated that modification which do not significantly decrease the function of the target moiety, for example antibody, antibody fragment, integrin ligand or integrin ligand fragment and/or the carrier particle are preferred.

[00189] In some embodiments where the carrier particle is a liposome or polymeric nanoparticle, an agent to be targeted to the islet cells can be captured within the carrier particle, for example liposomes or polymeric nanoparticle. For example, a suspension of an agent, e.g., anti-inflammatory agent, antibody, antibody fragment, integrin ligand or integrin ligand fragment or variant thereof can be encapsulated in micelles to form liposomes by conventional methods (U.S. Patent No. 5,043,164, U.S. Patent No. 4,957, 735, U.S. Patent No. 4,925,661 ; Connor and Huang, (1985) . Cell Biol. 101 : 581 ; Lasic D.D. (1992) Nature 355: 279; Novel Drug Delivery (eds. Prescott and Nimmo, Wiley, New York, 1989); Reddy et al. (1992) . Immunol. 148: 1585), which are incorporated herein in their entirety by reference.

[00190] In some embodiments, a carrier particle can be a protein, for example, a protein which binds nucleic acids or other agents. Such protein carrier particles include, but are not limited to nucleic acid binding domains, protamines and the like. In some embodiments, a protein carrier particle is a series of arginine, e.g., at least about 7 arginines (7R), or at least about 9 arginines (9R), which are effective at binding RNA {e.g., modRNA) and RNAi agents, to deliver the RNAi and RNA agents to the pancreatic islet endothelial cells targeted by the associated islet-targeting molecule.

[00191] In some embodiments, and in the event that the carrier particle or affinity binding moiety is a peptide or protein, and the islet-targeting molecule is a protein or peptide, they can be fused together, either in frame or out of frame to form an islet-targeting molecule-carrier particle and/or affinity binding moiety fusion protein. In general, an islet-targeting molecule, e.g., an islet-targeting peptide and carrier particle and/or affinity binding moiety can be fused directly or via one or more amino acid linkers. Any suitable amino acid linkers can be used to modify the stability, conformation, charge, or other structure features of the resulting fusion protein in order to facilitate its transport to target pancreatic islet endothelial cells. In some embodiments, fusion proteins can also be formed from the carrier particle and agent, where both the carrier particle and agents are proteins or contain amino acids as part of their structure, and preferably the activity of the agent is not compromised by being fused with the carrier particle.

[00192] The term "fusion protein" refers to a recombinant protein of two or more fused proteins. Fusion proteins can be produced, for example, by a nucleic acid sequence encoding one protein joined to the nucleic acid encoding another protein such that they constitute a single open-reading frame that can be translated in the cells into a single polypeptide harboring all the intended proteins. The order of arrangement of the proteins can vary. As a non-limiting example, a nucleic acid sequence encoding an islet-targeting molecule, e.g., an islet-targeting peptide can be fused to either the 5' or the 3' end of the nucleic acid sequence encoding a carrier particle and/or affinity binding moiety. In this manner, on expression of the nucleic acid construct, the islet-targeting molecule, e.g., an islet-targeting peptide or fragment thereof is functionally expressed and fused to the N-terminal or C-terminal end of the carrier protein and/or affinity binding moiety. In certain embodiments, the carrier peptide can be modified such that the function of the protein carrier particle (i.e., ability to associate with the agent) remains unaffected by fusion to the islet-targeting molecule, e.g., an islet-targeting peptide and vice versa, the islet-targeting molecule, e.g., an islet-targeting peptide can be modified so that the islet-targeting molecule, e.g., an islet- targeting peptide retains the ability to bind to a cell surface receptor on the target pancreatic islet endothelial cell even when fused with another protein, for example the carrier particle, and/or affinity binding protein.

[00193] In some embodiments, the carrier particle can comprise a liposome comprising multiple layers that assembled in a step-wise fashion, where each layer can comprise at least one islet-targeting molecule, e.g., an islet-targeting peptide. In one embodiment, the first step is the preparation of empty nano-scale liposomes. Liposomes may be prepared by any method known to the skilled artisan. The second step is the addition of a first layer of surface modification. The first layer is added to the liposome by covalent modification. The first layer comprises hyaluronic acid, or other cryoprotectant glucosaminoglycan. The liposome composition may also be lyophilized and reconstituted at any time after the addition of the first layer. The third step is to add a second surface modification. The second layer is added by covalent attachment to the first layer. The second layer comprises at least one islet-targeting molecule, e.g., an islet-targeting peptide. Further layers may add to the liposome and these layers may include additional islet-targeting molecule, e.g., an islet-targeting peptides. Alternatively, the second layer may include a heterogeneous mix of islet-targeting molecule, e.g., an islet-targeting peptides as well as agents. The liposome composition can be lyophilized after addition of the final targeting layer. An agent of interest (e.g., anti-inflammatory agent, or agent commonly used in the treatment of diabetes) can be encapsulated by the liposome by rehydration of the liposome with an aqueous solution containing the agent. In one embodiment, agents that are poorly soluble in aqueous solutions or agents that are hydrophobic may be added to the composition during preparation of the liposomes in step one.

[00194] In another embodiment, an islet-targeting molecule: carrier particle complex as disclosed herein can comprise a multi-layered liposome with cryoprotectant conjugated lipid particles. In such embodiments, a cryoprotectant can be covalently linked to the lipid polar groups of the phospholipids and it forms the first layer of surface modification on the liposome discussed supra. The islet-targeting molecule forms the second layer of coat and it is added on to the first layer of cryoprotectant. The multi- layered liposome may be lyophilized for storage. The agent of interest is encapsulated by the liposome by rehydration of the liposome with an aqueous solution containing the agent.

[00195] As used herein, the term "associated with" means that one entity is in physical association or contact with another. Thus, an islet-targeting molecule "associated with" a carrier particle moiety can be either covalently or non-covalently joined to the carrier particle. The association can be mediated by a linker moiety, particularly where the association is covalent. The term "association" or "interaction" or "associated with" are used interchangeably herein and as used in reference to the association or interaction of the islet-targeting molecule with a carrier particle or affinity binding moiety, and refers to any association between the islet-targeting molecule with the cell to be delivered, for example via an affinity binding moiety, or affinity binding moiety: binding partner: affinity binding moiety complex, either by a direct linkage or an indirect linkage.

[00196] The term "linked" refers to two or more entities that are joined by any means known by persons of ordinary skill in the art, for example an islet-targeting molecule, e.g., an islet-targeting peptide linked to a carrier particle, or an affinity binding moiety, e.g., an antibody or fragment thereof. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins to be linked. The linker can also be a non- covalent bond, e.g., an organometallic bond through a metal center such as platinum atom. For covalent linkages, various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like. To provide for linking, the islet-targeting molecule, e.g., an islet-targeting peptide, the carrier particle, or affinity binding moiety can be modified by oxidation, hydroxylation, substitution, reduction etc. to provide a site for coupling. It will be appreciated that modification which do not significantly decrease the function of any of the islet-targeting molecule, e.g., an islet-targeting peptide, the carrier particle, or affinity binding moiety are preferred.

[00197] Alternatively, in some embodiments two or more entities that are joined can be linked by indirect linkage. An indirect linkage includes an association between an islet-targeting molecule and the affinity binding moiety, e.g., an antibody of fragment thereof, wherein the islet-targeting molecule and the affinity binding moiety are attached via a "linker moiety", e.g., they are not directly linked. Linker moieties include, but are not limited to, chemical linker moieties or for example a peptide linker moiety. In some embodiments, a linker between a targeting moiety and the binding moiety is formed by reacting the polymer and a linker selected e.g., from the group consisting of p-nitrophenyl chloroformate, carbonyldiimidazole(CDI), Ν,Ν'-disuccinimidyl carbonate(DSC), cis-aconitic anhydride, and a mixture of these compounds. In some embodiments, the islet-targeting molecule, e.g., an islet-targeting peptide is associated with a first affinity binding moiety, which associates with a binding partner, where the binding partner associates with a second affinity binding moiety which is associated with the cell to be delivered to the islet endothelial cells, e.g., is associated with a stem cell or progenitor cell, such as a EPC.

[00198] A direct linkage includes any linkage wherein a linker moiety is not required. In one embodiment, a direct linkage includes a chemical or a physical interaction wherein the two moieties, i.e. the islet-targeting molecule and carrier particle, or affinity binding moiety interact such that they are attracted to each other. Examples of direct interactions include covalent interactions, non-covalent interactions, hydrophobic/hydrophilic, ionic (e.g., electrostatic, coulombic attraction, ion-dipole, charge- transfer), Van der Waals, or hydrogen bonding, and chemical bonding, including the formation of a covalent bond. Accordingly, in one embodiment, a targeting moiety, such as an antibody of fragment thereof and the binding moiety are not linked via a linker, e.g., they are directly linked. In a further embodiment, a targeting moiety and the binding moiety are electrostatically associated with each other.

[00199] As used herein, the term "conjugate" or "conjugation" refers to the attachment of two or more entities to form one entity. For example, the methods of the present invention provide conjugation of a islet-targeting molecule, e.g., an islet-targeting peptide of the present invention joined with another entity, for example a carrier particle or affinity binding moiety. The attachment can be by means of linkers, chemical modification, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion or by any means known to one skilled in the art. The joining can be permanent or reversible. In some embodiments, several linkers can be included in order to take advantage of desired properties of each linker and each protein in the conjugate. Flexible linkers and linkers that increase the solubility of the conjugates are contemplated for use alone or with other linkers as disclosed herein. Peptide linkers can be linked by expressing DNA encoding the linker to one or more proteins in the conjugate. Linkers can be acid cleavable, photocleavable and heat sensitive linkers. Methods for conjugation are well known by persons skilled in the art and are encompassed for use in the present invention.

[00200] According to the present invention, an islet-targeting molecule, e.g., an islet-targeting peptide can be linked to the affinity binding moiety such as an antibody, or antigen binding antibody fragment via any suitable means, as known in the art, see for example U.S. Patent Nos. 4,625,014, 5,057,301 and 5, 514,363, which are incorporated herein in their entirety by reference.

[00201] A large variety of methods for conjugation of an islet-targeting molecule, e.g., an islet-targeting peptide with a carrier particle and/or affinity binding moiety are known in the art. Such methods are e.g., described by Hermanson (1996, Bioconjugate Techniques, Academic Press), in U.S. 6,180,084 and U.S. 6,264,914 which are incorporated herein in their entirety by reference and include e.g., methods used to link haptens to carriers proteins as routinely used in applied immunology (see Harlow and Lane, 1988, "Antibodies: A laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). It is recognized that, in some cases, an islet-targeting molecule, e.g., an islet-targeting peptide, carrier particle and/or affinity binding moiety can lose efficacy or functionality upon conjugation depending, e.g., on the conjugation procedure or the chemical group utilized therein. However, given the large variety of methods for conjugation, the skilled person is able to find a conjugation method that does not or least affects the efficacy or functionality of the entities to be conjugated.

[00202] In some embodiments, an islet-targeting molecule, e.g., an islet-targeting peptide can be associated with a carrier particle and/or affinity binding moiety can be conjugated by cross-linking.

Crosslinking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), N-hydroxysuccinimide (NHS), and a water soluble carbodiimide, preferably 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). As is known to the skilled artisan, any crosslinking chemistry can be used, including, but not limited to, thioether, thioester, malimide and thiol, amine- carboxyl, amine-amine, and others listed in organic chemistry manuals, such as, Elements of Organic Chemistry, Isaak and Henry Zimmerman Macmillan Publishing Co., Inc. 866 Third Avenue, New York, N.Y. 10022.

[00203] Other linkage approaches to conjugate the islet-targeting molecule, e.g., an islet-targeting peptide with a carrier particle and/or affinity binding moiety include but are not limited to aminocaproic horse radish peroxidase (HRP) or a heterobiofunctional cross-linker, e.g., carbonyl reactive and sulfhydryl- reactive cross-linker. Heterobiofunctional cross linking reagents usually contain two reactive groups that can be coupled to two different function targets on proteins and other macromolecules in a two or three-step process, which can limit the degree of polymerization often associated with using homo- biofunctional cross-linkers. Such multistep protocols can offer a great control of conjugate size and the molar ratio of components.

Agents

[00204] One aspect of the present invention relates to a composition for the delivery of an agent associated with a carrier particle to target pancreatic islet endothelial cells, as disclosed herein. In some embodiments, it is envisioned that where the carrier particle comprises different layers, the islet-targeting molecule:carrier particle can be used for simultaneous delivery of an insoluble agent and a soluble agent to the target pancreatic islet endothelial cell, wherein the carrier particle can comprise an insoluble agent and/or a soluble agent, and wherein the carrier particle is attached or conjugated to at least one or more islet-targeting molecule, e.g., an islet-targeting peptide, where the islet-targeting molecule binds to and has specific affinity for cell surface-markers expressed by pancreatic islet endothelial cells.

[00205] In one embodiment, the islet-targeting molecule:carrier particle allows for delivery of at least two agents to a pancreatic islet endothelial, e.g., CE cell. Methods to generate such carrier particles loaded with two agents are disclosed herein. In one embodiment, one of the two agents is hydrophilic (i.e. a soluble agent) which is entrapped in the aqueous phase of the carrier particle, such as the center of a liposome. In another embodiment, the other agent is hydrophobic (or an insoluble agent) which is entrapped in the lipid phase of the carrier particle, for example a hydrophobic agent can be associated with a lipid layer of the liposome.

[00206] For purposes of the present invention, "agent" means any agent or compound that can affect the body therapeutically, or which can be used in vivo for diagnosis. Examples of therapeutic agents are any agent useful in the treatment of diabetes, e.g., Type 1 diabetes, and includes but is not limited to, antiinflammatory molecules such as genistein, cyclosporine A, prednisone, mesalamine (5-aminosalicylic acid), simvastatin (inhibitor of HMG-CoA reductase), Herbimycin A (a receptor tyrosine kinase inhibitor similar to Genistein), docosahexaenoic acid (DHA; n-3 fatty acid), as well as therapeutic nucleic acids including nucleic acid analogs, e.g., RNAi agents and synthetic modified RNA (modRNA).

[00207] An "agent" as used herein refers to an agent that is transported by the carrier particle and islet- targeting molecule to target islet endothelial cells. An agent can be a chemical molecule of synthetic or biological origin. In some embodiments, an agent is generally a molecule that can be used in a pharmaceutical composition, for example the agent is a therapeutic agent. An agent as used herein also refers to any chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition, and are herein referred to as "therapeutic agents".

[00208] In alternative embodiments, an agent can be a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject for imaging purposes in the subject, for example to monitor the presence or progression of disease or condition, or monitor the location of an islet-targeting molecule: carrier particle complex in the treatment of diabetes and are herein referred to as "imaging agents" or "diagnostic agents".

[00209] A chemical entity or biological product as disclosed herein is preferably, but not necessarily a low molecular weight compound, but can also be a larger compound, or any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNAi, such as siRNA, shRNA, miRNA, nucleic acid analogues, microRNA, miRNA analogues, synthetic modified RNA (modRNA), antigomirs, peptides, peptidomimetics, avimers, receptors, ligands, and antibodies, aptamers, polypeptides or analogues, derivatives or variants thereof. For example, oligomers of nucleic acids, amino acids, carbohydrates include without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications, derivatives and combinations thereof

[00210] A therapeutic agent as used herein is an agent useful in the treatment of diabetes. In some embodiments, the agent is a therapeutic agent useful in the treatment of Type 1 diabetes in a subject. In some embodiments, a therapeutic agent encompassed for use in the methods of treatment of Type 1 diabetes is cyclosporine A and prednisone (Feutren, G. et al, Lancet (1986) 2(8499): 119-24; Silverstein, J. et ah, N Engl J Med (1988) 319(10):599-604), or derivatives or analogues thereof. In some embodiments, a therapeutic agent is an agent useful in the treatment of Type 2 diabetes, where such agent is selected from any agent which promotes the survival of islet β cells.

[00211] Hydrophilic agents or Soluble agents

[00212] In some embodiments, where one can use the an islet-targeting molecule: carrier particle complex to simultaneously deliver at least one insoluble agent and at least one soluble agent to a target pancreatic islet endothelial cell. A soluble agent is also referred to as a water-soluble agent, a hydrophilic agent as that term is defined herein.

[00213] Any soluble agent is contemplated for delivery to a target cell using the methods and compositions as disclosed herein. Examples of soluble agents include, for example, but are not limited to, proteins, peptides, antibodies, antibody fragments, nucleic acids such as DNA and RNA and RNAi agents such as siRNA, miRNA and the like; nucleic acid analogs such PNA (peptide nucleic acid), LNA (locked nucleic acid), pcPNA (pseudo-complementary PNA) and the like, as other agents which are soluble as according to the term as defined herein. Typically, all globular proteins are soluble, which includes enzymes, enzyme fragments, and recombinant proteins. In some embodiments, a soluble protein useful for delivery using the compositions and methods as disclosed herein is a recombinant version or variant of a native protein which has been modified to increase its solubility and/or stability in solution. A soluble protein as disclosed herein is a protein which goes into solution. Stated another way, if 30% of a crude protein preparation (containing multiple proteins) goes into solution, 30% of the crude protein preparation comprises soluble proteins.

[00214] In some embodiments, an agent is a gene or polynucleotide, such as plasmid DNA, DNA fragment, oligonucleotide, oligodeoxynucleotide, antisense oligonucleotide, chimeric RNA/DNA oligonucleotide, RNA, siRNA, ribozyme, or viral particle.

[00215] In some embodiments, an agent is a nucleic acid, e.g., DNA, RNA, siRNA, plasmid DNA, short-hairpin RNA, small temporal RNA (stRNA), microRNA (miRNA), RNA mimetics, or

heterochromatic siRNA. The nucleic acid agent of interest has a charged backbone that prevents efficient encapsulation in the lipid particle. Accordingly, the nucleic acid agent of interest may be condensed with a cationic polymer, e.g., PEI, polyamine spermidine, and spermine, or cationic peptide, e.g., protamine and polylysine, prior to encapsulation in the lipid particle. In one embodiment, the agent is not condensed with a cationic polymer.

[00216] In some embodiments, an agent is a synthetic modified RNA (modRNA) molecule. Synthetic modified RNA's for use in the compositions, methods and kits as disclosed herein are described in U.S. Provisional Application 61/387,220, filed September 28, 2010, and U.S. Provisional Application

61/325,003, filed: April 16, 2010, both of which are incorporated herein in their entirety by reference.

[00217] In some embodiments, an agent functions as an RNA interference (RNAi) molecule. The term "RNAi" as used herein refers to interfering RNA, or RNA interference molecules are nucleic acid molecules or analogues thereof for example RNA-based molecules that inhibit gene expression. RNAi refers to a means of selective post-transcriptional gene silencing. RNAi can result in the destruction of specific mRNA, or prevents the processing or translation of RNA, such as mRNA.

[00218] In some embodiments, an agent is a siRNA. The term "short interfering RNA" (siRNA), also referred to herein as "small interfering RNA" is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi. An siRNA can be chemically synthesized, it can be produced by in vitro transcription, or it can be produced within a host cell. siRNA molecules can also be generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated.

[00219] In one embodiment, an siRNA agent is a double stranded RNA (dsRNA) molecule of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 30 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, and more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and can contain a 3' and/or 5' overhang on each strand having a length of about 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

[00220] An siRNA agent for use in the methods as disclosed herein also include small hairpin (also called stem loop) RNAs (shRNAs). In one embodiment, these shRNAs are composed of a short, e.g., about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501, incorporated by reference herein in its entirety).

[00221] The term "shRNA" as used herein refers to short hairpin RNA which functions as RNAi and/or siRNA species but differs in that shRNA species are double stranded hairpin-like structure for increased stability.

[00222] In some embodiments, the agent is an avimer. Avimer s are multi-domain proteins with binding and inhibiting properties and are comprised typically of multiple independent binding domains linked together, and as such creates avidity and improved affinity and specificity as compared to conventional single epitope binding proteins such as antibodies. In some embodiments, one can use an avimer that is a protein or polypeptide that can bind simultaneously to a single protein target and/or multiple protein targets, as known as multi-point attachment in the art. Avimers are useful as therapeutic agents which function son multiple drug targets simultaneously for the progenitor cell and/or treatment of multifactorial diseases or disorders, for example multifactorial cancer malignancies or inflammatory disorders or autoimmune diseases.

[00223] In some embodiments, the agent is an antigomir. Antigomirs are oligonucleotides, for example synthetic oligonucleotides capable of gene silencing endogenous miRNAs.

[00224] The term "association" or "interaction" as used herein in reference to the association or interaction of an agent, e.g., siRNA, with a carrier particle, refers to any association between the agent, e.g., siRNA, with a carrier particle, e.g., a peptide carrier, either by a direct linkage or an indirect linkage. An indirect linkage includes an association between an agent, e.g., siRNA, and a carrier particle wherein said agent, e.g., siRNA, and said carrier particle are attached via a linker moiety, e.g., they are not directly linked. Linker moieties include, but are not limited to, e.g., nucleic acid linker molecules, e.g., biodegradable nucleic acid linker molecules. A nucleic acid linker molecule can be, for example, a dimer, trimer, tetramer, or longer nucleic acid molecule, for example an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length.

[00225] A direct linkage includes any linkage wherein a linker moiety is not required. In one embodiment, a direct linkage includes a chemical or a physical interaction wherein the two moieties, the therapeutic agent, e.g., siRNA, and the carrier particle, interact such that they are attracted to each other. Examples of direct interactions include non-covalent interactions, hydrophobic/hydrophilic, ionic (e.g., electrostatic, coulombic attraction, ion-dipole, charge-transfer), Van der Waals, or hydrogen bonding, and chemical bonding, including the formation of a covalent bond. Accordingly, in one embodiment, an agent, e.g., siRNA, and the carrier particle are not linked via a linker, e.g., they are directly linked. In a further embodiment, the therapeutic agent, e.g., siRNA, and the carrier particle are electrostatically associated with each other.

[00226] In some embodiments, agents delivered to islet endothelial cells by the methods as disclosed herein include small molecules chemical and peptides to block intracellular signaling cascades, enzymes (kinases), proteasome function, lipid metabolism, cell cycle and membrane trafficking. Agents delivered by the methods of the present invention include agents that inhibit leukocyte extravasation or decrease vascular permeability. Such therapeutic agents can be useful in the treatment of, for example but not limited to, sustained inflammation, atherosclerosis, autoimmune diseases, ischemia-reperfusion injury and angiogenesis.

[00227] In another embodiment, an agent, for example a siRNA therapeutic agent as disclosed herein can be prepared to be delivered in a "prodrug" form. The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.

[00228] In one embodiment, an agent is a protein, or growth factor, cytokine, immunomodulating agent, or other protein, including proteins which when expressed present an antigen which stimulates or suppresses the immune system. [00229] In another embodiment, the agent is a diagnostic agent capable of detection in vivo following administration of a composition comprising an islet-targeting molecule: carrier particle complex.

Exemplary diagnostic agents include electron dense material, magnetic resonance imaging agents, radiopharmaceuticals and fluorescent molecules. Radionucleotides useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetium, including isotopes ^Cu, ⁶⁷Cu, ^mIn, ^99mTc, ⁶⁷Ga or ⁶⁸Ga. Imaging agents disclosed by Low et al. in U.S. Pat. No. 5,688,488, incorporated herein by reference, are useful in the liposomal complexes described herein.

[00230] In one aspect of the method, the liposome product is detectably labeled with a label selected from the group including a radioactive label, a fluorescent label, a non-fluorescent label, a dye, or a compound which enhances magnetic resonance imaging (MRI). In one embodiment, the liposome product is detected by acoustic reflectivity. The label may be attached to the exterior of the liposome or may be encapsulated in the interior of the liposome.

[00231] In some embodiments, an agent can be an imaging agent. In order to function as a suitable agent for medical imaging, the effector agent is useful in a molecular imaging diagnosis procedure, for example but not limited to, magnetic resonance (MR) imaging. Delivery of such imaging agents using the methods and compositions as disclosed herein can be used to image extent of leukocyte extravasation and/or vascular permeability by MRI or PET for example. Contrast enhancement can be provided by gadolinium, for example, gadolinium in the form of Gd-DTPA-aminohexanoic acid. Other imaging agents are useful in the methods as disclosed herein include, for example other lanthanide ion coordination complexes can allow for even greater enhanced relaxation at higher field strength (Aime, S., et al., Chem. Soc. Rev. 27:19-29, 1998; Aime et al., J. Mannet. Reson. Iman. 16:394-406, 2002). Paramagnetic CES T agents are useful as imaging agents in the methods and compositions as disclosed herein, for example as Eu+3, Tb+3, Dy+3, Er+3, Tm+3, or Yb+ 3 alter tissue contrast via chemical exchange saturation transfer of presaturated spins to bulk I water (Elst, L.V., et al. , Mann. Reson. Med. 47: 1121-1130, 2002). In some embodiments, more than one imaging agent can be used simultaneously in the composition and methods of the present invention, with techniques available for attachment of multiple imaging agents, for example Gd-DTPA to proteins to enhance the MR signal known by persons of ordinary skill in the art. The Tl acceleration and contrast enhancement of Gd and especially Fe have been shown to saturate at very high field strength, however, while these other lanthanides do not, thus taking full advantage of the increased resolution of very high field strengths.

[00232] In some embodiments, an imaging agent is useful as diagnostic agent capable of detection in vivo following administration. Exemplary imaging agents useful for diagnostic purposes include electron dense material, magnetic resonance imaging agents, radiopharmaceuticals and fluorescent molecules. Radionucleotides useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetium, including isotopes ⁶⁴Cu, ⁶⁷Cu, ^mIn, ^99mTc, ⁶⁷Ga or ⁶⁸Ga. Imaging agents disclosed by Low et al. in U.S. Pat. No. 5,688,488, incorporated herein by reference, are also useful in the compositions as disclosed herein.

Insoluble Agents

[00233] In one embodiment, the methods as disclosed herein allow islet-targeting molecule: carrier particle complexes to simultaneously deliver at least one insoluble agent and at least one soluble agent to a target pancreatic islet endothelial cell, as disclosed herein. An insoluble agent is also referred to as a water-insoluble agent, a hydrophobic agent or a lipophilic agent, as those terms are defined herein.

[00234] Any insoluble agent is contemplated for delivery to a target cell using the methods and compositions as disclosed herein. Examples of insoluble agents include, for example, but are not limited to anti-inflammatory agents, such as but not limited to genistein, cyclosporine A and prednisone.

[00235] In some embodiments, an insoluble agent useful in the methods as disclosed herein is a therapeutic agent for the treatment of diabetes which can be delivered to pancreatic islet endothelial cells by the methods as described herein. In some embodiments, an agent can be a therapeutic agent or a diagnostic agent as disclosed herein.

[00236] Accordingly in some embodiments, the methods to use the islet-targeting molecule, e.g., an islet-targeting peptide as disclosed herein can also be used for diagnostic purposes, for example but not limited to visualization of islet survival in response to treatment, or during disease progression. In further embodiments, the compositions and methods of the present invention are useful for monitoring the effect of a therapeutic intervention and/or for prognostic purposes. For example, in some embodiments the present invention can be used for monitoring the efficacy of a therapeutic treatment in a subject treated with a therapy for diabetes and monitoring the attenuation of pancreatic islet loss in the subject, and in some embodiments, where a islet-targeting molecule: affinity binding moiety: EPC cell complex is administered, for monitoring increase of pancreatic islet endothelial cells.

[00237] Accordingly, as disclosed herein the method provides a means to deliver islet-targeting molecule, e.g., an islet-targeting peptide: carrier particle complexes comprising agents such as siRNA, nucleic acids, nucleic acid analogues, miRNA, miRNA mimetics, antagomirs, synthetic modified RNAs (modRNA) and the like to pancreatic islet endothelial cells in vivo and in vivo. The methods as disclosed herein are useful for delivering agents to pancreatic islet endothelial cells, in vitro, in vivo or ex vivo for multiple purposes, such as (i) research purposes including but not limited to investigating or studying islet function and responses, increasing our understanding of islet death in diabetes, pancreatic islet regeneration, and response to agents as well as general assays for preventing islet cell death, and (ii) therapeutic purposes.

[00238] Other examples of some insoluble agents for use in the compositions and methods as disclosed herein include, but are not limited to, immunosuppressive and immunoactive agents, and anti- inflammatory agents, antibiotics, anti-epileptics, anesthetics, hormones, and nutrients. A detailed description of these and other suitable drugs may be found in Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing Co. Philadelphia, Pa. which is hereby incorporated by reference.

[00239] Insoluble agents or insoluble drugs can have pharmaceutical efficacy in a number of therapeutic and diagnostic imaging areas. Non-limiting classes of compounds and agents from which poorly water soluble drugs that melt without decomposition and are useful in this invention can be selected include anesthetic agents, ace inhibiting agents, antithrombotic agents, anti-allergic agents, anti- angiogenic agents, antibacterial agents, antibiotic agents, anticoagulant agents, anticancer agents, antidiabetic agents, antihypertension agents, antifungal agents, antihypotensive agents, antiinflammatory agents, antimicotic agents, antimigraine agents, antiparkinson agents, antirheumatic agents, antithrombins, antiviral agents, beta blocking agents, bronchospamolytic agents, calcium antagonists, cardiovascular agents, cardiac glycosidic agents, carotenoids, cephalosporins, contraceptive agents, cytostatic agents, diuretic agents, enkephalins, fibrinolytic agents, growth hormones, immunosuppressants, insulins, interferons, lactation inhibiting agents, lipid-lowering agents, lymphokines, neurologic agents, prostacyclins, prostaglandins, psycho-pharmaceutical agents, protease inhibitors, magnetic resonance diagnostic imaging agents, reproductive control hormones, sedative agents, sex hormones, somatostatins, steroid hormonal agents, vaccines, vasodilating agents, and vitamins.

[00240] Encapsulating or entrapping agents in carrier particles

[00241] In one embodiment, the methods as disclosed herein allow islet-targeting molecule: carrier particle complex to deliver a hydrophilic agent to target pancreatic islet endothelial cells, as disclosed herein. In some embodiments, the hydrophilic agent is encapsulated in the carrier particle. In some embodiments where the agent is a hydrophilic agent, for example a nucleic acid agent such as DNA, RNA, siRNA, RNAi, modified RNA (modRNA), plasmid DNA, short-hairpin RNA, small temporal RNA (stRNA), microRNA (miRNA), RNA mimetics, or heterochromatic siRNA, or where the agent is a nucleic acid agent that has a charged backbone that prevents efficient encapsulation in the lipid particle, such agents can be condensed with a cationic polymer, e.g., PEI, polyamine spermidine, and spermine, or cationic peptide, e.g., protamine and polylysine, prior to encapsulation in the lipid particle. In some embodiments, the agent is not condensed with a cationic polymer.

[00242] In some embodiments, an agent is encapsulated in the lipid particle or other polymeric nanoparticle in the following manner: The lipid particle or polymeric nanoparticle, in which can additionally comprise a cryoprotectant and/or a targeting moiety is provided lyophilized. The agent is in an aqueous solution. The agent in aqueous solution is utilized to rehydrate the lyophilized lipid particle or nanoparticle. Thus, the agent is encapsulated in the rehydrated lipid particle or polymeric nanoparticle. An example of encapsulation of a soluble agent within the lipid particle includes, but not limited to, soluble agents or anti-inflammatory agents, such as genistein as demonstrated in the Examples. Other antiinflammatory agents which can be delivered include, for example, but are not limited to cyclosporine A, prednisone, non-steroidal anti-inflammatory agent (NSAIDS), for example COX-1 inhibitors and COX -2 inhibitors, which are well known by persons of ordinary skill in the art.

[00243] In another embodiment, two or more agents can be delivered by carrier particle, for example a lipid particle or polymeric nanoparticles by the methods as disclosed herein and in the Examples. In such embodiments, one agent can be an insoluble (i.e. hydrophobic or lipophilic) agent and the other agent a soluble (i.e. hydrophilic) agent. An insoluble (or hydrophobic/lipophilic) agent can be added to the lipid particle during formation of the lipid particle and can associate with the lipid portion of the lipid particle. The soluble agent (i.e. hydrophilic agent) is associated with the lipid particle by being added in the aqueous solution during the rehydration of the lyophilized lipid particle, and therefore encapsulated in the carrier particle, such as genistein, as demonstrated in the Examples. An exemplary embodiment of two agent delivery can include a soluble agent, such as a nucleic acid, e.g., RNAi, modRNA etc., and/or antiinflammatory agent, which is encapsulated or entrapped in the aqueous interior of a carrier particle liposome, and where an insoluble (hydrophobic) agent and poorly soluble in aqueous solution is associated with the lipid portion of the liposome carrier particle. As used herein, "poorly soluble in aqueous solution" refers to a composition that is less that 10% soluble in water.

[00244] Any suitable lipid: pharmaceutical agent ratio that is efficacious is contemplated by the present invention. In some embodiments, the lipid: pharmaceutical agent molar ratios include about 2: 1 to about 30: 1, about 5: 1 to about 100: 1, about 10: 1 to about 40: 1, about 15: 1 to about 25: 1.

[00245] In some embodiments, the loading efficiency of therapeutic or pharmaceutical agent is a percent encapsulated pharmaceutical agent of about 50%, about 60%, about 70% or greater. In one embodiment, the loading efficiency for a soluble agent is a range from 50 -100%. In some embodiments, the loading efficiency of an insoluble agent to be associated with the lipid portion of the lipid particle, (i.e. a pharmaceutical agent poorly soluble in aqueous solution), is a percent loaded pharmaceutical agent of about 50%, about 60%, about 70%, about 80%, about 90%, about 100%. In one embodiment, the loading efficiency for a hydrophobic agent in the lipid layer is a range from 80 -100%.

[00246] In one aspect of the method, an islet-targeting molecule, e.g., an islet-targeting peptide: carrier particle complex can be detectably labeled, for example it can comprise a carrier particle such as a liposome or polymeric nanoparticle is detectably labeled with a label selected from the group including a radioactive label, a fluorescent label, a non-fluorescent label, a dye, or a compound which enhances magnetic resonance imaging (MRI). In one embodiment, the liposome product is detected by acoustic reflectivity. The label may be attached to the exterior of the liposome or may be encapsulated in the interior of the liposome.

Agents encapsulated in, or on the exterior of carrier particles

RNA interference inducing molecules [00247] In another embodiment, the invention provides a method of delivering an RNA interference (RNAi) agent into a target pancreatic islet endothelial cell, the method comprising administering an islet- targeting molecule: carrier particle complex to the subject, where the carrier particle is associated with the RNAi agent. Preferably, the double stranded RNA is an siRNA.

[00248] As used herein, "double stranded RNA" or "dsRNA" refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of a single RNA molecule that doubles back on itself to form a two-stranded structure. For example, the stem loop structure of the progenitor molecules from which the single-stranded miRNA is derived, called the pre-miRNA (B artel et al. 2004. Cell 116:281-297), comprises a dsRNA molecule.

[00249] As used herein, the terms "RNA interference inducing molecule" or "RNAi molecule" or

"RNAi agent" or "RNAi agent" are used interchangeably herein to refer to an RNA molecule, such as a double stranded RNA, which functions to inhibit gene expression of a target gene through RNA-mediated target transcript cleavage or RNA interference. Stated another way, the RNA interference inducing molecule induces gene silencing of the target gene. The overall effect of an RNA interference inducing molecule is gene silencing of the target gene. A double-stranded RNA, such as that used in siRNA, has different properties than single-stranded RNA, double-stranded DNA or single-stranded DNA. Each of the species of nucleic acids is bound by mostly non-overlapping sets of binding proteins in the cell and degraded by mostly non-overlapping sets of nucleases. The nuclear genome of all cells is DNA-based and as such is unlikely to produce immune responses except in autoimmune disease (Pisetsky. Clin Diagn Lab Immunol. (1998) Jan;51 : l-6). Single-stranded RNA (ssRNA) is the form endogenously found in eukaryotic cells as the product of DNA transcription. Cellular ssRNA molecules include messenger RNAs (and the progenitor pre-messenger RNAs), small nuclear RNAs, small nucleolar RNAs, transfer RNAs and ribosomal RNAs. Single-stranded RNA can induce interferon and inflammatory immune response via TLR7 and TLR8 receptors (Proc Natl Acad Sci. (2004). 101 :5598-603; Science. (2004) 303: 1526-9; Science (2004) 303: 1529-3). Double-stranded RNA induces a size-dependent immune response such that dsRNA larger than 30bp activates the interferon response, while shorter dsRNAs feed into the cell's endogenous RNA interference machinery downstream of the Dicer enzyme. MicroRNAs (miRNAs), including short temporal RNAs and small modulatory RNAs, are the only known cellular dsRNA molecules in mammals and were not discovered until 2001 (Kim. (2005) Mol Cells. 19: 1-15). Response to extracellular RNA in the bloodstream, double- or single-stranded of any length, is rapid excretion by the kidneys and degradation by enzymes (PLOS Biol. 2004. 2: 18-20).

[00250] As used herein, the term "effects RNA interference" refers to the initiation or causation of RNAi-mediated gene silencing, or to conditions that result in RNA interference-mediated gene silencing.

[00251] Numerous specific siRNA molecules have been designed that have been shown to inhibit gene expression (Ratcliff et al. Science 276: 1558-1560, 1997; Waterhouse et al. Nature 411 :834-842, 2001). In addition, specific siRNA molecules have been shown to inhibit, for example, HIV-1 entry to a cell by targeting the host CD4 protein expression in target cells thereby reducing the entry sites for HIV-1 which targets cells expressing CD4 (Novina et al. Nature Medicine, 8:681-686, 2002). Short interfering RNA have further been designed and successfully used to silence expression of Fas to reduce Fas-mediated apoptosis in vivo (Song et al. Nature Medicine 9:347-351, 2003).

[00252] It has been shown in plants that longer, about 24-26 nucleotides (nt) long siRNA correlates with systemic silencing and methylation of homologous DNA. Conversely, the about 21-22 nt short siRNA class correlates with mRNA degradation but not with systemic signaling or methylation (Hamilton et al. EMBO J. 2002 Sep 2;21(17):4671-9). These findings reveal an unexpected level of complexity in the RNA silencing pathway in plants that may also apply in animals. In higher order eukaryotes, DNA is methylated at cytosines located 5' to guanosine in the CpG dinucleotide. This modification has important regulatory effects on gene expression, especially when involving CpG-rich areas known as CpG islands, located in the promoter regions of many genes. While almost all gene-associated islands are protected from methylation on autosomal chromosomes, extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X-chromosomes of females. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. In this last situation, promoter region hypermethylation stands as an alternative to coding region mutations in eliminating tumor suppression gene function (Herman, et al.). The use of siRNA molecules for directing methylation of a target gene is described in U.S. Provisional Application No. 60/447,013, filed Feb. 13, 2003, referred to in U.S. Patent Application Publication No. 20040091918.

[00253] It is also known that the RNA interference does not have to match perfectly to its target sequence. Preferably, however, the 5' and middle part of the antisense (guide) strand of the siRNA is perfectly complementary to the target nucleic acid sequence.

[00254] The RNA interference -inducing molecule according to the present invention includes RNA molecules that have natural or modified nucleotides, natural ribose sugars or modified sugars and natural or modified phosphate backbone.

[00255] Accordingly, the RNA interference-inducing molecule referred to in the specification includes, but is not limited to, unmodified and modified double stranded (ds) RNA molecules including, short- temporal RNA (stRNA), small interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA

(miRNA), double-stranded RNA (dsRNA), (see, e.g., Baulcombe, Science 297:2002-2003, 2002). The dsRNA molecules, e.g., siRNA, also may contain 3' overhangs, preferably 3'UU or 3'TT overhangs. In one embodiment, the siRNA molecules of the present invention do not include RNA molecules that comprise ssRNA greater than about 30-40 bases, about 40-50 bases, about 50 bases or more. In one embodiment, the siRNA molecules of the present invention have a double stranded structure. In one embodiment, the siRNA molecules of the present invention are double stranded for more than about 25%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90% of their length.

[00256] The RNA interference as described herein also includes RNA molecules having one or more non-natural nucleotides, i.e. nucleotides other than adenine "A", guanine "G", uracil "U", or cytosine "C", a modified nucleotide residue or a derivative or analog of a natural nucleotide are also useful. Any modified residue, derivative or analog may be used to the extent that it does not eliminate or substantially reduce (by at least 50%) RNAi activity of the dsRNA. These forms thus include, but are not limited to, aminoallyl UTP, pseudo-UTP, 5-I-UTP, 5-I-CTP, 5-Br-UTP, alpha-S ATP, alpha-S CTP, alpha-S GTP, alpha-S UTP, 4-thio UTP, 2-thio-CTP, 2'NH₂ UTP, 2'NH₂ CTP, and 2'F UTP. Such modified nucleotides include, but are not limited to, aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine, 5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine, alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2'NH2 uridine, 2'NH2 cytidine, and 2' F uridine, including the free pho (NTP) RNA molecules as well as all other useful forms of the nucleotides.

[00257] RNA interference as referred to herein additionally includes RNA molecules which contain modifications in the ribose sugars, as well as modifications in the "phosphate backbone" of the nucleotide chain. For example, siRNA or miRNA molecules containing oc-D-arabinofuranosyl structures in place of the naturally-occurring oc-D-ribonucleosides found in RNA can be used in RNA interference according to the present invention (U.S. Pat. No. 5,177,196). Other examples include RNA molecules containing the o-linkage between the sugar and the heterocyclic base of the nucleoside, which confers nuclease resistance and tight complementary strand binding to the oligonucleotide molecules similar to the oligonucleotides containing 2'-0-methyl ribose, arabinose and particularly oc-arabinose (U.S. Pat. No. 5,177,196 which is incorporated herein in its entirety by reference). Also, phosphorothioate linkages can be used to stabilize the siRNA and miRNA molecules (U.S. Pat. No. 5,177,196). siRNA and miRNA molecules having various "tails" covalently attached to either their 3'- or to their 5'-ends, or to both, are also been known in the art and can be used to stabilize the siRNA and miRNA molecules delivered using the methods of the present invention. Generally speaking, intercalating groups, various kinds of reporter groups and lipophilic groups attached to the 3' or 5' ends of the RNA molecules are well known to one skilled in the art and are useful according to the methods of the present invention. Descriptions of syntheses of 3'- cholesterol or 3'-acridine modified oligonucleotides applicable to preparation of modified RNA molecules useful according to the present invention can be found, for example, in the articles: Gamper, H. B. et al., (1993) Nucleic Acids Res. 21 145-150; and Reed, M. W. et al., (1991) Bioconjugate Chem. 2:217-225.

[00258] Various specific siRNA and miRNA molecules have been described and additional molecules can be easily designed by one skilled in the art. For example, the miRNA Database at world-wide -web address: sanger.ac.uk, followed by /Software/Rfam/mirna/index provides a useful source to identify additional miRNAs useful according to the present invention (Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111 ; Ambros V, et al, RNA, 2003, 9(3):277-279). [00259] An "siRNA" as used herein relates to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is expressed in the same cell as the gene or target gene. "siRNA" thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[00260] siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs). In one embodiment, these shRNAs are composed of a short, e.g., about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow.

[00261] Short interfering RNA (siRNA)-complex or micro interfering RNA (miRN A) -complex as referred to herein is a complex wherein an islet-targeting molecule: carrier particle is associated or complexed or mixed with the RNA interference, such as siRNA. Suitable siRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,

polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine,

poly thiodiethylaminomethyle thy lene P(TDAE), polyaminostyrene (e.g., p-amino),

poly (methylcyanoacrylate) , poly (e thy Icy anoacrylate) , poly (buty Icy anoacrylate) ,

poly (isobutylcyanoacrylate) , poly(isohexylcynaoacrylate) , DE AE-methacrylate , DE AE-hexylacrylate ,

DEAE-acrylamide, DE AE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG), and polyethylenimine.

[00262] In another embodiment, siRNAs useful according the methods of the present invention are found in WO 05/042719, WO 05/013886, WO 04/039957, and U.S. Pat. App. No. 20040248296 which are incorporated in their entirety herein by reference. Other useful siRNAs useful in the methods of the present invention include, but are not limited to, those found in U.S. Pat. App. Nos. 20050176666,

20050176665, 20050176664, 20050176663, 20050176025, 20050176024, 20050171040, 20050171039, 20050164970, 20050164968, 20050164967, 20050164966, 20050164224, 20050159382, 20050159381, 20050159380, 20050159379, 20050159378, 20050159376, 20050158735, 20050153916, 20050153915, 20050153914, 20050148530, 20050143333, 20050137155, 20050137153, 20050137151, 20050136436, 20050130181, 20050124569, 20050124568, 20050124567, 20050124566, 20050119212, 20050106726, 20050096284, 20050080031, 20050079610, 20050075306, 20050075304, 20050070497, 20050054598, 20050054596, 20050053583, 20050048529, 20040248174, 20050043266, 20050043257, 20050042646, 20040242518, 20040241854, 20040235775, 20040220129, 20040220128, 20040219671, 20040209832, 20040209831, 20040198682, 20040191905, 20040180357, 20040152651, 20040138163, 20040121353, 20040102389, 20040077574, 20040019001, 20040018176, 20040009946, 20040006035, 20030206887, 20030190635, 20030175950, 20030170891, 20030148507, 20030143732, and WO 05/060721, WO 05/060721, WO 05/045039, WO 05/059134, WO 05/045041, WO 05/045040, WO 05/045039, WO 05/027980, WO 05/014837, WO 05/002594, WO 04/085645, WO 04/078181, WO 04/076623, and WO 04/04635, which are all incorporated herein in their entirety by reference.

[00263] The RNA interference according to the present invention can be produced using any known techniques such as direct chemical synthesis, through processing of longer double stranded RNAs by exposure to recombinant Dicer protein or Drosophila embryo lysates, through an in vitro system derived from S2 cells, using phage RNA polymerase, RNA-dependent RNA polymerase, and DNA based vectors. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, for example, about 21-23 nucleotide, siRNAs from the lysate, etc. Chemical synthesis usually proceeds by making two single stranded RNA -oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA. Other examples include methods disclosed in WO 99/32619 and WO 01/68836 that teach chemical and enzymatic synthesis of siRNA. Moreover, numerous commercial services are available for designing and manufacturing specific siRNAs (see, e.g., QIAGEN Inc., Valencia, CA and AMBION Inc., Austin, TX)

[00264] The RNA interference, useful in the methods of the present invention includes siRNAs that target gene expression of any protein encoded inside a eukaryotic cell. Examples of these proteins include endogenous mammalian proteins, parasitic proteins, viral proteins encoded by an eukaryotic cell after entry of a virus into the cell. Examples of methods of preparing such RNA interference are shown, for example in an international patent application Nos. PCT/US03/34424, PCT/US03/34686, and U.S.

provisional patent applications No. 60/488,501, 60/488,155 and 60/516,172 the contents and references of all of these patent applications are herein incorporated by reference in their entirety.

[00265] Unlike the siRNA delivery methods to treat diabetes described in the prior art, the method of the present invention allows targeting of the RNAi agents specifically to pancreatic islet endothelial cell to minimize or to avoid completely undesired potential side effects of siRNA therapy. The islet-targeting molecule, e.g., an islet-targeting peptide as disclosed herein specifically brings the carrier particle with the RNAi agent to the target pancreatic islet endothelial cells. Accordingly, the method and compositions as disclosed herein provides a system to deliver RNAi agents, or cells into a limited number of cells thereby limiting, for example, potential side effects of therapies.

Affinity binding moiety

[00266] In one embodiment, the present invention comprises an affinity binding moiety which binds to a cell-surface antigen on a delivery cell, e.g., stem cell or progenitor cell, such as an EPC to deliver the EPC to the islet endothelial cell mediated by the islet-targeting peptide. For example, one such cell- surface antigen is a receptor present on desired cell to be delivered, e.g., a cell-surface antigen on the EPC. Accordingly in one embodiment, the present invention relates an affinity binding moiety which is associated to the islet-targeting molecule, where the affinity binding moiety can be such as an antibody or antigen-binding fragment thereof which targets and binds to the stem cell, e.g., EPC.

[00267] In some embodiments, an affinity binding moiety which binds to a cell-surface antigen on a delivery cell, e.g., EPC cell or other stem cell is an antibody. The antibody is preferably a single chain antibody, a Fab portion of an antibody or a (Fab')₂ segment or scFv.

[00268] Any antibody with a known sequence can be used as an affinity binding moiety according to the methods as disclosed herein to prepare a construct as described above. As used herein, an "antibody" or "functional fragment" of an antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab')₂ fragments, F(ab) fragments, Fv fragments, single domain antibodies, dimeric and trimeric antibody fragment constructs, minibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule and/or which bind a cell surface antigen.

[00269] As described, the affinity binding moiety is associated with an islet-targeting molecule, e.g., an islet-targeting peptide. In one embodiment, one uses a single vector containing gene segments that will express both the islet-targeting molecule and the affinity binding moiety. However, one can use a vector system to co-transfect a cell with at least two vectors and select for cells expressing the fusion protein. Preferably, one uses a single vector. One preferably attaches the sequence encoding the affinity binding moiety to a nucleic acid sequence encoding the islet-targeting molecule by standard means. For example, a gene for human protamine (Balhorn, . of Cell. Biol. 93:298-305 (1982)).

[00270] If antibodies are used as an affinity binding moiety, the use of single chain antibodies as the affinity binding moiety is preferable. In some instances, the full antibody and (Fab')₂ segments are typically preferred. In one embodiment, one could synthesize the fusion protein so that the affinity binding moiety is attached to the carboxy-terminus of the light or heavy chain of an intact immunoglobulin, such [00271] In order to limit antigenic reaction, the affinity binding moiety is preferably selected to take into account the host animal whose cells will be targeted. Thus, if the target animal is a mouse, one preferably uses murine antibodies, whereas if the target animal is a human, one preferably uses a human antibody or a humanized antibody.

[00272] In some embodiments, the affinity binding moiety binds to membrane proteins on the cell surface of the delivery cell, e.g., EPC, including receptors and antigens which can be internalized by receptor mediated endocytosis after interaction with the ligand to the receptor or antibodies. (Dautry- Varsat, A., et al, Sci. Am. 250:52-58 (1984)). This endocytic process is exploited by the delivery system as disclosed herein. Because this process can damage an agent, e.g., RNAi agent that is being internalized, in some embodiments it may be desirable to use a segment containing multiple repeats of the RNAi agent of interest. In some embodiments, one can also include sequences or moieties that disrupt endosomes and lysosomes. See, e.g., Cristiano, R. J., et al., Proc. Natl. Acad. Sci. USA 90:11548-11552 (1993); Wagner, E., et al., Proc. Natl. Acad. Sci. USA 89:6099-6103 (1992); Cotten, M., et al., Proc. Natl. Acad. Sci. USA 89:6094-6098 (1992).

[00273] Antibodies that are affinity binding moieties can be reactive to, or bind specifically to cell surface antigens on the cell to be delivered, e.g., stem cell, progenitor cell, such as EPC cell, or such as antibodies or fragments that bind to EPCs can be readily raised in animals such as rabbits or mice by immunization with the antigen expressed on the surface of the EPC. Immunized mice are particularly useful for providing sources of B cells for the manufacture of hybridomas, which in turn are cultured to produce large quantities of monoclonal antibodies.

[00274] Antibodies provide high binding avidity and unique specificity to a wide range of target antigens and haptens. Monoclonal antibodies useful as targeting moieties in the practice of the present invention include whole antibody and fragments thereof and are generated in accordance with

conventional techniques, such as hybridoma synthesis, recombinant DNA techniques and protein synthesis.

[00275] Useful monoclonal antibodies and fragments can be derived from any species (including humans) or can be formed as chimeric proteins which employ sequences from more than one species. Human monoclonal antibodies or "humanized" murine antibody are also used in accordance with the present invention. For example, murine monoclonal antibody can be "humanized" by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) or the complementarily determining regions thereof with the nucleotide sequence encoding a human constant domain region and an Fc region. Humanized targeting moieties are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half- life and a reduction of the possibly of adverse immune reactions in a manner similar to that disclosed in European Patent Application No. 0,411,893 A2 which is incorporated herein in its entirety by reference. The murine monoclonal antibodies should preferably be employed in humanized form. Antigen binding activity is determined by the sequences and conformation of the amino acids of the six complementarily determining regions (CDRs) that are located (three each) on the light and heavy chains of the variable portion (Fv) of the antibody. The 25-kDa single -chain Fv (scFv) molecule is composed of a variable region (VL) of the light chain and a variable region (VH) of the heavy chain joined via a short peptide spacer sequence. Techniques have been developed to display scFv molecules on the surface of filamentous phage that contain the gene for the scFv. scFv molecules with a broad range of antigenic- specificities can be present in a single large pool of scFv-phage library. Some examples of high affinity monoclonal antibodies and chimeric derivatives thereof, useful in the methods of the present invention, are described in the European Patent Application EP 186,833; PCT Patent Application WO 92/16553; and US Patent No. 6,090,923, which are incorporated herein in their entirety by reference.

[00276] Chimeric antibodies are immunoglobin molecules characterized by two or more segments or portions derived from different animal species. Generally, the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as murine monoclonal antibody, and the immunoglobin constant region is derived from a human immunoglobin molecule. In some embodiments, both regions and the combination have low immunogenicity as routinely determined.

[00277] One limitation of scFv molecules is their monovalent interaction with target antigen. One of the easiest methods of improving the binding of a scFv to its target antigen is to increase its functional affinity through the creation of a multimer. Association of identical scFv molecules to form diabodies, triabodies and tetrabodies can provide molecules comprising a number of identical Fv modules. These reagents are therefore multivalent, but monospecific. The association of two different scFv molecules, each comprising a VH and VL domain derived from different parent Ig will form a fully functional bispecific diabody. A unique application of bispecific scFvs is to bind two sites simultaneously on the same target molecule via two (adjacent) surface epitopes. These reagents gain a significant avidity advantage over a single scFv or Fab fragments. A number of multivalent scFv-based structures has been engineered, including for example, miniantibodies, dimeric miniantibodies, minibodies, (scFv)₂, diabodies and triabodies. These molecules span a range of valence (two to four binding sites), size (50 to 120 kDa), flexibility and ease of production. Single chain Fv antibody fragments (scFvs) are predominantly monomeric when the VH and VL domains are joined by polypeptide linkers of at least 12 residues. The monomer scFv is

thermodynamically stable with linkers of 12 and 25 amino acids length under all conditions. The noncovalent diabody and triabody molecules are easy to engineer and are produced by shortening the peptide linker that connects the variable heavy and variable light chains of a single scFv molecule. The scFv dimers are joined by amphipathic helices that offer a high degree of flexibility and the miniantibody structure can be modified to create a dimeric bispecific (DiBi) miniantibody that contains two miniantibodies (four scFv molecules) connected via a double helix. Gene -fused or disulfide bonded scFv dimers provide an intermediate degree of flexibility and are generated by straightforward cloning techniques adding a C-terminal Gly4Cys sequence. scFv-CH3 minibodies are comprised of two scFv molecules joined to an IgG CH3 domain either directly (LD minibody) or via a very flexible hinge region (Flex minibody). With a molecular weight of approximately 80 kDa, these divalent constructs are capable of significant binding to antigens. The Flex minibody exhibits impressive tumor localization in mice. Bi- and tri-specific multimers can be formed by association of different scFv molecules. Increase in functional affinity can be reached when Fab or single chain Fv antibody fragments (scFv) fragments are complexed into dimers, trimers or larger aggregates. The most important advantage of multivalent scFvs over monovalent scFv and Fab fragments is the gain in functional binding affinity (avidity) to target antigens. High avidity requires that scFv multimers are capable of binding simultaneously to separate target antigens. The gain in functional affinity for scFv diabodies compared to scFv monomers is significant and is seen primarily in reduced off -rates, which result from multiple binding to two or more target antigens and to rebinding when one Fv dissociates. When such scFv molecules associate into multimers, they can be designed with either high avidity to a single target antigen or with multiple specificities to different target antigens. Multiple binding to antigens is dependent on correct alignment and orientation in the Fv modules. For full avidity in multivalent scFvs target, the antigen binding sites must point towards the same direction. If multiple binding is not sterically possible then apparent gains in functional affinity are likely to be due the effect of increased rebinding, which is dependent on diffusion rates and antigen concentration. Antibodies conjugated with moieties that improve their properties are also contemplated for the instant invention. For example, antibody conjugates with PEG that increases their half-life in vivo can be used as targeting moieties in accordance with the methods of the present invention. Immune libraries are prepared by subjecting the genes encoding variable antibody fragments from the B lymphocytes of naive or immunized animals or patients to PCR amplification. Combinations of oligonucleotides which are specific for immunoglobulin genes or for the immunoglobulin gene families are used. Immunoglobulin germ line genes can be used to prepare semisynthetic antibody repertoires, with the complementarity- determining region of the variable fragments being amplified by PCR using degenerate primers. These single -pot libraries have the advantage that antibody fragments against a large number of antigens can be isolated from one single library. The phage-display technique can be used to increase the affinity of antibody fragments, with new libraries being prepared from already existing antibody fragments by random, codon-based or site-directed mutagenesis, by shuffling the chains of individual domains with those of fragments from naive repertoires or by using bacterial mutator strains.

[00278] Alternatively, a SCID-hu mouse, for example the model developed by GENPHARM, can be used to produce antibodies, or fragments thereof. In one embodiment, a new type of high avidity binding molecule, termed peptabody, created by harnessing the effect of multivalent interaction is contemplated. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In some embodiments, proteins-binding agents can be targeted to tissue- or tumor-specific targets by using bispecific antibodies, for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of antibodies can be used for production of recombinant bispecific single-chain Abs directing ligands and/or chimeric inhibitors at cell surface molecules. Alternatively in some embodiments, two or more protein-binding molecules can be administered, for example in some embodiments a protein binding molecule can be an antibody that is conjugated to another, different antibody. Each antibody is reactive with a different target site epitope (associated with the same or a different target site antigen). The different antibodies or antibody fragments with the associated binding moieties and RNAi molecules attached accumulate additively at the desired target site. Antibody-based or non- antibody-based affinity binding moieties can be employed to attach the associated islet-targeting molecule to the cell to be delivered, e.g., stem cell or progenitor cell, e.g., EPC.

Treatment of Diabetes

[00279] In some embodiments, the islet-targeting agent as disclosed herein is useful for the treatment, including prophylactic treatment of diabetes, such as Type 1, Type 2 and Type 1.5 diabetes. In particular, as disclosed herein in the Examples, an islet-targeting molecule, e.g., an islet-targeting peptide which is associated with a carrier particle: agent complex can be used in the treatment of Type 1 diabetes, where the agent which is delivered to the pancreatic islet endothelial cells is an anti-inflammatory agent, such as but not limited to Genistein, cyclosporine A and prednisone. In other embodiments, an islet-targeting molecule, e.g., an islet-targeting peptide which is associated with a delivery cell, e.g., stem cell or progenitor, e.g., EPC in an islet-targeting: affinity binding moiety : EPC complex can be used in the treatment of Type 2 diabetes, where the cell to be delivered promotes survival of the pancreatic islet endothelial cells.

[00280] By "treatment", "prevention" or "amelioration" of a disease or disorder is meant delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing down or stopping the progression, aggravation or deterioration the progression or severity of a condition associated with such a disease or disorder. In one embodiment, the symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. In some embodiments, the terms "treat" or "treatment" typically refer to therapeutic treatment, however can in some embodiments refer to prophylactic or preventative measures, wherein the object is to delay the onset of the disease, or prevent or slow the development of the disease, such as slow down the development of diabetes.

[00281] Treatment is generally 'effective" if one or more symptoms or clinical markers of diabetes are reduced as that term is defined herein. Alternatively, treatment is "effective" if the progression of a diabetes disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already diagnosed with diabetes, or in some embodiments, those identified to be susceptible to developing diabetes.

[00282] Treatment of Diabetes is determined by standard medical methods. A goal of Diabetes treatment is to bring sugar levels down to as close to normal as is safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. A particular physician may set different targets for the patent, depending on other factors, such as how often the patient has low blood sugar reactions. Useful medical tests include tests on the patient's blood and urine to determine blood sugar level, tests for glycosylated hemoglobin level (HbAlc; a measure of average blood glucose levels over the past 2-3 months, normal range being 4-6%), tests for cholesterol and fat levels, and tests for urine protein level. Such tests are standard tests known to those of skill in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by having fewer patients in the program with complications relating to Diabetes, such as diseases of the eye, kidney disease, or nerve disease.

[00283] Delaying the onset of diabetes in a subject refers to delay of onset of at least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, and can include the entire lifespan of the subject.

[00284] In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of Type 1 diabetes, Type 2 Diabetes Mellitus, or pre -diabetic conditions. In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female. A subject can be one who has been previously diagnosed with or identified as suffering from or having Diabetes (e.g., Type 1 or Type 2), one or more complications related to Diabetes, or a pre-diabetic condition, and optionally, but need not have already undergone treatment for the Diabetes, the one or more complications related to Diabetes, or the pre-diabetic condition. A subject can also be one who is not suffering from Diabetes or a pre-diabetic condition. A subject can also be one who has been diagnosed with or identified as suffering from Diabetes, one or more complications related to Diabetes, or a pre-diabetic condition, but who show improvements in known Diabetes risk factors as a result of receiving one or more treatments for Diabetes, one or more complications related to Diabetes, or the pre- diabetic condition. Alternatively, a subject can also be one who has not been previously diagnosed as having Diabetes, one or more complications related to Diabetes, or a pre-diabetic condition. For example, a subject can be one who exhibits one or more risk factors for Diabetes, complications related to Diabetes, or a pre-diabetic condition, or a subject who does not exhibit Diabetes risk factors, or a subject who is asymptomatic for Diabetes, one or more Diabetes-related complications, or a pre-diabetic condition. A subject can also be one who is suffering from or at risk of developing Diabetes or a pre- diabetic condition. A subject can also be one who has been diagnosed with or identified as having one or more complications related to Diabetes or a pre-diabetic condition as defined herein, or alternatively, a subject can be one who has not been previously diagnosed with or identified as having one or more complications related to Diabetes or a pre-diabetic condition.

[00285] As used herein, the phrase "subject in need of diabetes treatment" refers to a subject who is diagnosed with or identified as suffering from, having or at risk for developing diabetes (e.g., Type 1, Type 1.5 or Type 2), one or more complications related to diabetes, or a pre-diabetic condition.

[00286] A subject in need of diabetes treatment can be identified using any method used for diagnosis of diabetes. For example, Type 1 diabetes can be diagnosed using a glycosylated hemoglobin (AIC) test, a random blood glucose test and/or a fasting blood glucose test. Parameters for diagnosis of diabetes are known in the art and available to skilled artisan without much effort.

[00287] In some embodiments, the methods of the invention further comprise selecting a subject identified as being in need of diabetes treatment. A subject in need of diabetes treatment can be selected based on the symptoms presented, such as symptoms of type 1, type 1.5 or type 2 diabetes. Exemplary symptoms of diabetes include, but are not limited to, excessive thirst (polydipsia), frequent urination (polyuria), extreme hunger (polyphagia), extreme fatigue, weight loss, hyperglycemia, low levels of insulin, high blood sugar (e.g., sugar levels over 250 mg, over 300 mg), presence of ketones present in urine, fatigue, dry and/or itchy skin, blurred vision, slow healing cuts or sores, more infections than usual, numbness and tingling in feet, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease,

cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, and combinations thereof.

[00288] In some embodiments, a composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide and its associated complex for administration to a subject can further comprise a pharmaceutically active agent, such as those agents known in the art for treatment of diabetes and or for having anti-hyperglycemic activities, for example, inhibitors of dipeptidyl peptidase 4 (DPP-4) (e.g., Alogliptin, Linagliptin, Saxagliptin, Sitagliptin, Vildagliptin, and Berberine), biguanides (e.g., Metformin, Buformin and Phenformin), peroxisome proliferator-activated receptor (PPAR) modulators such as thiazolidinediones (TZDs) (e.g., Pioglitazone, Rivoglitazone, Rosiglitazone and Troglitazone), dual PPAR agonists (e.g., Aleglitazar, Muraglitazar and Tesaglitazar), sulfonylureas (e.g., Acetohexamide,

Carbutamide, Chlorpropamide, Gliclazide, Tolbutamide, Tolazamide, Glibenclamide (Glyburide), Glipizide, Gliquidone, Glyclopyr amide, and Glimepiride), meglitinides ("glinides") (e.g., Nateglinide, Repaglinide and Mitiglinide), glucagon-like peptide- 1 (GLP-1) and analogs (e.g., Exendin-4, Exenatide, Liraglutide, Albiglutide), insulin and insulin analogs (e.g., Insulin lispro, Insulin aspart, Insluin glulisine, Insulin glargine, Insulin detemir, Exubera and NPH insulin), alpha-glucosidase inhibitors (e.g., Acarbose, Miglitol and Voglibose), amylin analogs (e.g. Pramlintide), Sodium-dependent glucose cotransporter T2 (SGLT T2) inhibitors (e.g., Dapgliflozin, Remogliflozin and Sergliflozin) and others (e.g. Benfluorex and Tolrestat).

[00289] In some embodiments, a complex associated with an islet-targeting molecule, e.g., an islet- targeting peptide can be, for example, an islet-targeting molecule: carrier particle: agent complex and/or islet-targeting molecule: affinity binding moiety: EPC complex. In some embodiments, a complex associated with an islet-targeting molecule can be, for example, an islet-targeting molecule: affinity binding moiety: carrier particle: EPC complex.

[00290] In type 1 diabetes, β-cells are undesirably destroyed by continued autoimmune response. This autoimmune response may also destroy an islet-targeting molecule: carrier particle: agent complex, and/or an EPC cell-associated with the islet-targeting molecule which administered into a subject. Thus, this autoimmune response can be attenuated by use of compounds that inhibit or block such an autoimmune response. In some embodiments, a composition comprising an islet-targeting molecule: carrier particle: agent complex and/or an islet-targeting molecule: affinity binding moiety: EPC complex can further comprise (in some embodiments, associated with the carrier particle) a pharmaceutically active agent which is an immune response modulator. As used herein, the term "immune response modulator" refers to compound (e.g., a small-molecule, antibody, peptide, nucleic acid, or gene therapy reagent) that inhibits autoimmune response in a subject. Without wishing to be bound by theory, an immune response modulator inhibits the autoimmune response by inhibiting the activity, activation, or expression of inflammatory cytokines (e.g., IL-12, IL-23 or IL-27), or STAT -4. Exemplary immune response modulators include, but are not limited to, members of the group consisting of Lisofylline (LSF) and the LSF analogs and derivatives described in U.S. Pat. No. 6,774,130, contents of which are herein incorporated by reference in their entirety.

[00291] A composition comprising an islet-targeting molecule: carrier particle: agent complex and/or islet-targeting molecule: affinity binding moiety: EPC complex can be administrated to the subject in the same time, of different times as the administration of another therapeutic agent, e.g., an immune response modulator. When administrated at different times, the compositions comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC can be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4, hours, 8 hours, 12 hours, 24 hours of administration of another therapeutic agent. When a composition comprising an islet- targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC are administered with an additional therapeutic agent in different pharmaceutical compositions, routes of administration can be different. In some embodiments, a subject is administered a composition comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC. In another embodiment, a subject is administered a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC and a composition comprising an additional therapeutic agent, where administration is substantially at the same time, or subsequent to each other.

[00292] Toxicity and therapeutic efficacy of administration of a compositions comprising an islet- targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Compositions comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC that exhibit large therapeutic indices are preferred.

[00293] The amount of a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC can be tested using several well- established diabetic animal models.

[00294] For example, but not limited to, the non-obese diabetic (NOD) mouse can be used, which carries a genetic defect that results in insulitis showing at several weeks of age (Yoshida et al. , Rev.

Immunogenet. 2: 140 (2000)). 60-90% of the females develop overt diabetes by 20-30 weeks. The immune -related pathology appears to be similar to that in human Type 1 diabetes. Other models of Type 1 diabetes are mice with transgene and knockout mutations (Wong et al., Immunol. Rev. 169:93 (1999)). A rat model for spontaneous Type 1 diabetes was reported by Lenzen et al. (Diabetologia 44: 1189 (2001)). Hyperglycemia can also be induced in mice (>500 mg glucose/dL) by way of a single intraperitoneal injection of streptozotocin (Soria et al , Diabetes 49:157, 2000), or by sequential low doses of streptozotocin (Ito et al, Environ. Toxicol. Pharmacol. 9:71, 2001). To test the efficacy of an islet- targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC, the mice are monitored for return of glucose to normal levels (<200 mg/dL).

[00295] Larger animals provide a good model for following the sequelae of chronic hyperglycemia. Dogs can be rendered insulin-dependent by removing the pancreas ( . Endocrinol. 158:49, 2001), or by feeding galactose (Kador et al., Arch. Opthalmol. 113:352, 1995). There is also an inherited model for Type 1 diabetes in keeshond dogs (Am. J. Pathol. 105:194, 1981). Early work with a dog model (Banting et al , Can. Med. Assoc. J. 22: 141, 1922) resulted in a couple of Canadians making a long ocean journey to Stockholm in February of 1925. [00296] By way of illustration, a pilot study can be conducted by administering an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC into the following animals: a) non- diabetic nude (T-cell deficient) mice; b) nude mice rendered diabetic by streptozotocin treatment; and c) nude mice in the process of regenerating islets following partial pancreatectomy. For non-diabetic mice, the endpoints of the efficacy of administration of islet-targeting molecule: affinity binding moiety: EPC complex can be the assessment of graft survival (histological examination) of the EPC cells and determination of insulin production by biochemical analysis, RIA, ELISA, and immunohistochemistry. Streptozotocin treated and partially pancreatectomized animals can also be evaluated for survival, metabolic control (blood glucose) and weight gain.

[00297] In some embodiments, data obtained from the cell culture assays and in animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[00298] The therapeutically effective dose of a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC can also be estimated initially from cell culture assays. A dose may be formulated in animal models in vivo to achieve a secretion of insulin at a concentration which is appropriate in response to circulating glucose in the plasma. Alternatively, the effects of any particular dosage can be monitored by a suitable bioassay.

[00299] With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment or make other alteration to treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the agents delivered by an islet-targeting molecule complexed with either a carrier particle-agent or the successful engraftment of EPCs delivered by the islet targeting peptide. The desired dose can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. Such subdoses can be administered as unit dosage forms. In some embodiments, administration is chronic, e.g., one or more doses daily over a period of weeks or months. Examples of dosing schedules are

administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.

[00300] In another aspect of the invention, the methods provide use of an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein. In one embodiment of the invention, an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein may be used for the production of a pharmaceutical composition, for the use in administration into subjects in need of treatment, e.g., a subject that has, or is at risk of developing diabetes, for example but not limited to subjects with congenital and acquired diabetes. In one embodiment, an EPC cell delivered using an islet-targeting: affinity binding moiety: EPC complex may be genetically modified. In another aspect, the subject may have or be at risk of diabetes and/or metabolic disorder. In some embodiments, an EPC cell delivered to pancreas islet cells using the islet-targeting molecule as disclosed herein may be autologous and/or allogenic. In some embodiments, the subject is a mammal, and in other embodiments the mammal is a human.

[00301] The use of an islet-targeting molecule, e.g., an islet-targeting peptide, complexed with either a carrier particle-agent and/or EPC as disclosed herein provides advantages over existing methods because it allows delivery of the agents specifically to the pancreatic islet endothelial cells, thus reducing off-target adverse side effects, and in some embodiments, the EPC cells can be obtained or harvested from the subject who is administered an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC. This is highly advantageous as it provides reduced complications associated with rejection of the EPC cells.

[00302] In another embodiment, an islet-targeting molecule complexed with either a carrier particle- agent and/or EPC can be used in models for studying properties of differentiation of endothelial cells into insulin-producing cells, e.g., to pancreatic β-cells or pancreatic β-like cells, or pathways of development of cells of endoderm origin into pancreatic β-cells.

[00303] In some embodiments, the EPC cells delivered by the islet-targeting molecules as disclosed herein can be genetically engineered to comprise markers operatively linked to promoters that are expressed when a marker is expressed or secreted, for example, a marker can be operatively linked to a suitable promoter, so that the marker is expressed when the EPC cell expresses the native protein associated with the promoter. In some embodiments, the EPC cells can be genetically-modified EPCs that are engineered to secrete insulin. In some embodiments, the EPC cells are genetically modified to secrete insulin in response to glucose levels and exhibit glucose sensitivity. In some embodiments, the EPC cells delivered to islet cells by the islet-targeting molecule can be used in a model for studying the survival of islet cells in diabetes.

[00304] In other embodiments, the islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC can be used in animal and in vitro models for studying the role of islet β-cells in the pancreas and in the development of diabetes and metabolic disorders. In some embodiments, the animal model can be an animal model of diabetes, or alternatively, the model can carry a mutation and/or polymorphism (e.g., in the gene Pdxl which leads to early-onset insulin-dependent diabetes mellitus (NIDDM), as well as maturity onset diabetes of the young type 4 (MODY4), which can be used to identify small molecules and other therapeutic agents that can be used to treat subjects with diabetes with a mutation or polymorphism in Pdxl.

[00305] In one embodiment of the invention relates to a method of treating diabetes in a subject comprising administering an effective amount of a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC as disclosed herein to a subject with diabetes and/or a metabolic disorder. In a further embodiment, the invention provides a method for treating diabetes, comprising administering a composition comprising an islet- targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC as disclosed herein to a subject that has, or has increased risk of developing diabetes in an effective amount sufficient to produce insulin in response to increased blood glucose levels.

[00306] In one embodiment of the above methods, the subject is a human and a delivery cell, e.g., stem cell or progenitor, e.g., EPC cell as disclosed herein are human cells. In some embodiments, the invention contemplates that a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC disclosed herein are administered directly to the pancreas of a subject, or is administered systemically. In some embodiments, an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC as disclosed herein can be administered to any suitable location in the subject, for example into blood vessel or the liver or any suitable site where the islet-targeting molecule comprising complexes can migrate and target pancreatic islet endothelial cells in the subject.

[00307] The present invention is also directed to a method of treating a subject with diabetes or a metabolic disorder which occurs as a consequence of genetic defect, physical injury, environmental insult or conditioning, bad health, obesity and other diabetes risk factors commonly known by a person of ordinary skill in the art. Efficacy of treatment of a subject administered a composition comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC can be monitored by clinically accepted criteria and tests, which include for example, (i) Glycated hemoglobin (AIC) test, which indicates a subjects average blood sugar level for the past two to three months, by measuring the percentage of blood sugar attached to hemoglobin, the oxygen-carrying protein in red blood cells. The higher your blood sugar levels, the more hemoglobin has sugar attached. An AIC level of 6.5 percent or higher on two separate tests indicates the subject has diabetes. A test value of 6-6.5% suggest the subject has prediabetes, (ii) Random blood sugar test. A blood sample will be taken from the subject at a random time, and a random blood sugar level of 200 milligrams per deciliter (mg/dL) - 11.1 millimoles per liter (mmol/L), or higher indicated the subject has diabetes, (iii) Fasting blood sugar test. A blood sample is taken from the subject after an overnight fast. A fasting blood sugar level between 70 and 99 mg/dL (3.9 and 5.5 mmol/L) is normal. If the subjects fasting blood sugar levels is 126 mg/dL (7 mmol/L) or higher on two separate tests, the subject has diabetes. A blood sugar level from 100 to 125 mg/dL (5.6 to 6.9 mmol/L) indicates the subject has prediabetes, (iv) Oral glucose tolerance test. A blood sample will be taken after the subject has fasted for at least eight hours or overnight and then ingested a sugary solution, and the blood sugar level will be measured two hours later. A blood sugar level less than 140 mg/dL (7.8 mmol/L) is normal. A blood sugar level from 140 to 199 mg/dL (7.8 to 11 mmol/L) is considered prediabetes. This is sometimes referred to as impaired glucose tolerance (IGT). A blood sugar level of 200 mg/dL (11.1 mmol/L) or higher may indicate diabetes.

[00308] In some embodiments, the effects of administration of a composition comprising an islet- targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein to a subject in need thereof is associated with improved exercise tolerance or other quality of life measures, and decreased mortality. The effects of an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC can be evident over the course of days to weeks after the procedure. However, beneficial effects may be observed as early as several hours after the procedure, and may persist for several years.

[00309] In some embodiments, a composition comprising an islet-targeting molecule complexed with an EPC as disclosed herein may be used for tissue reconstitution or regeneration in a human patient or other subject in need of such treatment. In some embodiments compositions an islet-targeting molecule complexed with an EPC can be administered in a manner that permits the islet-targeting molecule to deliver the EPC cells to pancreatic islet endothelial cells, and allow the EPC cells to graft and reconstitute or regenerate the functionally deficient area. Special devices are available that are adapted for administering compositions capable of reconstituting a population of β-cells in the pancreas or at an alternative desired location. Accordingly, in some embodiments, a composition comprising an islet- targeting molecule complexed with either a carrier particle-agent and/or EPC can be administered to a recipient subject's pancreas by injection, or administered by intramuscular injection.

[00310] In some embodiments, compositions comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein have a variety of uses in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, for example, a population of definitive endoderm cells and/or pdxl -positive pancreatic progenitors as disclosed herein may be administered to enhance insulin production in response to increase in blood glucose level for any perceived need, such as an inborn error in metabolic function, the effect of a disease condition (e.g.

diabetes), or the result of significant trauma (i.e., damage to the pancreas or loss or damage to islet β- cells). In some embodiments, a composition comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein are administered to the subject not only help restore function to damaged or otherwise unhealthy tissues, but also facilitate remodeling of the damaged tissues. For example, in particular embodiments, a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide associated with an EPC cell can be administered to a subject to facilitate survival of existing pancreas islet cells in type 2 diabetes, according to the methods as disclosed herein.

[00311] To determine the suitability of an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC for therapeutic administration, an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC can first be tested in a suitable animal model. At one level, the delivered EPC cells are assessed for their ability to survive and maintain their phenotype in vivo. An islet-targeting molecule, e.g., an islet-targeting peptide complexed with an EPC can be administered to immunodeficient animals (such as nude mice, or animals rendered immunodeficient chemically or by irradiation). Tissues are harvested after a period of regrowth, and assessed as to whether the administered cells or progeny thereof are still present.

[00312] In some embodiments, survival of EPC cells can be performed by administering EPC:islet targeting molecule, e.g., a islet targeting peptide to cells that comprise a detectable label (such as green fluorescent protein, or beta-galactosidase); that have been prelabeled (for example, with BrdU or [3H] thymidine), or by subsequent detection of a constitutive cell marker (for example, using human-specific antibody). The presence and phenotype of the administered population of delivered EPC cells can be assessed by immunohistochemistry or ELISA using human-specific antibody, or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for human

polynucleotides, according to published sequence data.

[00313] A number of animal models for testing diabetes are available for such testing, and are commonly known in the art, for example as disclosed in U.S. Patent 6,187,991 which is incorporated herein by reference, as well as rodent models; NOD (non-obese mouse), BB_DB mice, KDP rat and TCR mice, and other animal models of diabetes as described in Rees et al., Diabet Med. (2005) Apr;22(4):359- 70; Srinivasan K, et al., Indian J Med Res. (2007) Mar;125(3):451-7; Chatzigeorgiou A, et al. , In Vivo (2009) Mar-Apr;23(2):245-58, which are incorporated herein by reference.

[00314] In some embodiments, a composition comprising an islet-targeting molecule, e.g., an islet targeting peptide complexed with either a carrier particle-agent and/or EPC as disclosed herein may be administered in any physiologically acceptable excipient, where the islet-targeting molecule, e.g., an islet- targeting peptide delivers the carrier particle: agent or associated EPC cell to the pancreatic islet endothelial cells. In some embodiments, a composition comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein can be introduced by injection, catheter, or the like. In some embodiments, a composition comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC as disclosed herein can be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use on thawing. If frozen, a composition comprising an islet-targeting molecule complexed with an EPC will usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[00315] In some embodiments, a composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide complexed with either a carrier particle-agent and/or EPC as disclosed herein can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular

Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and

Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with either a carrier particle-agent and/or EPC as disclosed herein will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising an islet-targeting molecule complexed with either a carrier particle-agent and/or EPC can also comprise or be accompanied with one or more other ingredients that facilitate the functional mobilization of the islet-targeting molecule to the target pancreatic islet endothelial cells.

[00316] In some embodiments, the EPC cells delivered by the islet-targeting molecule as disclosed herein may be genetically altered in order to introduce useful genes, e.g., genes useful in insulin production by pancreatic β-cells, e.g., repair of a genetic defect in an individual, selectable marker, etc., or for the selective suicide of implanted EPC cells. In some embodiments, the EPC cells delivered by the islet-targeting molecule as disclosed herein can also be genetically modified to enhance survival, control proliferation, and the like. In some embodiments, the EPC cells delivered by the islet-targeting molecule as disclosed herein can be genetically altering by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a gene of interest. In one embodiment, EPC cells delivered by the islet-targeting molecule, e.g., an islet-targeting peptide as disclosed herein is transfected with genes encoding a telomerase catalytic component (TERT), typically under a heterologous promoter that increases telomerase expression beyond what occurs under the endogenous promoter, (see International Patent Application WO 98/14592, which is incorporated herein by reference). In other embodiments, a selectable marker is introduced, to provide for greater purity of the population the EPC cells delivered by the islet-targeting molecule, e.g., an islet-targeting peptide as disclosed herein. In some embodiments, the EPC cells delivered by the islet-targeting molecule as disclosed herein may be genetically altered using vector containing supernatants over an 8-16 h period, and then exchanged into growth medium for 1-2 days. Genetically altered EPC cells delivered by the islet- targeting molecule as disclosed herein can be selected using a drug selection agent such as puromycin, G418, or blasticidin, and then recultured.

[00317] Gene therapy can be used to either modify a cell to replace a gene product, to facilitate regeneration of tissue, to treat disease, or to improve survival of the cells following implantation into a subject (i.e., prevent rejection).

[00318] In an alternative embodiment, the EPC cells delivered by the islet-targeting molecule as disclosed herein as disclosed herein can also be genetically altered in order to enhance their ability to be involved in tissue regeneration, or to deliver a therapeutic gene to a site of administration. A vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in the differentiated cell type. Of particular interest are cells that are genetically altered to express one or more growth factors of various types, such as somatostatin, glucagon, and other factors.

[00319] Many vectors useful for transferring exogenous genes into the EPC cells delivered by the islet- targeting molecule, e.g., an islet-targeting peptide as disclosed herein are available. The vectors may be episomal, e.g., plasmids, virus derived vectors such as cytomegalovirus, adenovirus, etc., or may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus derived vectors such MMLV, HIV-1, ALV, etc. In some embodiments, combinations of retroviruses and an appropriate packaging cell line may also find use, where the capsid proteins will be functional for infecting the EPC cells delivered by the islet-targeting molecule as disclosed herein.

Usually, the EPC cells and virus will be incubated for at least about 24 hours in the culture medium. In some embodiments, the EPC cells are then allowed to grow in the culture medium for short intervals in some applications, e.g., 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis, and then association with the islet-targeting molecule as disclosed herein.

Commonly used retroviral vectors are "defective", i.e., unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line.

[00320] The host cell specificity of the retrovirus is determined by the envelope protein, env (pl20). The envelope protein is provided by the packaging cell line. Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g. MMLV, are capable of infecting most murine and rat cell types. Ecotropic packaging cell lines include BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearing amphotropic envelope protein, e.g. 4070A (Danos et al, supra.), are capable of infecting most mammalian cell types, including human, dog and mouse. Amphotropic packaging cell lines include PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902) GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelope protein, e.g., AKR env, are capable of infecting most mammalian cell types, except murine cells. In some embodiments, the vectors may include genes that must later be removed, e.g., using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g., by including genes that allow selective toxicity such as herpes virus TK, Bcl-Xs, etc.

[00321] Suitable inducible promoters are activated in a desired target cell type, either the transfected cell, or progeny thereof. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 100 fold, more usually by at least about 1000 fold. Various promoters are known that are induced in different cell types.

[00322] In one aspect of the present invention, a composition comprising islet-targeting molecule, e.g., an islet-targeting peptide complexed with a carrier particle: agent complex and/or EPC as disclosed herein are suitable for administering systemically or to a target anatomical site, e.g., the pancreas. A composition comprising islet-targeting molecule complexed with a carrier particle : agent complex and/or EPC can be administered into or nearby a subject's pancreas, for example, or may be administered systemically, such as, but not limited to, intra-arterial or intravenous administration. In alternative embodiments, a composition comprising islet-targeting molecule complexed with a carrier particle : agent complex and/or EPC of the present invention can be administered in various ways as would be appropriate for the homing of the islet targeting peptide to the pancreatic or secretory system, including but not limited to parenteral, including intravenous and intraarterial administration, intrathecal administration, intraventricular administration, intraparenchymal, intracranial, intracisternal, intrastriatal, and intranigral administration. Optionally, a composition comprising islet-targeting molecule, e.g., an islet-targeting peptide complexed with a carrier particle : agent complex and/or EPC are administered in conjunction with an

immunosuppressive agent.

[00323] In some embodiments, a composition comprising islet-targeting molecule, e.g., an islet-targeting peptide complexed with an carrier particle : agent complex and/or EPC can be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement, including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. A composition comprising an islet-targeting molecule complexed with an carrier particle : agent complex and/or EPC can be administered to a subject the following locations: clinic, clinical office, emergency department, hospital ward, intensive care unit, operating room, catheterization suites, and radiologic suites.

[00324] In other embodiments, a composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide complexed with a carrier particle : agent complex and/or EPC is stored for later administration. A composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide, complexed with a carrier particle : agent complex and/or EPC may be divided into more than one aliquot or unit such that part of a composition comprising islet-targeting molecule complexed with a carrier particle : agent complex and/or EPC is retained for later application while part is applied immediately to the subject. Moderate to long-term storage of all or part of the EPC cells in a cell bank is also within the scope of this invention, as disclosed in U.S. Patent Application Serial No. 20030054331 and Patent Application No. WO03024215, and is incorporated by reference in their entireties. At the end of processing, the concentrated cells may be loaded into a delivery device, such as a syringe, for placement into the recipient by any means known to one of ordinary skill in the art.

[00325] In some embodiments, a composition comprising an islet-targeting molecule complexed with an EPC can be applied alone or in combination with other cells, tissue, tissue fragments, growth factors such as VEGF and other known angiogenic or arteriogenic growth factors, biologically active or inert compounds, resorbable plastic scaffolds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the population. In some embodiments, a composition comprising an islet- targeting molecule complexed with an EPC may also be modified by insertion of DNA or by placement in cell culture in such a way as to change, enhance, or supplement the function of the cells for derivation of a structural or therapeutic purpose. For example, gene transfer techniques for stem cells are known by persons of ordinary skill in the art, as disclosed in (Morizono et al. , 2003; Mosca et al. , 2000), and may include viral transfection techniques, and more specifically, adeno-associated virus gene transfer techniques, as disclosed in (Walther and Stein, 2000) and (Athanasopoulos et al., 2000). Non-viral based techniques may also be performed as disclosed in (Murarnatsu et al., 1998).

[00326] In another aspect, in some embodiments, the EPC cells delivered by the islet-targeting molecule as disclosed herein could be combined with a gene encoding pro-angiogenic growth factor(s). Genes encoding anti-apoptotic factors or agents could also be applied. Addition of the gene (or combination of genes) could be by any technology known in the art including but not limited to adenoviral transduction, "gene guns," liposome-mediated transduction, and retrovirus or lentivirus-mediated transduction, plasmid' adeno-associated virus. Cells could be implanted along with a carrier material bearing gene delivery vehicle capable of releasing and/or presenting genes to the cells over time such that transduction can continue or be initiated. Particularly when the EPC cells and/or tissue containing the cells are administered to a patient other than the patient from whom the cells and/or tissue were obtained, one or more immunosuppressive agents may be administered to the patient receiving the cells and/or tissue to reduce, and preferably prevent, rejection of the transplant. As used herein, the term "immunosuppressive drug or agent" is intended to include pharmaceutical agents which inhibit or interfere with normal immune function. Examples of immunosuppressive agents suitable with the methods disclosed herein include agents that inhibit T-cell/B- cell costimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No 2002/0182211, which is incorporated herein by reference. In one embodiment, a immunosuppressive agent is

cyclosporine A. Other examples include myophenylate mofetil, rapamycin, and anti- thymocyte globulin. In one embodiment, the immunosuppressive drug is administered with at least one other therapeutic agent. The immunosuppressive drug is administered in a formulation which is compatible with the route of administration and is administered to a subject at a dosage sufficient to achieve the desired therapeutic effect. In another embodiment, the immunosuppressive drug is administered transiently for a sufficient time to induce tolerance to the cardiovascular stem cells of the invention.

[00327] Pharmaceutical compositions comprising effective amounts of an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC are also contemplated by the present invention. These compositions comprise an effective amount of an islet-targeting molecule, e.g., an islet- targeting peptide, complexed with a carrier particle: agent complex and/or EPC, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. In certain aspects of the present invention, a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC are administered to the subject in need of a transplant in sterile saline. In other aspects of the present invention, a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC are administered in Hanks Balanced Salt Solution (HBSS) or Isolyte S, pH 7.4. Other approaches may also be used, including the use of serum free cellular media. In one embodiment, a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC are administered in plasma or fetal bovine serum, and DMSO. Systemic administration of a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide, complexed with a carrier particle: agent complex and/or EPC to the subject may be preferred in certain indications, whereas direct administration at the site of or in proximity to the diseased and/or damaged tissue may be preferred in other indications.

[00328] In some embodiments, a composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide complexed with a carrier particle: agent complex and/or EPC can optionally be packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution or thawing (if frozen) a composition comprising an islet-targeting molecule complexed with an carrier particle: agent complex and/or EPC prior to administration to a subject.

[00329] In one embodiment, a composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide complexed with a carrier particle: agent complex and/or EPC are administered with a differentiation agent. In one embodiment, a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC are combined with the differentiation agent to administration into the subject. In another embodiment, islet-targeting molecule complexes can be administered separately to the subject from a differentiation agent. Optionally, if the islet-targeting molecule complexes are administered separately from the differentiation agent, there is a temporal separation in the administration of the islet-targeting molecule complexes and the differentiation agent. The temporal separation may range from about less than a minute in time, to about hours or days in time. The determination of the optimal timing and order of administration is readily and routinely determined by one of ordinary skill in the art.

Diagnosis of diabetes

[00330] Type 1 diabetes is an autoimmune disease that results in destruction of insulin-producing β cells of the pancreatic islets. Lack of insulin causes an increase of fasting blood glucose (around 70-120 mg/dL in nondiabetic people) that begins to appear in the urine above the renal threshold (about 190-200 mg/dl in most people). The World Health Organization defines the diagnostic value of fasting plasma glucose concentration to 7.0 mmol/1 (126 mg/dl) and above for Diabetes Mellitus (whole blood 6.1 mmol/1 or 110 mg/dl), or 2-hour glucose level of 11.1 mmol/L or higher (200 mg/dL or higher).

[00331] Type 1 diabetes can be diagnosed using a variety of diagnostic tests that include, but are not limited to, the following: (1) glycated hemoglobin (AlC) test, (2) random blood glucose test and/or (3) fasting blood glucose test.

[00332] The Glycated hemoglobin (AlC) test is a blood test that reflects the average blood glucose level of a subject over the preceding two to three months. The test measures the percentage of blood glucose attached to hemoglobin, which correlates with blood glucose levels (e.g., the higher the blood glucose levels, the more hemoglobin is glycosylated). An AIC level of 6.5 percent or higher on two separate tests is indicative of diabetes. A result between 6 and 6.5 percent is considered prediabetic, which indicates a high risk of developing diabetes.

[00333] The Random Blood Glucose Test comprises obtaining a blood sample at a random time point from a subject suspected of having diabetes. Blood glucose values can be expressed in milligrams per deciliter (mg/dL) or millimoles per liter (mmol/L). A random blood glucose level of 200 mg/dL (11.1 mmol/L) or higher indicates the subject likely has diabetes, especially when coupled with any of the signs and symptoms of diabetes, such as frequent urination and extreme thirst.

[00334] For the fasting blood glucose test, a blood sample is obtained after an overnight fast. A fasting blood glucose level less than 100 mg/dL (5.6 mmol/L) is considered normal. A fasting blood glucose level from 100 to 125 mg/dL (5.6 to 6.9 mmol/L) is considered prediabetic, while a level of 126 mg/dL (7 mmol/L) or higher on two separate tests is indicative of diabetes.

[00335] Type 1 diabetes can also be distinguished from type 2 diabetes using a C-peptide assay, which is a measure of endogenous insulin production. The presence of anti-islet antibodies (to Glutamic Acid

Decarboxylase, Insulinoma Associated Peptide -2 or insulin), or lack of insulin resistance, determined by a glucose tolerance test, is also indicative of type 1 , as many type 2 diabetics continue to produce insulin internally, and all have some degree of insulin resistance.

[00336] Testing for GAD 65 antibodies has been proposed as an improved test for differentiating between type 1 and type 2 diabetes, as it appears that the immune system is involved in Type 1 diabetes etiology.

[00337] In some embodiments, the present invention provides compositions for the use of islet-targeting molecule, e.g., an islet-targeting peptide, complexed with a carrier particle: agent complex and/or EPC produced by the methods as disclosed herein to restore islet function in a subject in need of such therapy. Any condition relating to inadequate production of a pancreatic endocrine (insulin, glucagon, or somatostatin), or the inability to properly regulate secretion may be considered for treatment with a composition comprising islet-targeting molecule, e.g., an islet-targeting peptide, complexed with a carrier particle: agent complex and/or EPC prepared according to this invention, as appropriate. Of especial interest is the treatment of Type 1 (insulin-dependent) or Type 2 diabetes mellitus.

[00338] Subjects in need thereof can be selected for treatment based on confirmed long-term dependence on administration of exogenous insulin, and acceptable risk profile. A composition comprising islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC can be infused through a catheter in the portal vein. The subject can then be subjected to abdominal ultrasound and blood tests to determine liver function. Daily insulin requirement is tracked, and the subject is given a second transplant if required. Follow-up monitoring includes frequent blood tests for drug levels, immune function, general health status, and whether the patient remains insulin independent. [00339] General approaches to the management of the diabetic patient are provided in standard textbooks, such as the Textbook of Internal Medicine, 3rd Edition, by W. N. Kelley ed., Lippincott-Raven, 1997; and in specialized references such as Diabetes Mellitus: A Fundamental and Clinical Text 2nd Edition, by D. Leroith ed., Lippincott Williams & Wilkins 2000; Diabetes (Atlas of Clinical

Endocrinology Vol. 2) by C. R. Kahn et al. eds., Blackwell Science 1999; and Medical Management of Type 1 Diabetes 3rd Edition, McGraw Hill 1998. Use of islet cells for the treatment of Type 1 diabetes is discussed at length in Cellular Inter-Relationships in the Pancreas: Implications for Islet Transplantation, by L. Rosenberg et al., Chapman & Hall 1999; and Fetal Islet Transplantation, by C. M. Peterson et al. eds., Kluwer 1995.

[00340] As always, the ultimate responsibility for subject selection, the mode of administration, and dosage of a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC is the responsibility of the managing clinician. For purposes of commercial distribution, a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC as disclosed herein are typically supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. This invention also includes sets of a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC that exist at any time during their manufacture, distribution, or use. The sets of compositions comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC can comprise any combination of two or more agents described in this disclosure. Each set comprising a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC can be packaged together, or in separate containers in the same facility, or at different locations, under control of the same entity or different entities sharing a business relationship.

[00341] For general principles in medicinal formulation of cell compositions, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996. The composition is optionally packaged in a suitable container with written instructions for a desired purpose, such as the treatment of diabetes.

Formulations

[00342] In some embodiments, a composition of the present invention can comprise a plurality of islet- targeting molecules, e.g., islet-targeting peptides, complexed with a carrier particle: agent complex and/or EPC, where the agents present in the composition that are associated with the carrier particle can also be different, or where the cells associated with the islet-targeting molecule are different cell types, therefore allowing a delivery of a heterologous population of cells. For instance in some embodiments, an agent associated with the carrier particle can be a different type of effector agent, for example nucleic acid agent or a peptide agent. In some embodiments, an agent can be different variant of the same type of agent, for example if the agent is a nucleic acid, the composition can comprise both RNA and DNA agents. In further embodiments, the composition can comprise a plurality of agents that are variants of the same type of agent, for example variants or derivatives of siRNA. By way of a non-limiting example, the composition can comprise a plurality of RNAi agents that associate with the carrier peptide, where the RNAi agents are different, for example the RNAi agent silences different gene targets or targets different regions on the same gene.

[00343] Compositions as disclosed herein comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC can be administered by any convenient route, including parenteral, enteral, mucosal, topical, e.g., subcutaneous, intravenous, topical, intramuscular,

intraperitoneal, transdermal, rectal, vaginal, intranasal or intraocular. In one embodiment, the

compositions as disclosed herein are not topically administered. In one embodiment, the delivery is by oral administration of the composition formulation. In one embodiment, the delivery is by intranasal administration of the composition, especially for use in therapy of the brain and related organs (e.g., meninges and spinal cord). Along these lines, intraocular administration is also possible. In another embodiment, the delivery means is by intravenous (i.v.) administration of the composition, which is especially advantageous when a longer-lasting i.v. formulation is desired. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference.

[00344] Compositions comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with a carrier particle: agent complex and/or EPC can be administered in prophylactically or

therapeutically effective amounts. A composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide complexed with a carrier particle: agent complex and/or EPC as disclosed herein can be administered along with a pharmaceutically acceptable carrier. A prophylactically or therapeutically effective amount means that amount necessary, at least partly, to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular disease or disorder being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose can be administered for medical reasons, psychological reasons or for virtually any other reasons.

[00345] In the preparation of pharmaceutical formulations comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC as disclosed herein in the form of dosage units for oral administration the compound selected can be mixed with solid, powdered ingredients, such as lactose, saccharose, sorbitol, mannitol, starch, arnylopectin, cellulose derivatives, gelatin, or another suitable ingredient, as well as with disintegrating agents and lubricating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes. The mixture is then processed into granules or pressed into tablets.

[00346] Soft gelatin capsules can be prepared with capsules containing a mixture of the active compound or compounds of the invention in vegetable oil, fat, or other suitable vehicle for soft gelatin capsules. Hard gelatin capsules can contain granules of the active compound. Hard gelatin capsules can also contain the targeted delivery composition including the targeting moiety and the carrier particle as well as the therapeutic agent in combination with solid powdered ingredients such as lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, arnylopectin, cellulose derivatives or gelatin.

[00347] Dosage units for rectal or vaginal administration can be prepared (i) in the form of

suppositories which contain the active substance mixed with a neutral fat base; (ii) in the form of a gelatin rectal capsule which contains the active substance in a mixture with a vegetable oil, paraffin oil or other suitable vehicle for gelatin rectal capsules; (iii) in the form of a ready-made micro enema; or (iv) in the form of a dry micro enema formulation to be reconstituted in a suitable solvent just prior to

administration.

[00348] Liquid preparations for oral administration can be prepared in the form of syrups or suspensions, e.g., solutions or suspensions containing from 0.2% to 20% by weight of the active ingredient and the remainder consisting of sugar or sugar alcohols and a mixture of ethanol, water, glycerol, propylene glycol and polyethylene glycol. If desired, such liquid preparations can contain coloring agents, flavoring agents, saccharin and carboxymethyl cellulose or other thickening agents. Liquid preparations for oral administration can also be prepared in the form of a dry powder to be reconstituted with a suitable solvent prior to use.

[00349] Solutions for parenteral administration can be prepared as a solution of a compound of the invention in a pharmaceutically acceptable solvent, preferably in a concentration from 0.1% to 10% by weight. These solutions can also contain stabilizing ingredients and/or buffering ingredients and are dispensed into unit doses in the form of ampoules or vials. Solutions for parenteral administration can also be prepared as a dry preparation to be reconstituted with a suitable solvent extemporaneously before use.

[00350] The methods to deliver a composition comprising an islet-targeting molecule, e.g., an islet- targeting peptide, complexed with a carrier particle: agent complex and/or EPC as disclosed herein can also be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.

[00351] Furthermore, local administration of a composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide, complexed with a carrier particle: agent complex and/or EPC as disclosed herein to treat diabetes using surgically implanted, biodegradable implants is known. For example, a polyanhydride polymer, Gliadel® (Stolle R & D, Inc., Cincinnati, OH) a copolymer of poly- carboxyphenoxypropane and sebacic acid in a ratio of 20:80 has been used to make implants, intracranially implanted to treat malignant gliomas. Polymer and BCNU can be co-dissolved in methylene chloride and spray-dried into microspheres. The microspheres can then be pressed into discs 1.4 cm in diameter and 1.0 mm thick by compression molding, packaged in aluminum foil pouches under nitrogen atmosphere and sterilized by 2.2 megaRads of gamma irradiation. The polymer permits release of carmustine over a 2-3 week period, although it can take more than a year for the polymer to be largely degraded. Brem, H., et al., Placebo-Controlled Trial of Safety and Efficacy of Intraoperative Controlled Delivery by Biodegradable Polymers of Chemotherapy for Recurrent Gliomas, Lancet

345;10081012: 1995.

[00352] In addition to polymeric implants, osmotic pumps can also be utilized for delivery of a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC of the present invention by continuous infusion. An osmotic minipump contains a high- osmolality chamber that surrounds a flexible, yet impermeable, reservoir filled with the targeted delivery composition-containing vehicle. Subsequent to the subcutaneous implantation of this minipump, extracellular fluid enters through an outer semi-permeable membrane into the high-osmolality chamber, thereby compressing the reservoir to release leukocyte delivery agent at a controlled, pre-determined rate. The leukocyte delivery agent composition, released from the pump, is directed via a catheter to a stereotaxically placed cannula for infusion into the cerebroventricular space, as described herein.

[00353] For the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models so as to achieve a circulating concentration range that includes the IC₅₀ as determined in cell culture. Such information can then be used to more accurately determine useful doses in humans.

[00354] Toxicity and therapeutic effective amount of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC₅₀ and the LD₅₀. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

[00355] Dosage amount and interval can be adjusted individually to provide plasma levels of agent or EPC delivered by the islet-targeting molecule to trigger a response. These plasma levels are referred to as minimal effective concentrations (MECs). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. [00356] Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.

[00357] In cases of local administration or selective uptake, the effective local concentration of the leukocyte delivery agent cannot be related to plasma concentration. In such cases, other procedures known in the art can be employed to determine the correct dosage amount and interval.

[00358] The amount of a pharmaceutical composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with a carrier particle: agent complex and/or EPC of the present invention administered will, of course be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[00359] A pharmaceutical composition comprising an islet-targeting molecule, e.g., an islet-targeting peptide, complexed with a carrier particle: agent complex and/or EPC agent can, if desired, be presented in a suitable container {e.g., a pack or dispenser device), such as an FDA approved kit, which can contain one or more unit dosage forms containing the carrier portion containing the targeting and immune response triggering portions.

[00360] The method can further comprise administering to a subject a second therapy, wherein the second therapy is therapy for the treatment of diabetes related conditions. The second therapy can be administered to the subject before, during, after or a combination thereof relative to the administration of a composition comprising an islet-targeting molecule complexed with a carrier particle: agent complex and/or EPC as disclosed herein.

[00361] Pharmaceutical compositions comprising an islet-targeting molecule, e.g., an islet-targeting peptide complexed with a carrier particle: agent complex and/or EPC as disclosed herein can be administered by any convenient route, including parenteral, enteral, mucosal, topical, e.g., subcutaneous, intravenous, topical, intramuscular, intraperitoneal, transdermal, rectal, vaginal, intranasal or intraocular. In one embodiment, the lipid particles of the present invention are not topically administered. In one embodiment, the delivery is by oral administration of the particle formulation. In one embodiment, the delivery is by intranasal administration of the particle formulation, especially for use in therapy of the brain and related organs {e.g., meninges and spinal cord) that seeks to bypass the blood-brain barrier (BBB). Along these lines, intraocular administration is also possible. In another embodiment, the delivery means is by intravenous (i.v.) administration of the particle formulation, which is especially advantageous when a longer-lasting i.v. formulation is desired. Suitable formulations can be found in Remington's Pharmaceutical Sciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and 1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia (1985), each of which is incorporated herein by reference.

Kits [00362] Encompassed in the invention is an islet-selective delivery kit, comprising ready-to-use lyophilized composition comprising islet-targeting molecule complexed with a carrier particle: agent complex as disclosed herein, where in the islet-targeting molecule is associated with carrier particles and is ready for drug or agent encapsulation. The lyophilized islet-targeting molecule: carrier particle complex of the kit can be rehydrated directly in the drug or agent solution for drug or agent encapsulation respectively.

[00363] Also encompassed in the invention islet-selective delivery kit, comprising ready-to-use lyophilized composition comprising islet-targeting molecule complexed with an affinity binding moiety. For example, islet-targeting molecule associated with the affinity binding moiety is ready to be associated with a delivery stem cell or progenitor cell, e.g., EPC to be delivered to the pancreatic islet endothelial cells of the subject. The lyophilized islet-targeting molecule: affinity binding moiety complex of the kit can be added to EPC cells to attach the EPC cells to the islet-targeting molecule: affinity binding moiety complex. In some embodiments, the kit can comprise an affinity binding partner, and additional affinity binding moieties to attach to the EPCs, such that the islet-targeting molecule can be indirectly associated with the EPC via an affinity binding moiety: binding partner: affinity binding moiety-EPC linkage.

[00364] In some embodiments, the kits can optionally comprise instructions and reagents for proper reconstitution of the agent with the islet-targeting molecule: carrier particle complex, or the attachment of a population of EPC cells or other delivery cell of interest (e.g., pancreatic progenitor cells, stem cells other progenitor cells) to the islet-targeting: affinity binding moiety complex.

[00365] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[00366] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean ±1 %. The present invention is further explained in detail by the following examples, but the scope of the invention should not limit thereto. EXAMPLES

[00367] Throughout this application, various publications are referenced. The disclosures of all of the publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention. The Examples presented herein relate to methods, compositions and formulations of an islet-targeting molecule, e.g., an islet-targeting peptide to deliver nanoparticles (e.g., carrier particles) comprising agents and cells to pancreatic islet endothelial cells for the treatment of diabetes, e.g., Type 1 and Type 2 diabetes.

Materials and Methods

[00368] Polymer Nanoparticle Formulation. PLGA-PEG-COOH (75:25, PLGA M_w: 17000 kDa, PEG M_w: 3400 Da, Advanced Polymer Materials Inc., QC, Canada), was dissolved at a concentration of 1 mg/ml in DMSO with a trace amount of a fluorescent dye, Coumarin 6 (Sigma Aldrich, MO, USA). Nanoparticles were obtained by a simple solvent displacement method that involved dialyzing the polymer-dye solution against water at room temperature. The morphology and size distribution of the nanoparticles were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), as described below.

[00369] Nanoparticle-Peptide (NP-Pep) Conjugation. The cyclic islet-homing peptide CHVLWSTRC (Pep I) (SEQ ID NO: 2), discovered previously using phage display (Wang, X. et al, Genet Vaccines Ther (2008) 6: 7), and the scrambled sequence CVHWTLSRKC (Pep X) (SEQ ID NO: 3) were synthesized by Tufts University Peptide Core Facility, MA, USA. During peptide synthesis, a lysine residue (K) was inserted between the arginine and cysteine residues of Pep I to facilitate its covalent conjugation to PLGA- PEG-COOH using the carbodiimide chemistry, to render CHVLWSTRKC (SEQ ID NO: 1). Peptides were either directly conjugated to the polymer chain prior to nanoparticle formation or functionalized to the surface of the preformed nanoparticles using the conventional EDC/NHS chemistry. Briefly, the carboxyl end group of the PLGA-PEG-COOH was first activated with l-(3-dimethylamino-propyl)-3- ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) or N- hydroxysuccinimide (NHS) at a 1:5:10 molar ratio for 2 hours at room temperature either in phosphate buffer saline (PBS) or DMSO, depending on whether the modification was done to the nanoparticle surface or polymer chain. The pre-activated polymer/nanoparticles were then reacted with Pep I or Pep X in a 2:1 molar ratio for 4 hours at room temperature either in PBS or in PBS/DMSO, as per the reaction scheme. The reactions were then purified by either centrifugation/wash or simple dialysis. The polymer- peptide conjugation reaction was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy. ^!H NMR spectral measurements were performed using a Variance 600 MHz VNMRS spectrometer (Agilent, Palo Alto, CA, USA) in d-DMSO at 25 °C. The polymer-peptide conjugation efficiency was calculated by comparing the area under the tryptophan and lactide -CH peaks.

[00370] Genistein-loaded Nanoparticles (NP-Gen) and Drug Release Kinetics. Genistein (EMD Chemical, USA) was added to a 1 mg/ml PLGA-PEG-COOH solution (in DMSO) at 1%, 5% or 50% by weight of the co-polymer. Following 30 minutes of mixing, the genistein-polymer mix was dialyzed against water at room temperature to obtain genistein-loaded nanoparticles (NP-Gen) and remove excess genistein. The size distribution of NP-Gen was characterized by dynamic light scattering (DLS). To measure the release kinetics of incorporated genistein, 250 μg of NP-Gen was suspended in 1.5 ml PBS and incubated at 37°C for pre-determined time durations (n=4 per time point). At the end of each time point, NP-Gen nanoparticles were pelleted by spinning down the suspension at 10,000 rcf for 10 min and the pellets were dissolved in acetonitirile/methanol for UV-Vis spectral measurement of residual genistein using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, CA, USA). The O.D. measurements were converted into concentration using a standard curve generated from pure genistein of known concentrations (n=3 per concentration). To measure total drug loading, freshly prepared NP-Gen was immediately dissolved in acetonitirile/methanol for UV-Vis spectral measurement (n=3).

[00371] Transmission Electron Microscopy (TEM). A JEOL 1400 TEM microscope (JEOL, Peabody, MA, USA) was used to characterize the morphology of the nanoparticles. About 5 ul of nanoparticle solutions was added onto Formvar 400 mesh copper grids. After ~5 minutes, the excess solution was wicked by filter paper and the sample was washed with H₂0. Next, the sample was stained with 0.75% uranyl formate (Polysciences Inc, PA, USA) and air dried for 5 min prior to imaging.

[00372] Dynamic Light Scattering (DLS). A zeta particle size analyzer (Malvern instruments, UK) operating with a HeNe laser, and a 173° back scattering detector was used to determine the size distribution of the nanoparticles. Samples were prepared at 100 μg/ml in water and filtered through a 0.8 μπι or 5 μπι filter prior to the dynamic light scattering measurement (n=3 per condition). Malvern instrument software or Microsoft Excel was used to analyze the acquired data.

[00373] Nanoparticle (NP) binding and uptake. Mouse pancreatic islet CE cells were a gift from Judah Folkman while the mouse skin CE cells were isolated from the dermis of TRAMP mice. CE cells were cultured on gelatin-coated tissue culture dishes, grown in culture medium composed of low glucose DMEM, 10% fetal bovine serum, 10% Nu Serum IV, basic fibroblast growth factor (6 ng/ml), heparin salt (0.1 mg/ml), 1% insulin-transferrin-selenium and antibiotic/mycotic mixture, and were used between passages 12-19. For nanoparticle binding and uptake studies, CE cells were grown on gelatin-coated glass coverslips and NP-Pep I or NP-Pep X nanoparticles suspended in cell culture medium at 10 μg/ml were added to cells for 30 minutes at 37°C (n=3). Next, the unbound nanoparticles were removed by PBS rinsing and the cells were fixed with 4% paraformaldehyde (PFA). Fluorescent images of the samples were acquired with a Nikon Eclipse TE 2000-E microscope (Nikon, Japan) fitted with a CoolSnap HQ digital camera (Photometries). The fluorescence intensity of cell-bound nanoparticles (n>30 per condition) was measured using IP Lab imaging software (Becton Dickinson, NJ, USA). To determine whether cell- bound nanoparticles were internalized by an endocytic mechanism, CE cells treated with nanoparticles were stained with lysotracker^™ red (Invitrogen, CA, USA) to label intracellular acidic organelles

(lysosomes and endosomes). Quantitative analysis of the degree of colocalization between green (nanoparticle) and red (organelles) fluorescent images was performed using Volocity^* imaging software (PerkinElmer, MA, USA).

[00374] For flow studies, microfluidic channels were prepared from polydimethylsiloxane (PDMS) using conventional soft lithography (Xia, Y. and Whitesides, G. M. Annual review of materials science (1998) 28(1): 153-184). A master mold was designed using a CAD program and prepared by utilizing 80 micron thick features formed using a cutter plotter (CE5000, Graphtec, CA). The PDMS channels were sealed with a glass microslide (170 μπι thick) using plasma bonding. Microfluidic devices were then sterilized using oxygen plasma and coated with fibronectin (50ug/ml for 30 min) to support cell adhesion. Each PDMS device comprises two identical channels (80 μπι high x 500 μπι wide x 30 mm long), as shown in Figure 3. Islet CE cells (> 2xl0⁶ cells/ml) were introduced to one microchannel, followed by introduction of skin CEs, at a similar density, to the second channel in the same device. The devices were then placed in a tissue culture incubator (37°C, 5% C0₂) and the cells were allowed to adhere under static conditions for 2 hr. Following the static incubation, culture medium was infused at a flow rate of 50 uL/hr using a conventional syringe pump (Braintree Scientific, Braintree, MA). The cells were cultured in the devices for 1- 2 days until cell monolayer was formed. Nanoparticle suspension (10 μg/ml in culture medium) was then infused for 30 min through the channels at a flow rate of 800ul/hr (wall shear stress of ~2 dyne/cm²). Unbound nanoparticles were flushed away by infusing PBS through the channels at the same flow rate for more than 10 min. The samples were then fixed by infusing PFA for 5 min. Phase contrast and fluorescence microscopic images of cells and bound nanoparticles were acquired using a Nikon Eclipse TE 2000-E microscope (Nikon, Japan) fitted with a CoolSnap HQ digital camera

(Photometries).

[00375] Leukocyte Adhesion Assay. After islet CE cells were grown to confluence on gelatin-coated 24- well plates, the culture medium was replaced with low-serum assay medium composed of low glucose DMEM, 5% fetal bovine serum and antibiotic/mycotic mixture. Free genistein or genistein-loaded nanoparticles (NP-Gen) were then added to the confluent cells in assay medium to obtain a final free genistein concentration of 10, 50 or 100 μΜ or NP-Gen concentration of 10 μg/ml. Free genistein was added to cells for 18 hours prior to leukocyte adhesion while the NP-Gen solution was removed after 30 minutes, followed by rinsing with PBS and addition of fresh assay medium. After 18 hours of treatment with free genistein or bound NP-Gen, islet CE cells were stimulated with tumor necrosis factor-a (TNF; 10 ng/ml) for 5 hours. Separately, leukocytes were isolated from freshly collected mouse blood using a red blood cell lysis buffer (BD Pharm Lyse^™, Becton Dickinson, NJ, USA), as per the manufacturer's instructions, and labeled with a green fluorescence live cell tracker dye (Green CMFDA, Invitrogen, CA, USA). After 5 hours of stimulation, TNF solution was removed from islet CE cell cultures and fluorescently-labeled leukocytes added at a density of 200,000 cells/well. After 30 minutes of leukocyte/islet CE cell interaction, the leukocyte suspension was removed and the islet CE cells were rinsed 3-4 times with PBS prior to PFA fixation. Fluorescent images of labeled leukocytes were acquired using a Nikon Eclipse TE200 microscope (Nikon, Japan) fitted with a Spot RT Monochrome camera (Diagnostic Instruments, MI, USA) and leukocyte adhesion was quantified using Image J software (NIH).

[00376] Cell Viability. Islet CE cells were grown to confluence in gelatin-coated 96-well plates, following which they were either left untreated or treated with blank or genistein-loaded (NP-Gen) nanoparticles (10 μg/ml) for 18 hours. CellTiter-Blue^® reagent was then added to each well and, following 4 hour incubation at 37°C, the fluorescence signal was measured using a fluorescence multiwell plate reader (Victor3^™, PerkinElmer, MA, USA). All fluorescent intensity measurements were then normalized with respect to the untreated islet CE cells.

[00377] Statistical Analysis. All data are obtained from multiple replicates, as indicated in the respective procedures, and expressed as mean ± SEM. Statistical significance was determined using analysis of variance (ANOVA; InStat^®, GraphPad Software Inc.). Results were considered significant if p<0.01.

EXAMPLE 1

[00378] The inventors herein have demonstrated a proof -of-concept for use of islet-targeting nanoparticles for insulitis treatment that preferentially bind to islet capillary endothelial (CE) cells and locally deliver an anti-inflammatory agent to inhibit leukocyte adhesion to these cells. The active islet- targeting ability of these polymeric nanoparticles is conferred by a unique islet-homing peptide that is conjugated to their surface. These nanomaterials also function as superior drug delivery vehicles, as indicated by a significant increase in the immunosuppressive effect on leukocyte adhesion to islet endothelial cells exhibited by an encapsulated anti-inflammatory drug. By abrogating the use of deleterious anti-leukocyte proliferative agents and leveraging the excellent drug delivery properties of nanomaterials, this new islet-targeted immunomodulatory approach may create new therapeutic opportunities for preventing or significantly delaying the onset of type 1 diabetes in high-risk individuals.

[00379] As building block for these nanomaterials, the inventors used an amphiphilic poly(D,L-lactide- co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) co-polymer that spontaneously self- assembles in aqueous solution to form nanoscale particles (Figure 1 A). Both PLGA and PEG are FDA- approved for use in a variety of clinical products (Lively, T. N. et al, J Allergy Clin Immunol (2008) 121(l):88-94; Huang, H. Y. et al, Gene Ther (2008)15(9):660-7); thus their block co-polymer is considered to be safe for use in humans. To render these nanoparticles suitable for active islet targeting, the inventors employed the conventional carbodiimide chemistry to covalently conjugate to PLGA-b- PEG-COOH a cyclic peptide sequence (CHVLWSTRC; Pep I) that was previously identified to home specifically to pancreatic islet microvessels (Wang, X. et al, Genet Vaccines Ther (2008) 6:7) (Figure 1A). This approach to pre-functionalize the constitutive co-polymer blocks prior to their self-assembly into nanoparticles simplifies their optimization and large-scale production (Feutren, G. et al. Lancet (1986) 2(8499): 119-24; Silverstein, J. et al, N Engl J Med (1988) 319(10):599-604). Successful polymer- peptide conjugation was confirmed by the appearance of peptide-derived tryptophan peak in the 'H-NMR spectrum (Figure IB). Further analyses of the areas under the tryptophan and lactide -CH peaks revealed that 3 out of 10 polymer chains become modified with the islet-homing peptide. The size distribution profile of these nanoparticles determined by dynamic light scattering revealed an average diameter (d_avg) of 190 nm, which was independently confirmed by imaging the nanoparticles with a transmission electron microscope (Figure 1C).

[00380] To demonstrate their ability to selectively bind to pancreatic islet vessels, the inventors added nanoparticle-peptide (NP-Pep I) conjugates labeled with the fluorescent tag to cultured mouse islet and skin CE cells. Coumarin incorporation did not alter nanoparticle size, as indicated by dynamic light scattering (Figure 14). Quantitative measurement of fluorescence intensity revealed 3-fold greater binding of NP-Pep I conjugates to islet CE cells compared to the skin cells (p<0.001 ; Figure 2A). The preferential binding of these nanomaterials to islet CE cells is attributed to the islet-targeting Pep I sequence since nanoparticles modified with a similar sized scrambled peptide (CVHWTLSRKC; Pep X) at a similar surface density (~ 3 out of 10 polymer chains modified with the peptide) did not exhibit islet CE cell specificity (Figures 2A and 15). Past work has implicated EphA4 receptors as mediators of this preferential NP-Pep I binding as the immunofluorescent staining pattern of EphA4 colocalizes with nanoparticle distribution in islets in vivo.

[00381] Quantitative fluorescence measurement of unbound nanoparticles revealed that approximately 32% of the added nanoparticles were taken up by islet CE cells. To determine whether and how these bound nanoparticles were taken up by islet CE cells, the inventors stained the NP-Pep I-bound islet CE cells with lysotracker™ or Mitotracker™ to label intracellular acidic lysosomes, endosomes, or mitochondria, respectively. Quantitative analysis of the green (NP-Pep I) and red (lysotracker^™or Mitotracker™ ) fluorescence images revealed -100% co-localization, whereas there was no colocalization with mitochondria (Figure 16). These data indicate that these nanomaterials are taken up via endocytosis and processed in intracellular endocytic compartments (Figure 2B). EXAMPLE 2

[00382] To mimic the binding of NP-Pep I conjugates to islet vessels in vivo where islet CE cells will encounter these nanomaterials under flow, the inventors designed a simple microfluidic device where islet and skin CE cells were grown in parallel channels and fluorescently-labeled NP-Pep I conjugates were flowed over them at a physiological flow rate (2 dyne/cm²; Figure 3). Quantitative analyses of fluorescence intensity revealed that, similar to static culture conditions, NP-Pep I conjugates exhibit a 3- fold greater binding affinity to islet CE cells compared to skin CE cells (p<0.001; Figure 3), thus, confirming the robust islet-homing capability of these engineered nanoparticles.

[00383] During insulitis, blood leukocytes infiltrating the pancreatic islets produce various cytokines, such as tumor necrosis factor a (TNF) and interleukin-ΐβ, that render islet CE cells more adhesive, thereby creating a self-amplifying loop of autoimmune β cell destruction (Eizirik, D. L. et al, Nat Rev Endocrinol (2009) 5(4):219-26; Xia, Y. and Whitesides, G. M. Annual review of materials science (1998) 28(1): 153-184). To demonstrate the promise of islet-targeting nanomaterials as a therapeutic intervention for insulitis, the inventors tested their ability to inhibit leukocyte adhesion to TNF-stimulated cultured islet CE cells. This in vitro leukocyte/endothelial cell adhesion assay has been widely used as an inflammation model for both basic mechanistic and drug discovery studies (Mitragotri, S. and Lahann, J. Nat Mater (2009) 8(l):15-23; Kumar, P. et al, Expert Rev Mol Med (2009)1 l:el9). In the present study, the inventors used genistein, a protein tyrosine kinase inhibitor that is known to impair leukocyte binding to TNF-stimulated endothelial cells, as a model drug for incorporation within the islet-targeting

nanoparticles.

[00384] The inventors first confirmed the anti-inflammatory effect of genistein by showing that islet CE cells treated with soluble genistein exhibit dose-dependent inhibition of leukocyte adhesion (Figure 4A). Next, the inventors incorporated genistein into the nanoparticles at 5% by weight of the added co-polymer and evaluated its release kinetics over a 48-hour period. UV-Vis spectral measurement of the released genistein revealed a typical temporal profile where an initial burst release lasting approximately 8 hours was followed by a gradual release over 48 hours, with approximately 75% of the incorporated genistein being released over two days (Figure 4B). The drug release kinetics were similar to that reported previously for hydrophobic drugs incorporated within PLGA-PEG-COOH co-block polymers of similar molecular weight (Silverstein, J. et al., N Engl J Med (1988) 319(10): 599-604). From these drug release measurements, the inventors also determined that the incorporation efficiency of genistein was -40% (w/w). Thus, these islet-targeting nanomaterials can be efficiently used as controlled drug delivery vehicles.

[00385] To demonstrate the therapeutic efficacy of the genistein-loaded islet-targeting nanoparticles (NP-Gen) for insulitis treatment, the inventors treated islet CE cells with NP-Gen or blank nanoparticles (10 μg/ml) for approximately 18 hours prior to TNF stimulation and quantified the adhesion of fluorescently-labeled leukocytes. Less than half the number of leukocytes (p<0.001) adhered to islet CE cells treated with NP-Gen compared to those treated with blank nanoparticles or untreated cells (Figure 4C) and there was no detectable change in cell viability (Figure 6C). Further, islet CE cells treated with blank or NP-Gen nanoparticles exhibited no apparent loss in viability (Figure 6), indicating that the decrease in leukocyte/islet CE cell adhesion seen with NP-Gen resulted from the immunosuppressive effect of genistein. In addition, the islet-targeting nanoparticles did not exert any detrimental effect on insulin-producing islet β cells (Figure 6C in Supporting Information), thus further indicating the therapeutic potential of these islet-targeting nanoparticles.

[00386] Surprisingly, the finding that only 32% of the nanoparticles are taken up by islet CE cells indicates that the amount of genistein required to produce 50% inhibition of leukocyte/islet CE cell adhesion was found to be approximately 200-fold lower when incorporated within nanoparticles (0.04 μg genistein or 0.064 μg/ml genistein) than when added in free solution (2.7 μg genistein or 13.5 μg/ml genistein). This profound increase in the therapeutic efficacy of genistein when used as NP-Gen formulation is apparently due to the rapid preferential uptake of NP-Gen by islet CE cells, which likely creates a higher intracellular genistein concentration than that caused by free diffusion of soluble genistein. This superior therapeutic efficacy of the islet-targeting nanomaterials can be leveraged to achieve site-specific delivery and potent local effect of powerful anti-inflammatory drugs (e.g., cyclosporine A and prednisone) that are currently limited in their clinical use because they cause severe side effects upon systemic administration.

[00387] To determine whether greater immunosuppressive effect could be achieved by raising the dose of islet targeting NP-Gen nanoparticles administered, the inventors treated islet CE cells with increasing concentrations of NP-Gen and evaluated its effect on inhibition of leukocyte/islet CE cell adhesion.

Surprisingly, the inventors discovered that higher NP-Gen concentrations were less effective in inhibiting leukocyte adhesion to islet CE cells (Figure 5 A). This discovery was counterintuitive as the levels of intracellular genistein, and thus its inhibitory effect, were expected to increase with increasing concentrations of NP-Gen. The inventors assessed if the polymeric nanoparticles were themselves pro- inflammatory at higher concentrations, thus neutralizing the anti-inflammatory property of genistein.

Indeed, TNF-stimulated islet CE cells that were treated with blank nanoparticles at higher concentrations (50 μg/ml) exhibited greater leukocyte binding than those treated with a lower dose (10 μg/ml) of nanoparticles or left untreated (Figure 5A). Thus, nanoparticle concentration appears to be an important determinant of vascular inflammation, thereby underscoring the need to optimize nanoparticle dosage for immunomodulatory therapy.

[00388] The inventors next assessed if the immunosuppressive effect of NP-Gen could be regulated by varying genistein loading in the nanoparticles. When TNF-stimulated islet CE cells were treated with NP- Gen containing 1%, 5% and 50% (w/w) genistein, the inventors determined that greater inhibition of leukocyte/islet CE cell adhesion with increasing genistein loading (Figure 5B). However, in addition to enhancing the anti-inflammatory effect of NP-Gen, the increased drug loading also produced an overall increase in NP-Gen size. Specifically, the relatively homogeneous (d_avg 190 nm) nanoparticle population seen at low (0% and 1%) genistein loading was gradually replaced by a biphasic distribution at higher genistein concentrations (Figure 5C). For instance, at 5% genistein loading, two distinct NP-Gen populations emerged - a smaller NP-Gen population showing a characteristic peak at 190 nm and a second, larger NP-Gen population exhibiting a peak size of 458 nm. Increasing genistein loading to 50% (w/w) caused a further increase in overall NP-Gen size, with the largest fraction averaging at 615 nm and a secondary minor peak at 2300 nm.

[00389] This drug dose-dependent control of nanoparticle size has important implications for their effectiveness as vascular-targeting agents as it determines both their rate of clearance from blood and vascular retention/extravasation. Specifically, past studies have shown that particles larger than 500 nm are likely to get phagocytosed by circulating macrophages (Mitragotri, S. and Lahann, J. Nat Mater (2009) 8(1): 15-23) whereas particles smaller than 200 nm typically extravasate from hyperpermeable vessels that are characteristic of ischemic tissues and tumors (Peer, D. et al., Nat Nanotechnol (2007) 2(12):751-60). Since inflammation is also marked by an increase in vascular permeability (Kumar, P. et al., Expert Rev Mol Med (2009) 11 :el9), nanoparticles that range between 200-500 nm will likely be most suitable for active vascular targeting by virtue of their maximal vessel retention and minimal phagocytic clearance. Based on these design criteria, the 5% NP-Gen nanoparticles appear to be the most optimal formulation for active islet vessel targeting and immunotherapeutic delivery as they combine potent anti-inflammatory effects with suitable dimensions.

EXAMPLE 3

[00390] Type 2 diabetes, in contrast to Type 1 diabetes, is characterized by a progressive decline in islet mass and function resulting from insulin de-sensitization and, consequently, chronic hyperglycemia in the body. The inventors herein have demonstrated that endothelial progenitor cells (EPCs) can significantly enhance islet function in vitro (Fig. 7). To selectively deliver EPCs to pancreatic islets for in situ islet normalization in type 2 diabetics, the inventors have tethered the islet-targeting nanoparticles onto the surface of EPCs using biotin-streptavidin linkage (Figures 8-10). These EPC-nanoparticle conjugates exhibit significantly stronger binding to islet capillary endothelial cells compared to unmodified EPCs in vitro (Fig. 11). The inventors have demonstrated optimal density of islet-targeting nanoparticles on EPC surface to achieve maximal binding to islet endothelium in vitro. The inventors are performing in vivo studies to demonstrate that these nanoengineered EPCs can home selectively to pancreatic islets when injected systemically. Accordingly, herein the inventors have demonstrated that the nanomaterials-based approach for targeted delivery of drugs or EPCs to pancreatic islets has direct therapeutic implications for both type 1 and type 2 diabetes. Further, this nanoengineering approach can be adapted for the delivery of such therapeutic agents to other tissues and organs in the body.

EXAMPLE 4

[00391] In summary, the inventors have demonstrated a proof-of-concept for islet-targeting

nanoparticles useful as a new therapeutic intervention for insulitis in individuals that are at high risk of developing type 1 diabetes. In addition to exhibiting selective islet-targeting capability, these

nanomaterials offer a tremendous advantage over systemic drug delivery by virtue of their ability to elicit a similar immunosuppressive response with a 200-fold lower drug concentration. The inventors also demonstrate that maximizing the immunotherapeutic potential of these nanomaterials requires precise control of both their physicochemical properties and administered drug dose. [00392] To realize the full impact of the field of nanomedicine, smart nanoscale materials can be developed that can selectively home and deliver therapeutic payloads to any tissue and organ of interest. The success of such tissue -targeted nanotherapeutics will depend on: 1) identification of unique tissue- targeting moieties (peptides, aptamers, antibodies) using high-throughput techniques such as phage display, 2) design of long-circulating nanoparticles of appropriate size using biodegradable polymers, and 3) identification of pathological conditions that will particularly benefit from this active targeting strategy. The incidence of diabetes has reached epidemic proportions worldwide. Thus, the inventors have demonstrated herein that nanomaterials that can selectively home to pancreatic islets and are of particular interest for diabetes therapy, as demonstrated herein, as well as for early diagnostic screening of pre- diabetic subjects through incorporation of imaging agents that can permit real-time monitoring of pancreatic islets.

[00393] Stem and progenitor cells are also being increasingly explored as therapeutics for pancreatic islet regeneration. Thus, by conjugating the islet-targeting nanomaterials to the surface of these regenerative cells, the inventors demonstrate that it is possible to selectively guide the attached cells to diseased islets following systemic delivery, thereby substantially improving their islet engraftment and therapeutic efficacy in the future.

Claims

1. A composition for targeted delivery of an agent to an islet endothelial cell, the composition

comprising at least one islet-targeting molecule, at least one carrier particle, and at least one agent associated with the carrier particle, wherein the islet-targeting molecule is associated with the carrier particle.

2. The composition of claim 1 , wherein the islet-targeting molecule is selected from a peptide, an

antibody, an antibody fragment, an aptamer or other moiety which binds to pancreatic endothelial cells with high specificity.

3. The composition of claim 1 or 2, wherein the islet-targeting molecule is a peptide comprising SEQ ID NO: 1 or a fragment or variant thereof.

4. The composition of any one of claims 1-3, wherein the antibody or aptamers binds a cell surface receptor expressed on the islet endothelial cell.

5. The composition of any one of claims 1-4, wherein the cell surface receptor is selected from the group consisting of: ephrin A4 (Eph A4), Willebrand factor, CD86 (B7-2), ICOS ligand (ICOS-L), CD105, CD31, CD 146, endothelial cell leucocyte adhesion molecule- 1, acetylated low density lipoprotein, endostatin and pigment epithelial-derived factor.

6. The composition of any one of claims 1-5, wherein the carrier particle is a nanoparticle.

7. The composition of any one of claims 1-6, wherein the carrier particle is selected from the group consisting of: a liposome, a dendrimers, nanocrystals, quantum dots, nanoshell and nanorods,

8. The composition of any one of claims 1-7, wherein the islet-targeting molecule is covalently attached to a block co-polymer, wherein a plurality of co-polymer forms the carrier particle.

9. The composition of any one of claims 1-8, wherein the block co-polymer is [PLGA-b-PEG-COOH]n, or a biodegradable or non-biodegradable polymer.

10. The composition of any one of claims 1-9, wherein the agent associated with the carrier particle is encapsulated in the carrier particle.

11. The composition of any one of claims 1-10, wherein the agent is an anti-inflammatory agent.

12. The composition of any one of claims 1-11, wherein the anti-inflammatory agent is selected from the group consisting of Genistein, cyclosporine A, prednisone, mesalamine (5-aminosalicylic acid), simvastatin (inhibitor of HMG-CoA reductase), Herbimycin A, docosahexaenoic acid (DHA).

13. The composition of any one of claims 1-12, for use in a method for the treatment of an inflammatory disorder.

14. The composition of any one of claims 1-13 for use in a method for the treatment of Type 1 diabetes in a subject.

15. A method of treating Type 1 diabetes in a subject, the method comprising administering to the subject the composition of claim 1.

16. A method to promote β-islet cell survival in a subject, the method comprising administering to the subject the composition of claim 1.

17. The method of claims 15 or 16, wherein the agent is an anti-inflammatory agent.

18. The method of any one of claims 15-17, wherein the anti-inflammatory agent is selected from the group consisting of Genistein, cyclosporine A, prednisone mesalamine (5-aminosalicylic acid), simvastatin (inhibitor of HMG-CoA reductase), Herbimycin A, docosahexaenoic acid (DHA).

19. The method of any one of claims 15-18, wherein the agent which promotes β-islet cell survival is an anti-apoptotic agent.

20. The method of any one of claims 15-19, wherein the agent which promotes β-islet cell survival is a metabolic agent that enhances insulin secretion by islet β cells.

21. The method of any one of claims 15-20, wherein the metabolic agent which enhances insulin

secretion by islet β cells is selected from the group consisting of insulinotropic polypeptide (GIP- gastric inhibitory peptide), GLP-1 (glucagon-like peptide-1), exendin-4 (a GLP-1 analog) or homologues thereof.

22. The method of any one of claims 15-21, wherein the subject has Type 1 diabetes.

23. The method of any of claims 15 to 22, wherein the subject has juvenile diabetes.

24. The method of any of claims 15 to 23, wherein the subject is a human subject.

25. The method of any of claims 15 to 24, wherein the subject is an animal subject.

26. A method for preparing a composition of claim 1 for targeted delivery of an agent to an islet

endothelial cell, comprising associating an islet-targeting molecule with a block co-polymer, wherein the block-copolymer forms a carrier particle in aqueous solutions, and wherein the aqueous solution comprises an agent for delivery to an islet endothelial cell.

27. The composition of claim 26, wherein the islet-targeting molecule is selected from a peptide, an antibody, an antibody fragment, an aptamer or other moiety which binds to pancreatic endothelial cells with high specificity.

28. The composition of claim 26 or 27, wherein the islet-targeting molecule is a peptide comprising SEQ ID NO: 1 or a fragment or variant thereof.

29. The composition of claim 26, 27, or 28, wherein the antibody, antibody fragment or aptamer binds a cell surface receptor expressed on the islet endothelial cell.

30. The composition of any one of claims 26-29, wherein the cell surface receptor is selected from the group consisting of: ephrin A4 (Eph A4), Willebrand factor, CD86 (B7-2), ICOS ligand (ICOS-L), CD 105, CD31, CD 146, endothelial cell leucocyte adhesion molecule- 1, acetylated low density lipoprotein, endostatin and pigment epithelial-derived factor.

31. The method of any one of claims 26-30, wherein the block polymer is [PLGA-b-PEG-COOH]n or a biodegradable or non-biodegradable polymer.

32. The method of any one of claims 26-31 , wherein the agent is an anti-inflammatory agent.

33. The method of any one of claims 26-32, wherein the anti-inflammatory agent is selected from the group consisting of Genistein, cyclosporine A, prednisone, mesalamine (5-aminosalicylic acid), simvastatin (inhibitor of HMG-CoA reductase), Herbimycin A, docosahexaenoic acid (DHA;) or analogues or derivatives thereof.

34. A composition for targeted delivery of an endothelial progenitor cell (EPC) to an islet endothelial cell, the composition comprising at least one islet-targeting molecule, and at least one affinity binding molecule to attach the islet targeting peptide to the surface of the endothelial progenitor cell (EPC).

35. The composition of claim 34, wherein the islet-targeting molecule is selected from a peptide, an

36. The composition of claim 34 or 35, wherein the islet-targeting molecule is a peptide comprising SEQ ID NO: 1 or a fragment or variant thereof.

37. The composition of any one of claims 34-36, wherein the antibody, antibody fragment or aptamer binds a cell surface receptor expressed on the islet endothelial cell.

38. The composition of any one of claims 34-37, wherein the cell surface receptor is selected from the group consisting of: ephrin A4 (Eph A4), Willebrand factor, CD86 (B7-2), ICOS ligand (ICOS-L), CD 105, CD31, CD 146, endothelial cell leucocyte adhesion molecule- 1, acetylated low density lipoprotein, endostatin and pigment epithelial-derived factor.

39. The composition of any one of claims 34-38, wherein the islet targeting molecule is non-covalently attached to the surface of the endothelial progenitor cell (EPC).

40. The composition of any one of claims 34-39, further comprising a carrier particle, wherein the islet- targeting molecule is associated with the carrier particle, and wherein the carrier particle is associated with the affinity binding molecule.

41. The composition of any one of claims 34-40, wherein the affinity binding molecule is part of an

affinity binding complex comprising a protein affinity binding molecule and an affinity binding partner, wherein the protein affinity binding molecule associates with the islet-targeting molecule or the surface of an endothelial progenitor cell (EPC), and the affinity binding partner associates with at least two protein affinity binding molecules to indirectly link the islet-targeting molecule with the endothelial progenitor cell (EPC).

42. The composition of any one of claims 34-41, wherein the protein affinity binding molecule is biotin and the affinity binding partner is streptavidin.

43. The composition of any one of claims 34-42, wherein the carrier particle is a nanoparticle.

44. The composition of any one of claims 34-43, wherein the carrier particle is selected from the group consisting of: a liposome, a micelle, dendrimers, nanocrystals, quantum dots, nanoshell and nanorods.

45. The composition of any one of claims 34-44, wherein the islet-targeting molecule is covalently attached to a block co-polymer, wherein a plurality of co-polymer forms the carrier particle.

46. The composition of any one of claims 34-45, wherein the block polymer is [PLGA-b-PEG-COOH]n or a biodegradable or non-biodegradable polymer.

47. The composition of any one of claims 34-46, wherein an agent can be associated with the carrier particle.

48. The composition of any one of claims 34-47, wherein the agent is encapsulated in the carrier particle.

49. The composition of any one of claims 34-48, wherein the agent is an anti-inflammatory agent.

50. The composition of any one of claims 34-49, for treatment of Type 2 diabetes in a subject.

51. The composition of any one of claims 34-50, for a medicament to increase the activity or number of β-islet cells, or activity and number of β-islet cells in a subject.

52. A method of treating Type 2 diabetes in a subject, the method comprising administering to the subject a composition of claim 34.

53. A method to promote β-islet cell survival in a subject, the method comprising administering to the subject a composition of claim 34.

54. A method for preparing a composition of claim 34 for targeted delivery of an endothelial progenitor cell (EPC) to an islet endothelial cell, comprising:

a. attaching a protein affinity binding molecule to a population of endothelial progenitor cells (EPC),

b. contacting the population of endothelial progenitor cells (EPC) with the attached protein affinity binding molecule with an affinity binding partner, wherein the protein affinity binding molecule associates with the affinity binding partner,

c. providing at least one islet-targeting molecule with a direct or indirectly associated protein affinity binding molecule.

55. The method of claim 54, wherein the islet-targeting molecule is selected from a peptide, an antibody, an antibody fragment, an aptamer or other moiety which binds to pancreatic endothelial cells with high specificity.

56. The method of claim 54 or 55, wherein the islet-targeting molecule is a peptide comprising SEQ ID NO: 1 or a fragment or variant thereof.

57. The method of any one of claims 54-56, wherein the antibody, antibody fragment or aptamer binds a cell surface receptor expressed on the islet endothelial cell.

58. The method of any one of claims 54-57, wherein the cell surface receptor is selected from the group consisting of: ephrin A4 (Eph A4), Willebrand factor, CD86 (B7-2), ICOS ligand (ICOS-L), CD105, CD31, CD 146, endothelial cell leucocyte adhesion molecule- 1, acetylated low density lipoprotein, endostatin and pigment epithelial-derived factor.

59. The method of any one of claims 54-58, wherein the protein affinity binding molecule is biotin and the affinity binding partner is streptavidin.

60. The method of any one of claims 54-59, wherein the at least one islet-targeting molecule is indirectly associated with an affinity binding molecule via at least one carrier particle.

61. The method of any one of claims 54-60, wherein the islet-targeting molecule is covalently attached to a block co-polymer, wherein a plurality of co-polymers forms the carrier particle.

62. The method of any one of claims 54-61, wherein the block polymer is [PLGA-b-PEG-COOH]n or a biodegradable or non-biodegradable polymer.

63. The method of any one of claims 54-60, wherein an agent can be associated with the carrier particle.

64. The method of any one of claims 54-63, wherein the agent is encapsulated in the carrier particle.

65. The method of any one of claims 43-64, wherein the agent is an anti-inflammatory agent.

66. The method of any one of claims 54-65, wherein the anti-inflammatory agent is selected from the group consisting of Genistein, cyclosporine A, prednisone, mesalamine (5-aminosalicylic acid), simvastatin (inhibitor of HMG-CoA reductase), Herbimycin A, docosahexaenoic acid (DHA) or derivatives or analogues thereof.

67. The method of any one of claims 54-61, wherein the carrier particle attached to at least one islet- targeting molecule and is produced by the method of any of claims 26 to 33.

68. A islet targeting peptide comprising SEQ ID NO: 1.

69. A kit comprising an islet-targeting molecule and a carrier particle.

70. The kit of claim 69, wherein the islet-targeting molecule is a peptide comprising SEQ ID NO: 1 or a fragment or variant thereof.

71. The kit of claim 69 or 70, wherein the carrier particle is a nanoparticle.

72. The kit of any one of claims 69-71, wherein the carrier particle is selected from the group consisting of: a liposome, a micelle, dendrimers, nanocrystals, quantum dots, nanoshell and nanorods.

73. The kit of any one of claims 69-72, wherein the islet-targeting molecule is covalently attached to a block co-polymer, wherein a plurality of co-polymer forms the carrier particle.

74. The kit of any one of claims 69-73, wherein the block polymer is [PLGA-b-PEG-COOH]n or a

biodegradable or non-biodegradable polymer.

75. A kit comprising:

a. an islet targeting molecule;

b. a first affinity binding molecule which can associate with the islet targeting molecule, c. a second affinity binding molecule which can associate with an endothelial progenitor cell (EPC), d. an affinity binding partner which can associate with the first and the second affinity binding molecule.

76. The kit of claim 75, wherein the first and the second affinity binding molecule are the same.

77. The kit of claim 75 or 76, wherein the protein affinity binding molecule is biotin and the affinity binding partner is streptavidin.

78. The kit of claim 75, 76, or 77, wherein the islet-targeting molecule is a peptide comprising SEQ ID NO: 1 or a fragment or variant thereof.