METHODS OF DELIVERING HEPARIN BINDING EPIDERMAL GROWTH FACTOR USING STEM CELL GENERATED EXOSOMES

Abstract

The invention provides for methods of delivering heparin binding epidermal growth factor (HB-EGF) to sites of intestinal injury using stem cell derived exosomes. In particular, the invention provides for methods of delivering HB-EGF loaded stem cell-derived exosomes to sites of intestinal injury and methods of protecting a subject and method of treating intestinal injury, such as necrotizing enterocolitis (NEC).

Claims

1. A stem cell derived exosome comprising heparin binding epidermal growth factor (HB-EGF) product or a fragment thereof wherein the exosome is produced by a stem cell transfected to express HB-EGF product or a fragment thereof.

2. The stem cell derived exosome of claim 1 wherein the exosome is derived from a neural stem cell.

3. The method of claim 1, wherein the HB-EGF product comprises amino acids of 74-148 of SEQ ID NO: 2.

4. A composition comprising a stem cell derived exosome of claim 1 and a carrier.

5. A method of delivering a heparin binding epidermal growth factor (HB-EGF) product or a fragment thereof to a site of intestinal injury comprising administering stem cell-derived exosomes comprising HB-EGF to a subject suffering from an intestinal injury.

6. (canceled)

7. A method of treating an intestinal injury comprising administering a stem cell-derived exosome comprising administering a heparin binding epidermal growth factor (HB-EGF) product or a fragment thereof to a subject suffering from an intestinal injury in an amount effective to reduce the severity of the intestinal injury.

8-10. (canceled)

11. The method of claim 5 wherein the stem cell is a neural stem cell.

12. The method of claim 11 wherein the stem cells are transfected to express HB-EGF or a fragment thereof.

13. The method of claim 5, wherein the HB-EGF product comprises amino acids of 74-148 of SEQ ID NO: 2.

14. The method of claim 5, wherein the intestinal injury is caused by necrotizing enterocolitis, hemorrhagic shock, resuscitation, ischemia/reperfusion injury, intestinal inflammatory conditions or intestinal infections.

15. The method of claim 5, wherein the subject is suffering from Hirschprung's Disease, intestinal dysmotility disorders, intestinal pseudo-obstruction (Ogilvie's Syndrome), inflammatory bowel disease, irritable bowel syndrome, radiation enteritis or chronic constipation, Crohn's Disease, bowel cancer, or colorectal cancers.

16. The method of claim 5 wherein the subject is an infant.

17. The method of claim 5, wherein the exosomes are administered intravenously or intraperitoneally.

18. The method of claim 5, wherein the exosomes are administered immediately following the intestinal injury or within 1-5 hours following the intestinal injury.

19-29. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0138] FIG. 1 depicts characterization of NSC-derived exosomes (A) Enteric NSC were harvested from intestines of E11.5 mice and expanded as neurospheres. (B) Condition medium was collected and exosomes purified with the PureExo Exosome Isolation Kit and stained with the red fluorescent dye PKH 26. (C) Transmission EM. Scale bar=100 nm. (D) Dynamic light scattering and zeta potential analysis. (E) Western blot shown positive expression for exosome markers CD9 and Flotilin.

[0139] FIG. 2 depicts NSC-derived target enteric NSC and injured neurons. Panel (A) shows exosomes stained with PKH26 dye, and applied to a mixed culture of rat intestinal LMMP cells. Red fluorescent dye labeled exosomes were localized only in culture NSCs 24 hours after application. Tuj-1 (mature neuron marker) green; exosomes, red; GFAP (glia cells) green; SMA (smooth muscle cell) green; Nestin (NSCs) green; DAPI (nuclei), LMMP: Longitudinal Muscle-Myenteric Plexus. aSMA: Alpha-smooth muscle actin. Panel (B) shownsPKH26 labeled NSC derived exosomes applied to a mixed culture of rat intestinal LMMP cells 30 min prior to anoxia/Reoxygenation (24 h/24 h) injury. Red cytoplasmic staining in target cells confirms uptake of NSC-derived exosomes in injured intestinal neurons. Panel (C) shows immunohistology specific for cleaved caspase-3 shown decreased neuronal apoptosis in cells transferred with exosomes.

[0140] FIG. 3 depicts NSC-derived exosomes can be loaded with HB-EGF and target injured enteric neurons. (a-c) Exosomes were isolated from enteric NSC, stained with PKH26 dye, and applied to a mixed culture of rat intestinal cells 30 min prior to A/R (4 h/24 h) injury. Tuj-1 (mature neuron marker) green; exosomes, red; DAPI (nuclei), blue. Red cytoplasmic staining in target cells confirms uptake of NSC-derived exosomes in injured neurons. (d-g) Enteric NSC were transfected with pGFP-hHB-EGF, exosomes harvested 48 h later were stained with PKH26, and exosomes applied to a mixed culture of rat intestinal cells 30 min prior to A/R (4 h/24 h). Tuj-1 (mature neuron marker) grey; exosomes, red; HB-EGF, green; DAPI (nuclei), blue. Green staining of exosomes confirms HB-EGF loading; yellow merged cytoplasmic staining in target cells confirms that exosomal HB-EGF was transferred to injured neurons.

[0141] FIG. 4 depicts NSC-derived exosomes targeting NEC injured intestine and protect against experimental NEC. Panel (A) Pups were subjected to NEC for 24 hours, and then control vehicle or PKH 26-stained exosomes were administered IP. Panel (B) Intestines were removed 48 hours later and examined with Xenon fluorescent imaging. Labeled exosomes (red) localized to injured intestine. Panel (C) Histologic intestinal sections were graded for NEC using the scoring system described before. Panel (D) Representative H&E images demonstrating exosome treatment restores intestinal integrity

[0142] FIG. 5 depicts the effect of treatment with mesenchymal stem cells or exosomes has on the severity of NEC.

DETAILED DESCRIPTION

[0143] The following examples illustrate the invention wherein Example 1 describes isolation and characterization of NSC-derived exosomes. Example 2 describes that NSC-derived exosomes target enteric NSC and injured neurons. Example 3 describes a neonatal rat model of experimental necrotizing enterocolitis. Example 4 describes that NSC-derived exosomes target NEC injured intestine and protect against experimental NEC. Example 5 describes that NSC-derived exosomes can be loaded with HB-EGF and target injured enteric neurons in culture. Example 6 describes that SC-derived exosomes can protect the intestines from NEC. Example 7 describes direct transfer of exosomes from donor SC to recipient intestinal/ENS cells protects recipient cells from injury.

EXAMPLES

Example 1

Isolation and Characterization of NSC-Derived Exosomes

[0144] Enteric NSCs were isolated from mid gestational guts of embryonic mice. The harvested cells were immunoselected using magnetic beads conjugated with anti-P75 antibody and cultured in NSC medium. Neurosphere-like bodies were allowed to form and were then passed repeatedly. NSC cultured condition medium were collected and spin down to remove debris.

[0145] Initially, NSC-derived exosomes were purified from neurosphere conditioned medium (CM) using the PureExo Exosome Isolation kit (101Bio) according to the manufacturer's instructions. Exosomes were then stained with the red fluorescent cell link dye PKH 26 (Sigma) and characterized for size, size distribution, charge, and morphology using dynamic light scattering, zeta potential analysis, and transmission electron microscopy. Electron microscopy (EM) confirmed the presence of 50-150 nm diameter bi-membrane vesicles. Zeta potential analysis revealed a high negative charge of 24 mV. Dynamic light scattering revealed a mean diameter of 157n. Western blot analysis confirmed the presence of the well-characterized marker of murine exosomes tetraspanin CD9, as well as the membrane associated cytoskeletal lipid raft protein flotilin. These results are depicted in FIG. 1.

Example 2

NSC-Derived Exosomes Target Enteric NSC and Injured Neurons

[0146] The anoxia/reoxgenation cell injured model (Watkins et al., J Surg Res 2012; 177:359-64) was used to demonstrate that the NSC-derived exosomes target enteric NSC and localized to injured neurons. Briefly, intestinal LMMP strips were dissected out from P3-P5 rat pups and were treated with enzymatic digestion to obtain mixed cells which includes: myenteric neurons, glial cell, neural stem cells (NSC), smooth muscular cells. Mixed cells were cultured in DMEM/F12: NeuroBasal medium (v/v=1:1) supplemental with 1B27 and 10% FBS (Gibco) for 10 days. PKH26 labeled NSC derived exosomes were added to the culture medium of the mixed cells and the targeted cells with exosomes were visualized 24 hours later under fluorescent microcopy. In addition, NSC-derived exosomes were applied to the cultured cells 30 minutes prior to the exposure of the mixed cells to 24 h/24 h anoxia/reoxygenation injury (anoxia chamber was filled with 95% N.sub.2 and 5% CO.sub.2). Cultured cells were then fixed in 4% PFA and exosomes transfer was observed under fluorescent microcopy.

[0147] As shown in FIG. 2, NSC-derived exosomes target and protect enteric NSC and injured neurons. The red fluorescent dye labeled exosomes were localized only in culture NSCs 24 hours after application. The PKH26 labeled NSC-derived exosomes were applied to a mixed culture of rat intestinal LMMP cells 30 min prior to anoxia/Reoxygenation (24 h/24 h) injury, and red cytoplasmic staining in target cells confirms uptake of NSC-derived exosomes in injured intestinal neurons. Immunohistochemistry specific for cleaved caspase-3 demonstrated decreased neuronal apoptosis in cells treated with exosomes.

Example 3

Neonatal Rat Model of Experimental Necrotizing Enterocolitis

[0148] The studies described herein utilize a neonatal rat model of experimental NEC. These experimental protocols were performed according to the guidelines for the ethical treatment of experimental animals and approved by the Institutional Animal Care and Use Committee of Nationwide Children's Hospital (#04203AR). Necrotizing enterocolitis was induced using a modification of the neonatal rat model of NEC initially described by Barlow et al. (J Pediatr Surg 9:587-95, 1974). Pregnant time-dated Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, Ind.) were delivered by C-section under CO2 anesthesia on day 21.5 of gestation. Newborn rats were placed in a neonatal incubator for temperature control. Neonatal rats were fed via gavage with a formula containing 15 g Similac 60/40 (Ross Pediatrics, Columbus, Ohio) in 75 mL Esbilac (Pet-Ag, New Hampshire, Ill.), a diet that provided 836.8 kJ/kg per day. Feeds were started at 0.1 mL every 4 hours beginning 2 hours after birth and advanced as tolerated up to a maximum of 0.4 mL per feeding by the fourth day of life. Animals were also exposed to a single dose of intragastric lipopolysaccharide (LPS; 2 mg/kg) 8 hours after birth, and were stressed by exposure to hypoxia (100% nitrogen for 1 minute) followed by hypothermia (4 C. for 10 minutes) twice a day beginning immediately after birth and continuing until the end of the experiment. In all experiments, pups were euthanized by cervical dislocation upon the development of any clinical signs of NEC. All remaining animals were sacrificed at the end of experiment at 96 hours after birth.

[0149] The HB-EGF used in all experiments was GMP-grade human mature HB-EGF produced in P. pastoris yeast (KBI BioPharma, Inc., Durham, N.C.). EGF was produced in E. coli and purchased from Vybion, Inc. (Ithaca, N.Y.).

[0150] To assess the histologic injury score, immediately upon sacrifice, the gastrointestinal tract was carefully removed and visually evaluated for typical signs of NEC including areas of bowel necrosis, intestinal hemorrhage and perforation. Three pieces each of duodenum, jejunum, ileum, and colon from every animal were fixed in 10% formalin for 24 hours, paraffin-embedded, sectioned at 5 m thickness, and stained with hematoxylin and eosin for histological evaluation of the presence and/or degree of NEC using the NEC histologic injury scoring system described by Caplan et al. (Pediatr Pathol 14:1017-28, 1994). Histological changes in the intestines were graded as follows: grade 0, no damage; grade 1, epithelial cell lifting or separation; grade 2, sloughing of epithelial cells to the mid villus level; grade 3, necrosis of the entire villus; and grade 4, transmural necrosis. All tissues were graded blindly by two independent observers. Tissues with histological scores of 2 or higher were designated as positive for NEC. Fisher's exact test was used for comparing the incidence of NEC between groups with no adjustments made for multiple comparisons. P-values less then 0.05 were considered statistically significant. All statistical analyses were performed using SAS, (version 9.1,SAS Institute, Cary, N.C.).

Example 4

NSC-Derived Exosomes Target NEC Injured Intestine and Protect Against Experimental NEC

[0151] Exosomes were stained with the red fluorescent cell linker dye PKH26 to allow their distribution to be tracked. Neonatal mice exposed to experimental NEC for 24 hours as described in Example 3. Newborn rat pups were delivered by C-section and subjected to repeated exposure to hypoxia and hypothermia. These pups were administered subjected to hypertonic feeding for 24 hours and then control vehicle or PKH26-stained exosomes were administered intraperitoneally (IP). Intestines were removed 48 hours later or when NEC clinical signs were observed in these rat pups. The pups were randomly assigned to the following groups: (1) breast feeding only; (2) BF+exosomes IP; (4) NEC; (4) NEC+exosomes IP.

[0152] Histologic intestinal sections were graded for NEC using the scoring system. Representative H&E images demonstrating that exosome treatment restores intestinal integrity are shown in FIG. 3.

[0153] This experiment demonstrates that NSC-derived exosomes protect intestines from experimental NEC injury. NSC-derived exosomes is a novel non-cell based therapy that protects the ENS from injury during NEC.

Example 5

NSC-Derived Exosomes can be Loaded with HB-EGF and Target Injured Enteric Neurons in Culture

[0154] Exosomes were isolated from enteric NSC, stained with PKH26 dye, and applied to a mixed culture of rat intestinal cells 30 minutes prior to anoxia/reoxygenation (A/R) (4 h/24 h) injury. The cells were stained for the mature neuron marker Tuj-1 (green); stained for exosomes (red) and the nuclei were stained with DAPI (blue). Red cytoplasmic staining in target cells confirmed uptake of NSC-derived exosomes in injured neurons as shown in FIG. 4.

[0155] Enteric NSC were transfected with pGFP-hHB-EGF and exosomes were harvested 48 hours later. The exosomes were subsequently stained with PKH26 and applied to a mixed culture of rat intestinal cells 30 min prior to A/R (4 h/24 h). The cells were stained as described above for the mature neuron marker Tuj-1 (grey); exosomes (red) HB-EGF (green) and the nuclei were stained with DAPI (blue). As shown in FIG. 4, green staining of exosomes confirms HB-EGF loading; yellow merged cytoplasmic staining in target cells confirms that exosomal HB-EGF was transferred to injured neurons.

[0156] The addition of native or HB-EGF-enriched NSC-derived exosomes to mixed cultures of rat intestinal cells exposed to A/R resulted in exosomal localization and HB-EGF delivery to injured enteric neurons.

Example 6

SC-Derived Exosomes can Protect the Intestines from NEC

[0157] Protective SC were harvested from mice, and exosomes were purified from conditioned medium of the SC using the PureExo Isolation kit and characterized as described in Example 1. Exosomes were stained with the red fluorescent cell linker dye PKH26 to allow their distribution to be tracked. Neonatal mice were exposed to experimental NEC as described in Example 3. Pups were randomly assigned to the following groups: (1) breast feeding only; (2) NECno treatment, (3) NEC+PBS, (4) NEC+mesenchymal stem cells (MSC) and (5) NEC+exosomes IP.

[0158] Equal numbers of exosomes to be delivered are determined using the small particle detection capabilities of the BD Influx flow Cytometer, calibrated down to 50 nm. Exosomes were administered intraperitoneally 8 hours after the pups were exposed to experimental NEC. Pups were sacrificed upon development of signs of NEC (bloody stools, abdominal distention, respiratory difficulty, lethargy) or at the end of the experiment at 72 hours, and analyzed for specific endpoints related to the ENS, as well as generalized endpoints related to NEC.

[0159] As shown in FIG. 5, the pups that were breast fed were found to have not experimental NEC. The pups which were exposed to only the experimental NEC model has 46% incidence of NEC. and the pups exposed to the experimental NEC model and only received the vehicle (PBS) had 41% NEC. The administration of stem cells and exosomes from those stem cells results in a statistically significant decrease in the incidence of NEC. There was no statistical difference between the incidence of NEC between the control group, nor was there a statistical difference between the treatment groups (MCS or Exosomes).

Example 7

Direct Transfer of Exosomes from Donor SC to Recipient Intestinal/ENS Cells Protects Recipient Cells from Injury

[0160] A co-culture system comprising silicone micro-culture devices containing two wells (0.22 cm.sup.2/well) is used to culture cells that are physically separated from each other by a central silicone wall. One well receives 3,000 donor SC which are allowed to adhere for 12 hours, and then the other well is seeded with recipient cells (using cell lines if available or using primary cells purified directly from intestine with immunoaffinity techniques). The silicone wall is then removed to allow secreted exosomes to move from donor to recipient cells. First, donor SC are transfected with pGFP/CD9 to allow exosome-associated CD9 to be tracked. GFP fluorescence in recipient cells is demonstrate that GFP-tagged CD9 transcript and/or protein was transferred from donor to recipient cells. To confirm that exosomes mediate this process, donor cells are pre-treated with GW4869 (0-100 g/ml), which is a well-characterized exosome inhibitor that blocks neutral sphingomyelinase 2 (nSMAse2), which is required for the biosynthesis of ceramide on which exosome production is dependent or with siRNA to nSMAse2 to block the production of exosomes. To confirm that HB-EGF mRNA is shuttled from donor to recipient cells via exosomes, donor SC is transfected with the pEGFP/hHB-EGF vector (Origene) or mock-transfected. Recipient cells are analyzed by RT-PCR for hHB-EGF using human-specific primers. To demonstrate that the transfer is dependent on exosomes, some wells of donor SC will be pre-treated with GW4868 (0-100 M) or nSMase si-RNA, either of which will reduce the level of GFP/hHB-EGF transcripts in both exosomes and recipient cells.

[0161] To establish that donor SC deliver HB-EGF protein to recipient cells, recipient cells are analyzed by direct fluorescence for GFP, and by ICC/Western blot for GFP or HB-EGF. To determine whether HB-EGF is delivered by exosomes in an already-translated form as protein or is translated in the recipient cells after delivery of the transcript, the following steps are used. First, recipient cells are treated with cyclohexamide to block protein synthesis. Second, recipient cells are pre-treated with pRFP/si-HB-EGF to deplete their endogenous HB-EGF mRNA and protein levels and to block their ability to translate protein from the delivered GFP/hHB-EGF transcript. For cyclohexamide- or si-HB-EGF-treated cells, any HB-EGF or GFP signal in recipient cells are due to direct transfer of GFP/hHB-EGF protein rather than mRNA. Conversely, GFP signal that is lost are attributable to protein translation from delivered GFP/hHB-EGF mRNA. Third, the time course of appearance of GFP/hHB-EGF in recipient cells are more rapid if delivered as protein vs. transcript since the latter must first be translated. Thus, recipient cells are examined hourly over 12 hours for HB-EGF or GFP protein, or for GFP/hHB-EGF transcript. Early HB-EGF and GFP protein signals correspond to delivery of GFP/hHB-EGF protein and are followed several hours later by a wave of HB-EGF and GFP protein signals if the GFP/hHB-EGF transcript is subsequently translated. This analysis demonstrates that that: (i) treatment of the donor SC with nSMase2 si-RNA or GW4869 ablates GFP/hHB-EGF protein in recipient cells thereby proving involvement of exosomes; or (ii) neutralizing anti-HB-EGF IgG added to the medium of donor or recipient cells does not block the appearance of GFP/hHB-EGF protein in recipient cells, thereby ruling out uptake of soluble (secreted) GFP/hHB-EGF by recipient cells. These experiments show that HB-EGF can be transferred from SC to one or more of the recipient cell types as part of a normal exosomal shuttling mechanism between the cells.

[0162] To demonstrate that exosomal HB-EGF protects recipient cells from injury, studies are performed on control recipient cells and on recipient cells that were first exposed to anoxia (95% N2/5% CO2) for 4 hours followed by reoxygenation for 24 hours. Exosomes are enriched for HB-EGF by transfection of donor SC with pEGFP/hHB-EGF or pCMV/hHB-EGF and exosomes isolated using PureExo. Exosomes are added to recipient cells for 3, 6, 12, or 24 hours prior to exposure of the cells to anoxia, or at 0, 3, 6, 12, or 24 h after anoxia. To assess their response to injury, recipient cells will be examined for morphological changes, apoptosis as determined by TUNEL and anti-caspase-3 staining, and necrosis as determined by LDH cytotoxicity assay. To confirm that protection of recipient cells is due to transfer of HB-EGF mRNA or protein from donor SC, HB-EGF-depleted exosomes are collected from SC in which HB-EGF has been silenced by si-HB-EGF have a compromised effect on recipient cells. Alternatively, exosomes are purified from donor SC harvested from HB-EGF KO mice. All of these techniques, as well as HB-EGF KO mice, are routine in the art.

Example 8

Therapeutic Value of Exosomally-Delivered HB-EGF in Experimental NEC

[0163] To load exosomes with HB-EGF, protective SC are transfected with a human HB-EGF plasmid, as we have described (James et al., J Surg Res 2010; 163:86-95; Fagbemi et al. Early Hum Dev 2001; 65:1-9). Exosomes are purified from control and HB-EGF-overexpressing SC using the PureExo kit and stained with the red fluorescent cell linker dye PKH26. Increased levels of HB-EGF mRNA and protein in exosomes from HB-EGF-overexpressing SC are confirmed by RT-PCR and Western blot. Neonatal mice are exposed to experimental NEC as described in Example 3. Pups will be randomly assigned to the following groups: (1) BF; (2) NEC; (3) NEC+control exosomes; and (4) NEC+HB-EGF-loaded exosomes. Exosome quantification is performed using the BD Influx flow Cytometer. Exosomes are delivered by either IP or IV injection immediately after birth or after 24 hours of exposure to stress. Pups are sacrificed upon development of signs of NEC (bloody stools, abdominal distention, respiratory difficulty, lethargy) or at the end of the experiment at 72 hours, and analyzed for specific endpoints related to the ENS and generalized endpoints related to NEC.