WO2019139762A1

WO2019139762A1 - Exosome compositions and use thereof for joint disorders and diseases

Info

Publication number: WO2019139762A1
Application number: PCT/US2018/066654
Authority: WO
Inventors: John W. Ludlow; Benjamin Buehrer; Peter Pieraccini
Original assignee: Zen-Bio, Inc.
Priority date: 2018-01-09
Filing date: 2018-12-20
Publication date: 2019-07-18

Abstract

Compositions containing isolated exosomes derived from stem cells are provided for uses including treatment of joint disorders caused by infection, immunological disturbances, trauma and degenerative joint diseases such as, for example, rheumatoid arthritis, osteoarthritis, overuse and traumatic joint injury. The isolated exosomes contain increased levels of stress-response molecules, including heat shock molecules. Methods are provided that include administering, such as by injection, an effective amount of a composition containing isolated stem cell exosomes having increased levels of stress-response molecules to the joint of the subject.

Description

EXOSOME COMPOSITIONS AND USE THEREOF FOR JOINT DISORDERS AND

DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No.

62/615,050, filed on January 9, 2018, the entire contents of which are all hereby incorporated herein by reference. This application also claims the benefit of U.S. Provisional Application No. 62/712,583, filed on July 31, 2018, the entire contents of which are all hereby incorporated herein by reference.

TECHNICAL FIELD

[2] The present disclosure relates to stem cell exosome compositions, and

preparation thereof, for uses including treatment of joint disorders caused by infection, immunological disturbances, trauma and degenerative joint diseases such as, for example, rheumatoid arthritis, osteoarthritis, overuse and traumatic joint injury.

BACKGROUND

[3] Joint disorders and diseases, including arthritis, commonly involve chronic inflammatory responses resulting in the degradation of cartilage, degeneration of bone and the loss of normal joint function. Healthy joints contain a balance of non-activated cell types, including chondrocytes (cartilage) and synoviocytes making up the surrounding synovial tissue. Whereas the synoviocytes secrete proteases and activators of cartilage degradation in a normal joint, these processes become activated in the case of arthritis, leading to a net degradation of cartilage, pain and impaired joint function. Treatment of musculoskeletal disorders and degenerative diseases has traditionally relied upon the natural ability of cartilage and bone tissue to repair themselves. However, in many instances the body is unable to repair such defects in a reasonable time, if at all, and most treatments are ineffective in the long term.

[4] As exemplified in rheumatoid arthritis, a systemic autoimmune disease, joints are infiltrated by inflammatory cells, including activated peripheral blood mononuclear cells (PBMCs) which secrete inflammatory cytokines into the synovium. This in turn activates resident cells to secrete proteinases and other factors that degrade cartilage and bone. Once a chronic inflammatory cascade is established in a joint, it is difficult to eliminate with existing treatments. Similarly, chronic knee pain is long-term pain, swelling, or sensitivity in one or both knees that can be caused by a number of conditions. Chronic knee pain rarely goes away without treatment.

[5] Those suffering from joint disorders commonly use systemic non-steroidal anti-inflammatory drugs (NSAIDs) to relieve the pain; however, these drugs can also inhibit cartilage synthesis and repair, exacerbating their condition. Due to limited circulation, many systemic treatments do not effectively penetrate the joint capsule; this has led to specific targeting of the affected joint by direct injection. Corticosteroid injections into the joint are another common short term treatment to reduce pain and inflammation· These injections are effective in reducing inflammation without inducing systemic steroidal side effects.

Unfortunately, site-specific damage to tendons, ligaments and articular cartilage can occur with repeated injections, limiting the timing and number of injections to a single joint.

Hyaluronic acid (HA), a lubricating component of synovial fluid, is routinely injected directly into arthritic joints with the intent of providing pain relief when other treatments have failed. Typically this approach entails weekly injections into the same joint over the course of three to five weeks but has shown variable results. In the best cases, symptoms are relieved for up to six months.

[6] Adult stem cells from different tissues, including bone marrow-derived

mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs) are capable of propagating and differentiating into several phenotypes such as osteoblasts, chondrocytes, myocytes, tendon-ligament fibroblasts, and adipocytes. In addition, these stem cells secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities. These secreted bioactive factors can suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate mitosis and differentiation of tissue-intrinsic reparative or stem cells. These trophic effects are distinct from the direct differentiation of stem cells into repair tissue.

[7] Adult stem cells have shown therapeutic potential in pre-clinical models of inflammatory diseases and are a promising candidate cell type for tissue engineering and cell therapy to repair damaged structures in various arthritic conditions. Stem cells possess anti inflammatory and immunosuppressive properties modulated by the secretion of biologically active molecules and since many joint disorders, including arthritis, are derived from a chronic inflammatory response involving tissue destruction, the anti-inflammatory and regenerative functions of stem cells could be exploited as a therapy. Several groups have demonstrated therapeutic effects of intra-articular injections of MSCs in experimental arthritis models, and these studies showed anti-inflammatory and reparative effects on cartilage using both bone marrow-derived MSCs and ASCs. Based on these pre-clinical studies, several clinical trials have been performed using a similar technique and have shown some promise.

[8] ASCs, specifically, have been shown to exhibit immune suppressive properties and release anti-inflammatory molecules. As an example, in an animal model system a single injection of ASCs into an arthritic knee joint attenuated inflammation in the synovial membrane, reduced thickening of the lining layer, reduced proteolytic enzyme expression levels, and protected against cartilage and meniscus damage. Apart from inhibition of pro- inflammatory cytokines by macrophages, ASCs may also stimulate macrophages to produce elevated levels of growth factors. Mechanistically, paracrine factors appear to be the main contributors of stem cell-mediated tissue regeneration in a number of preclinical and clinical studies rather than direct tissue cell replacement.

[9] More recently, extracellular vesicles (EVs), including exosomes, released from stem cells emerged as key paracrine messengers that can also participate in the healing process through influencing the local microenvironment with anti-inflammatory effects.

[10] There remains an unmet need for more effective and long lasting treatments for joint disorders and diseases, including rheumatoid arthritis, osteoarthritis, and arthritis resulting from overuse and traumatic injury.

[11] The presently disclosed subject matter provides improved exosome

compositions and methods for treating joint disorders and diseases.

SUMMARY

[12] In one embodiment, a method is provided for treatment of a joint in a subject, the method comprising: administering an effective amount of a composition comprising isolated stem cell exosomes having increased levels of stress-response molecules to the joint of the subject. The administering may be by injection. The isolated exosomes may be isolated from a culture medium conditioned by culturing stem cells under conditions that include a heat shock of the culture medium. The joint may be damaged, injured, or inflamed and the damage, injury, or inflammation can be associated with a disease or disorder, comprising one or a combination of osteoarthritis, rheumatoid arthritis, gout, reactive arthritis, psoriatic arthritis, or juvenile arthritis BRIEF DESCRIPTION OF THE DRAWINGS

[13] FIG. 1 is a graph showing PBMCs activated by P. Acnes and the effect of exosomes having increased levels of stress-response molecules on IL-6 cytokine release according to one or more embodiments of the present disclosure.

[14] FIG. 2 is a graph showing PBMCs activated by P. Acnes and the effect of exosomes having increased levels of stress-response molecules on TNFa cytokine release according to one or more embodiments of the present disclosure.

[15] FIG. 3 is a graph showing the reduction in the total number of LPS -induced air pouch infiltrating cells by treatment with various amounts of human ASC-derived exosomes according to one or more embodiments of the present disclosure.

[16] FIG. 4A is a graph showing the reduction in the number of LPS-induced

eosinophils infiltrating the air pouch by treatment with various amounts of human ASC- derived exosomes according to one or more embodiments of the present disclosure.

[17] FIG. 4B is a graph showing the reduction in the number of LPS-induced

neutrophils infiltrating the air pouch by treatment with various amounts of human ASC- derived exosomes according to one or more embodiments of the present disclosure.

[18] FIG. 4C is a graph showing the reduction in the number of LPS-induced Pan-T cells infiltrating the air pouch by treatment with various amounts of human ASC-derived exosomes according to one or more embodiments of the present disclosure.

[19] FIG. 5 is a graph showing the reduction in induced paw edema by injection of various amounts of human ASC-derived exosomes over the course of the study compared to vehicle injection according to one or more embodiments of the present disclosure. Sham animals were not induced and did not develop arthritis. 25, 125 and 250 million are injections of 25, 125 and 250 million heat shocked ASC-derived exosomes, respectively. *p<0.05; **r<0.01; ***p<0.00l compared to vehicle using Student’s T-test.

[20] FIG. 6 is a graph showing the reduction in mean arthritis score by injection of various amounts of human ASC-derived exosomes during the course of the study compared to vehicle injection according to one or more embodiments of the present disclosure. 25, 125 and 250 million are injections of 25, 125 and 250 million heat shocked ASC-derived exosomes, respectively. *p<0.05; **r<0.01; ***p<0.00l compared to vehicle using Student’s T-test. [21] FIG. 7A is a graph showing the reduction in arthritic pain by injection of various amounts of human ASC-derived exosomes during the course of the study compared to vehicle injection according to one or more embodiments of the present disclosure. The graph shows an increase in peak force tolerated by rats injected with various amounts of human ASC-derived exosomes. 25, 125 and 250 million are injections of 25, 125 and 250 million heat shocked ASC-derived exosomes, respectively. *p<0.05; **r<0.01; ***p<0.00l compared to vehicle using Student’s T-test.

[22] FIG. 7B is a graph showing the reduction in arthritic pain by injection of various amounts of human ASC-derived exosomes during the course of the study compared to vehicle injection according to one or more embodiments of the present disclosure. The graph shows an increase in the time the peak force was tolerated by rats injected with various amounts of human ASC-derived exosomes. 25, 125 and 250 million are injections of 25, 125 and 250 million heat shocked ASC-derived exosomes, respectively. *p<0.05; **p<0.0l; ***p<0.00l compared to vehicle using Student’s T-test.

[23] FIG. 8 is a graph showing the reduction in histopathological severity of

induced arthritis in joints injected with various amounts of human ASC-derived exosomes according to one or more embodiments of the present disclosure. 25, 125 and 250 million are injections of 25, 125 and 250 million heat shocked ASC-derived exosomes, respectively. *p<0.05; **p<0.0l compared to vehicle using Student’s T-test.

DETAILED DESCRIPTION

[24] For the purposes of promoting an understanding of the principles of the

present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[25] There is an unmet need for more effective injectable formulations for

regulating joint conditions such as the treatment of arthritis, and cartilage, bone, and synovial damage, and other defects. To solve this unmet need, the presently disclosed subject matter provides methods that include administering an effective amount of a composition containing isolated stem cell-derived exosomes having increased levels of stress-response molecules to the affected joints of a subject. The methods can be useful to regulate joint disorders and diseases and repair joint-related tissue damage. The isolated stem cell-derived exosomes may be derived from mesenchymal stem cells (MSC-derived exosomes). The mesenchymal stem cells may be of placental or adipose (ASC-derived) origin.

[26] Exosomes represent a compelling therapeutic for a range of indications,

especially those requiring delivery to tissues with reduced vasculature or prominent necrosis. Exosomes, unlike stem cells, do not require an oxygenated blood supply to exert their impact. And, because exosomes fuse with cell membranes directly, there is no requirement for receptor mediated uptake of their cargos.

[27] The methods of the present invention utilize stem cell-derived exosomes that have increased levels of stress response molecules. The improved stem cell-derived exosome- containing compositions of the present disclosure are based on the context-dependency of the loading of exosomes. In one example exemplified in EXAMPLE 1 herein, stem cell cultures are subjected to high temperature (otherwise known as“heat shock”) to produce exosomes having the increased levels of stress-response molecules, including the stress-response protein, HSP70. It is demonstrated herein that the isolated exosomes having increased stress- response molecules can have enhanced anti-inflammatory activity in cell cultures and in a rodent model (see EXAMPLES 2 & 3).

[28] The terms“exosomes”,“microvesicles”,“secreted microvesicles”,

“extracellular vesicles", and“secreted vesicles” are used interchangeably herein for the purposes of the specification and claims.

[29] The terms“freeze drying” and“lyophilization” are used interchangeably

herein for the purposes of the specification and claims.

[30] The terms“stress-response molecules,”“heat shock stress-response

molecules,” and“heat shock molecules” are used interchangeably herein for the purposes of the specification and claims. These terms are meant to include molecules that are produced by the stem cells and secreted via exosomes by the cultured stem cells as a result of a stress event. The stress event can include heat shock, such as the heat shock procedure described herein in EXAMPLE 1.

[31] The terms“a,”“an,” and“the” refer to“one or more” when used in this

application, including the claims.

[32] Throughout this specification and the claims, the terms“comprise,”

“comprises,” and“comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term“include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[33] For the purposes of this specification and claims, the term“about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[34] In one embodiment, a method is provided to improve joint disorders and

diseases in a subject. This method comprises administering stem cell-derived exosomes having increased levels of stress-response molecules, including heat shocked exosomes, to the joint. The animal may be a mammal, and in particular, may be a human or non-human primate. In one embodiment, a method is provided for treatment of a joint in a subject, the method comprising: administering an effective amount of a composition comprising isolated stem cell exosomes having increased levels of stress-response molecules to the joint of the subject.

[35] The administering may be by injection into the joint.

[36] The exosomes may be derived from stem cells allogeneic to the subject or autologous to the subject.

[37] The isolated exosomes may be isolated from a culture medium conditioned by culturing stem cells under conditions that include a heat shock of the culture medium. The heat shock of the culture medium may be at a temperature of about 4l°C to about 43 °C for about 1 hour to about 3 hours.

[38] The isolated stem cell exosomes having increased levels of stress-response molecules may be administered to the joint in conjunction with an acceptable pharmaceutical carrier. A suitable carrier is within the knowledge of one skilled in the art. Suitable pharmaceutical carriers include, but are not limited to, hyaluronan, chemically modified hyaluronan, saline, phosphate buffered saline, chondroitin sulfate, glucosamine,

mannosamine, proteoglycan, proteoglycan fragments, chitin, chitosan, or other

polysaccharaide or polymer material. [39] The isolated exosomes having increased levels of stress-response molecules may be administered to a joint to provide for the improvement or stabilization of damaged, injured, or inflamed joints. The damage, injury, or inflammation may be associated with a disease or disorder, such as osteoarthritis, rheumatoid arthritis, gout, reactive arthritis, psoriatic arthritis, or juvenile arthritis, for example. It also may result from an osteoarthrosis or chronic disease of the joint of noninflammatory character.

[40] Joints which may be improved or stabilized, and/or in which inflammation may be reduced, include, but are not limited to, knee joints, hip joints, shoulder joints, elbow joints, ankle joints, tarsal and metatarsal joints, wrist joints, spine, carpal and metacarpal joints, and the temporal mandibular joint.

[41] The stem cell exosomes having increased levels of stress-response molecules may be administered in an amount effective to improve and/or stabilize a joint in the subject. In general, the exosomes are administered in an amount ranging from about 1 x l0^A5/mL to 1 x l0^Al0/mL, preferably ranging from 1 x 10^A6 to 1 x lO^A9/mL. The exact number of exosomes is dependent upon a variety of factors, including, but not limited to, the age, weight, and sex of the subject, the extent and severity of the joint disease or disorder, the degree of exudation within the joint, the joint space, and other anatomical characteristics that will influence the delivery.

[42] Example 2 herein describes the ability of ASC-derived exosomes having

increased levels of stress response molecules to inhibit IL6 and TNFa protein secretion in PBMCs. In example 2, ASC-derived exosomes having increased levels of stress response molecules were produced by heat shocking according to EXAMPLE 1. Peripheral blood mononuclear cells (PBMCs) were concomitantly exposed to P. acnes soluble lysate

(44pg/ml) and the isolated exosomes from heat shocked ASCs. The results are shown in the graphs in FIG. 1 (IL-6) and FIG. 2 (TNFa). The results illustrate a statistically significant reduction in P. acnes-indaced elevation of both IL-6 and TNFa secretion by the isolated heat shocked ASC-derived exosomes. These data demonstrate that the isolated exosomes of the present disclosure can inhibit the production of inflammatory cytokines including IL6 and TNFa that act locally to recruit monocytes to the site of inflammation.

[43] Example 3 herein describes the ability of ASC-derived exosomes having

increased levels of stress response molecules to inhibit an induced inflammatory response in a rodent model simulating a joint capsule. In Example 3, the anti-inflammatory effects of ASC-derived exosomes having increased levels of stress response molecules (such as the heat shocked exosomes described in Example 1) is investigated by concomitant injection with LPS in an air pouch created under the skin of a mouse. The rodent air pouch model is an established small animal model that recapitulates the synovial environment and allows for the investigation of agents that may improve joint disorders through an anti-inflammatory activity. The total number of infiltrating cells in the air pouches was significantly reduced in a dose dependent manner using heat shocked ASC-derived exosomes as shown in FIG. 3. Additionally, data in FIG’s. 4A-4C (or Table 1) show that the heat shocked ASC-derived exosomes significantly reduced the number of infiltrating eosinophils, neutrophils and Pan-T cells. In addition, Table 2 shows the significant reduction in several pro-inflammatory cytokines in the lavage fluid. These data illustrate the anti-inflammatory activity of heat shocked ASC-derived exosomes and the efficacy of these exosomes in a rodent model of joint capsular inflammatory response.

[44] Example 4 herein describes the ability of ASC-derived exosomes having

increased levels of stress response molecules to inhibit arthritis in a collagen-induced arthritis rat model. The anti-inflammatory effects of the ASC-derived exosomes having increased levels of stress response molecules was investigated by intra-articular injection in rats induced with bovine collagen type II. The rats were injected in the hind right paw weekly and progression of arthritis followed by assessing paw edema, arthritic score (swelling, redness of paw, digits or joints), pain measurement (Von Frey test) and histopathology at day 40.

Several arthritis parameters were significantly reduced by treating with intra-articular injections of heat shocked ASC-derived exosomes. Paw edema was significantly reduced from day 25 to the end of the study by injection of 125 million heat shocked ASC-derived exosomes compared to vehicle control as shown in FIG. 5. This same dose of heat shocked ASC-derived exosomes significantly lowered the arthritis score from day 29 forward (FIG.

6.) indicating a significant reduction in the joint disorder. Consistent with the decreased severity of the disease, FIGs. 7A & 7B show a reduction in pain measures by a significant increase in peak force tolerated (FIG. 7A) and time of peak force tolerated (FIG. 7B) by the injection of 125 million heat shocked ASC-derived exosomes. Histopathological analysis of the injected joints shows a significant decrease in the severity and extent of arthritis in the heat shocked ASC-derived exosome injected joints as shown in FIG 8.

EXAMPLES [45] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Example 1

Preparation of Exosomes with Increased Levels of Stress-Response Molecules using

Heat Shock

[46] The following experiments describe the production of isolated exosomes having increased levels of stress-response molecules.

[47] Mesenchymal stem cells (placental or adipose origin) were cultured in a hollow fiber cartridge bioreactor (FIBERCELL BIOSYSTEMS) to produce exosomes having increased levels of heat shock stress-response molecules as follows. Prior to seeding, the bioreactor was conditioned with complete culture medium (DMEM/F12 containing 10%

FBS) for 24 hr at 37°C in a humidified, 5% C0₂ containing atmosphere. The bioreactor was seeded with 300 x 10⁶ mesenchymal stem cells (placental or adipose origin) and maintained at 37°C in a humidified, 5% CO2 containing atmosphere. Cells were grown for 2 weeks before beginning exosome harvest. Prior to harvesting exosome-containing medium, the bioreactor was washed 5 times with serum-free DMEM/F12 to remove bovine exosomes. After washing, the cells were subjected to a heat shock step as follows. The medium in the bioreactor was replaced with serum-free DMEM/F12 medium warmed to 4l°C, and the bioreactor was placed in a 4l°C, humidified, 5% CO2 containing atmosphere for 1 hr. Next the 4l°C medium was replaced with the same medium warmed to 37°C, and the bioreactor was placed in a 37°C, humidified, 5% CO2 containing atmosphere for 48 hr. After the 48 hr incubation, the conditioned serum-free DMEM/F12 medium was recovered, and in some instances, stored at -80°C for future processing.

[48] In separate preparations of isolated exosomes having increased levels of stress-response molecules, the same procedure as described above was followed except that the temperature of the medium used in the heat shock step was about 42°C in some preparations and about 43 °C in other preparations and the time period of heat shock ranged from as short as about 1 hour to as long as about 3 hours. [49] After thawing or fresh collection of the conditioned medium described above, the exosomes were isolated from the conditioned media by centrifugation of the medium at 3000 xg for 20 min at room temperature to pellet cell debris (in 50, 250, or 500 mL screw cap vessels). The clarified supernatant was collected and centrifuged at 100,000 xg (Avg. RCF) for 2 hrs at 4°C. The supernatant was aspirated and the pellet(s) resuspended in minimum volume of DPBS (300-1000 pL). Manufacturer’s instructions were followed to estimate protein and RNA concentration using a NANODROP (THERMO FISHER, Corp) spectrophotometer. The number of particles (exosomes) per mL and the particle (exosome) size were determined using the QNANO (IZON SCIENCE, Ltd) following manufacturer’s instructions. The isolated exosomes were aliquoted into appropriate volumes into 1.5 mL screw cap tubes.

Example 2

Heat Shocked ASC Exosomes inhibit Production of inflammatory Cytokines

[50] The potential of ASC-derived exosomes having increased levels of stress response molecules to inhibit IL6 and TNFa protein secretion in PBMCs was examined as described below.

[51] In this experiment, ASC-derived exosomes having increased levels of stress response molecules were produced by heat shocking according to EXAMPLE 1. P. acnes is one of the bacterial species known to contribute to inflammation by secreting various toxins lethal to soft tissue cells. Previous reports indicate the induction of inflammatory cascades in PBMCs in response to P. acnes soluble lysates, including the inflammatory molecules IL6 and TNFa [Rossol, 1990]. To evaluate the anti-inflammatory impact of treatment with the isolated exosomes prepared from heat shocked ASCs, PBMCs were concomitantly exposed to P. acnes soluble lysate (44pg/ml) and the isolated exosomes from heat shocked ASCs.

[52] For the experiment, isolated exosomes produced by heat shocked ASCs were tested. Specifically, to measure inflammatory response, multiplexed quantification of secreted cytokine protein levels for the inflammatory cytokines IL6 and TNFa was performed using a MagPix and Milliplex assay kits. PBMCs were seeded in 96-well plates and incubated overnight prior to treatments. Cells were treated without P. acnes soluble lysate and without exosomes (Media Only), with 44pg/ml P. acnes soluble lysate and without exosomes (P. acnes), with 44pg/ml P. acnes soluble lysate and lOOnM dexamethasone (P. acnes + lOOnM Dex), and with 44pg/ml P. acnes soluble lysate in combination with increasing amounts of heat shocked ASC-derived isolated exosomes (ASC Exosomes). The quantified cytokine data are shown in the graphs in FIG. 1 (IL-6) and FIG. 2 (TNFa). The results illustrate a statistically significant reduction in P. acnes- induced elevation of both IL-6 and TNFa secretion by the isolated heat shocked ASC-derived exosomes. These data demonstrate that the isolated exosomes of the present disclosure can inhibit the production of inflammatory cytokines including IL6 and TNFa that act locally to recruit monocytes to the site of inflammation·

Example 3

Heat Shocked ASC-derived Exosomes Reduce Inflammation in a Mouse Air Pouch

Model Simulating a Joint Capsule Response

[53] The potential of heat shocked ASC-derived exosomes produced according to EXAMPLE 1 to inhibit an induced inflammatory response in a rodent model simulating a joint capsule is examined as described below.

[54] Infiltration of inflammatory cell types into the joint capsule and synovium, and their secretion of pro-inflammatory cytokines is a hallmark of many joint disorders. The rodent air pouch model is an established small animal model that recapitulates the synovial environment and allows for the investigation of agents that may improve joint disorders through an anti-inflammatory activity. The anti-inflammatory effects of ASC-derived exosomes having increased levels of stress response molecules (such as the heat shocked exosomes described in Example 1) is investigated by concomitant injection with LPS in an air pouch created under the skin of a mouse. After six hours, the air pouches are lavaged with sterile saline and the number and types of infiltrating cells determined by flow cytometry. Additionally, the levels of thirty-one cytokines are quantified by multiplexed assay.

[55] For this study, ten Balb/c mice per treatment group were injected with

0.2mL/g sterile air under the skin on each mouse’s back to induce the air pouch. Three days later, 0.l2mL/g sterile air was injected into the same space to maintain the air pouch. After another three days lmL of air was removed from each pouch and treatments were applied as follows: Group 1 received sterile saline (lOmL/kg); Groups 2, 3 and 4 received 25, 125 and 250 million exosome particles per air pouch (lOmL/kg volume), respectively. Thirty minutes later, all animals received an air pouch injection containing 0.5pg/animal LPS in endotoxin- free PBS (lmL/mouse). Six hours after the LPS challenge, the animals were anesthetized and their pouches washed with 5mL of sterile saline and the lavage fluid analyzed for total cellular content, specific inflammatory cell populations, and the presence of cytokines by flow cytometry and multiplexed ELISA.

[56] The total number of infiltrating cells was significantly reduced in a dose dependent manner using heat shocked ASC-derived exosomes as shown in FIG. 3.

Additionally, data in FIG. 4 (or Table 1) show that of heat shocked ASC-derived exosomes significantly reduced the number of infiltrating eosinophils, neutrophils and Pan-T cells. In further support of the anti-inflammatory activity of heat shocked ASC-derived exosomes, Table 2 shows the significant reduction in several pro-inflammatory cytokines in the lavage fluid. These data illustrate the anti-inflammatory activity of heat shocked ASC-derived exosomes and the efficacy of the exosomes in a rodent model of joint capsular inflammatory response.

Table 1. Inflammatory Cell Types in Lavage Fluid (thousands of cells ± SEM)

* p<0.05, Student’s T-test

Table 2. Effect of Exosome Treatment on Cytokine Levels in Lavage Fluid (pg/mL ± SEM)

*p<0.05: **p<0.005, Dunnett’s multiple comparisons test

Example 4 Heat Shocked ASC-derived Exosomes Reduce Arthritis in a Rat Collagen Induced Arthritis Model Simulating Rheumatoid Arthritis

[57] The potential of heat shocked ASC-derived exosomes produced according to EXAMPLE 1 to inhibit arthritis in a collagen-induced arthritis rat model is examined as described below.

[58] Rheumatoid arthritis is characterized by a strong inflammatory response

within the joints including the secretion of inflammatory cytokines, activation of innate immune cells, bone and articular degradation, reduced cartilage repair, and pain. The administration of bovine collagen type II in an adjuvant induces a disease in rats that models rheumatoid arthritis in humans and allows for the investigation of agents that may improve joint disorders through an anti-inflammatory activity. The anti-inflammatory effects of ASC- derived exosomes having increased levels of stress response molecules (such as the heat shocked exosomes described in Example 1) is investigated by intra-articular injection in rats induced with bovine collagen type II. The rats are injected in the hind right paw weekly and progression of arthritis followed by assessing paw edema, arthritic score (swelling, redness of paw, digits or joints), pain measurement (Von Frey test) and histopathology at day 40.

[59] For this study, ten male Lewis rats per treatment group were injected

intradermally at the base of the tail with 300 pg of bovine collagen type II in 100 pL

Incomplete Freund’s Adjuvant (IFA) on Day 0. On day 21, a second injection of 100 pg of bovine collagen type II in IFA was administered. Treatments were administered once a week for 5 weeks by intra-articular injection in 20 pL final volume as follows: Groups 1, 2 and 3 received 25, 125 and 250 million exosome particles; Group 4 received 80 pg of

dexamethasone; Group 5 received vehicle injections; Group 6 was untreated without intra- articular injections; Group 7 consisted of 5 rats naive to the collagen induced arthritis as a negative control group. Rats were weighed on the day of adjuvant injection and two times per week until the end of the study. The arthritic score was determined daily on a treatment blinded basis using the scoring of 0 (no sign of arthritis), 1 (swelling and/or redness of the paw or one digit), 2 (involvement of two joints), 3 (involvement of greater than two joints) and 4 (severe arthritis of the entire paw and digits). The arthritic index was calculated for each rat by summing the scores for the individual paws. Paw thickness (edema) was determined weekly by plethysmometry; pain (mechanical allodynia) was determined weekly using an electronic Von Frey Hair Aesthesiometer. Histopathology was performed at the end of the study and included examining the knees and ankles after fixation and sectioning followed by hematoxylin and eosin staining of slides. Histological severity of arthritis was scored as follows: 0 (no damage), 1 (edema), 2 (presence of inflammatory cells), and 3 (bone resorption).

[60] Several arthritis parameters were significantly reduced by treating with intra- articular injections of heat shocked ASC-derived exosomes. Paw edema was significantly reduced from day 25 to the end of the study by injection of 125 million heat shocked ASC- derived exosomes compared to vehicle control as shown in FIG. 5. This same dose of heat shocked ASC-derived exosomes significantly lowered the arthritis score from day 29 forward (FIG. 6.) indicating a significant reduction in the joint disorder. Consistent with the decreased severity of the disease, FIG. 7 shows a reduction in pain measures by a significant increase in peak force tolerated and time of peak force tolerated by the injection of 125 million heat shocked ASC-derived exosomes. Histopathological analysis of the injected joints showed a significant decrease in the severity and extent of arthritis in the heat shocked ASC-derived exosome injected joints as shown in FIG 8. A reduction in inflammatory cell infiltration and bone resorption with the 125 million heat shocked ASC-derived exosome dose injections supports the use of ASC exosomes as a therapeutic for joint disorders.

[61] Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. These patents and publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[62] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present Examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.

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Claims

THAT WHICH IS CLAIMED:

1. A method for treatment of a joint in a subject, the method comprising: administering an effective amount of a composition comprising isolated stem cell exosomes having increased levels of stress-response molecules to the joint of the subject.

2. The method of claim 1, wherein the stem cells are mesenchymal stem cells.

3. The method of claim 2, wherein the mesenchymal stem cells are of placental or adipose origin.

4. The method of claim 1, wherein the administering is by injection.

5. The method of claim 1, wherein the isolated exosomes are isolated from a culture medium conditioned by culturing stem cells under conditions that include a heat shock of the culture medium.

6. The method of claim 5, wherein the heat shock of the culture medium is at a temperature of about 4l°C to about 43 °C for about 1 hour to about 3 hours.

7. The method of claim 1, wherein the exosomes are derived from stem cells allogeneic to the subject or autologous to the subject.

8. The method of claim 1, wherein the subject is an animal, a mammal, a human, or a non human primate.

9. The method of claim 1, wherein the composition further comprises a pharmaceutical carrier.

10. The method of claim 9, wherein the carrier comprises one or more of hyaluronan, chemically modified hyaluronan, saline, phosphate buffered saline, chondroitin sulfate, glucosamine, mannosamine, proteoglycan, proteoglycan fragments, chitin, chitosan, polys accharaide, or synthetic or non-synthetic polymer material.

11. The method of claim 1, wherein the joint is damaged, injured, or inflamed and the treatment is to provide for the improvement or stabilization of the damaged, injured, or inflamed joint.

12. The method of claim 11, wherein the damage, injury, or inflammation is associated with a disease or disorder, comprising one or a combination of osteoarthritis, rheumatoid arthritis, gout, reactive arthritis, psoriatic arthritis, or juvenile arthritis.

13. The method of claim 12, wherein the damage or injury is associated with an osteoarthrosis or chronic disease of the joint of noninflammatory character.

14. The method of claim 1, wherein the joint is one or a combination of knee joints, hip joints, shoulder joints, elbow joints, ankle joints, tarsal and metatarsal joints, wrist joints, spine, carpal and metacarpal joints, or temporal mandibular joints.

15. The method of claim 1, wherein the isolated exosomes are administered in an amount

effective to improve and/or stabilize the joint in an amount ranging from 1 x l0^A5/mL to 1 x l0^Al0/mL or from 1 x 10^A6 to 1 x lO^A9/mL.