Healthy extracellular vesicles for treatment of preeclampsia

Abstract

Provided herein are methods for treating preeclampsia or reducing preeclampsia symptoms by administering extracellular vesicles extracted from healthy placental mesenchymal stem cells. The preeclampsia symptoms may be characterized by increased levels of p21, CDK4, p53, and TDG in placental mesenchymal stem cells. Levels of these proteins are reduced with extracellular vesicle treatment.

Description

Atty. Dkt. No. 183161.00029HEALTHY EXTRACELLULAR VESICLES FOR TREATMENT OF PREECLAMPSTACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 740,798 filed on December 31, 2024, the content of which is incorporated by reference in its entirety.BACKGROUND

[0002] Preeclampsia (PE) affects up to 5-8% of pregnancies worldwide with significant effects on pregnancy-related morbidity and mortality. It is the leading cause of fetal growth restriction. Inflammation has been implicated as key condition leading to PE. Aspirin is used to prevent the incidence of preeclampsia in patients at risk, but no current treatment for reversal of preeclampsia is available. Therapeutic strategies are of interest.SUMMARY

[0003] In an aspect, provided herein is a method for treating preeclampsia a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of extracellular vesicles extracted from healthy placental mesenchymal stem cells. Between about 109and about 1014extracellular vesicles may be administered.

[0004] In another aspect, provided herein is a method for reducing placental inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of extracellular vesicles extracted from healthy placental mesenchymal stem cells. Between about 109and about 1014extracellular vesicles may be administered. The subject may have or be suspected of having preeclampsia.

[0005] In another aspect, provided herein is a method for improving anti-inflammatory function in preeclampsia placental mesenchymal stem cells (PE-MSCs), the method comprising contacting the PE-MSCs with extracellular vesicles extracted from healthy placental mesenchymal stem cells. The PE-MSCs may be contacted with about 103to about 1014extracellular vesicles. Thee PE-MSCs may be contacted with about 1 CP to about 108extracellular vesicles. The PE-MSCs may be contacted with the extracellular vesicles for between about 12 hours and about 72 hours. The PE-MSCs may be contacted for between about 24 hours and about 48 hours.Atty. Dkt. No. 183161.00029

[0006] The method may further comprise performing a mixed lymphocyte recti on, wherein a reduced inflammation index indicates improved anti-inflammatory function.

[0007] The method may further comprise measuring levels of CDK4, p53, and TDG, wherein reduced levels of at least one of CDK4, p53, and TDG indicate improved anti-inflammatory function.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1. Human mixed lymphocyte reaction (MLR) test to confirm immunomodulation capacity of preeclamptic placental mesenchymal stem cells (PE-MSC). Placental mesenchymal stem cells (MSC) were isolated from two placentas, one from a pregnancy complicated by preeclampsia and the other from a healthy pregnancy. Irradiated peripheral blood mononuclear cells (PBMC) (A(i)) and non-irradiated peripheral blood mononuclear cells (B) were cocultured with MSCs after treatment with (+) different concentrations of extracellular vesicles (exo) collected from healthy placental derived mesenchymal stem cells (P-MSC) and time exposures (24 vs. 48 hours) or aspirin (ASA) or no treatment (NT).

[0009] FIG. 2. Human mixed lymphocyte reaction (MLR) test to confirm immunomodulation capacity of preeclamptic placental mesenchymal stem cells (PE-MSC).

[0010] FIG. 3. Western blot results for cell cycle proteins p21, CDK4, and p53, and epigenetic regulator thymine-DNA glycosylase (TDG in preeclamptic MSCs and healthy MSCs before and after treatment with healthy extracellular vesicles.DETAILED DESCRIPTION

[0011] Described herein is a method for using extracellular vesicles derived from healthy placental mesenchymal stem cells to treat preeclampsia. Preeclampsia is a disorder characterized by the new onset of high blood pressure and often a significant amount of protein in the urine or significant end-organ damage during pregnancy. Preeclampsia increases the risk of undesirable and potentially lethal outcomes for both the mother and the fetus including preterm labor. The inventors have previously shown the dysfunctional role of preeclampsia placental mesenchymal stem cells in inflammation, cell cycle regulation, and the diminished production of immune-suppressive cytokines. The restoration of these cells to normal function by exposure to healthy placental mesenchymal stem cells is demonstrated herein.Atty. Dkt. No. 183161.00029

[0012] In a first aspect, provided herein is a method for treating preeclampsia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of extracellular vesicles extracted from healthy placental mesenchymal stem cells.

[0013] The terms “extracellular vesicle” and “EV” are used herein to refer to a vesicle of about 10 nm to 10 pm in size composed of fluid, macro-molecules, solutes, and metabolites from a cell contained by a lipid bilayer or micelle. These terms encompass both exosomes, ectosomes, macrovesicles, and apoptotic vesicles, and are within the broad microvesicles. Exosomes are released on the exocytosis of multivesicular bodies. Ectosomes are vesicles assembled at and released from the plasma membrane. In some cases, the EV is about 20 nm to 10 pm, 20 nm to 1 pm, 20 nm-500 nm, 30 nm-100 nm, 30 nm-160 nm, or 80-160 nm in size. In some embodiments, the EVs are exosomes that are about 20 to 150 nm in size. The EVs used herein are extracted from healthy placental mesenchymal stem cells. The term “healthy” refers to cells not afflicted with preeclampsia. Accordingly, the EVs extracted from these cells are also referred to herein as “healthy extracellular vesicles.”

[0014] The terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state, disorder, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). "Treatment,” encompasses any administration or application of a therapeutic or technique for a disorder (e.g., in a mammal, including a human), and includes inhibiting the disorder, arresting its development, relieving the disorder, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.

[0015] The terms "effective amount" or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The amount of the healthy extracellular vesicles that is therapeutically effective may vary depending on the route of administration of the condition of the subject. An effective amount may comprise between about 109and about 1014healthy extracellular vesicles.

[0016] As used herein, the term "administering" refers to dispensing, delivering or applying the substance to the subject. In terms of a composition of healthy extracellular vesicles, the term "administering" is intended to refer to delivering or applying it to the subject by any suitable route for delivery of the extracellular vesicles to the desired location within the subject.Atty. Dkt. No. 183161.00029

[0017] The healthy EVs may be administered by any appropriate route of administration, including but not limited to, the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, and administration by the intranasal or respiratory tract route.

[0018] The terms “subject” and “patient” are used herein interchangeably to refer to a mammal, such as a human, to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. In embodiments, the subject is a human.

[0019] In a second aspect, provided herein is a method for reducing placental inflammation in a subject having or suspected of having preeclampsia, the method comprising administering to the subject a therapeutically effective amount of extracellular vesicles extracted from healthy placental mesenchymal stem cells. The subject may have or be suspected of having preeclampsia.

[0020] In a third aspect, provided herein is a method for improving anti-inflammatory function in preeclampsia placental mesenchymal stem cells (PE-MSCs), the method comprising contacting the PE-MSCs with extracellular vesicles extracted from healthy placental mesenchymal stem cells. Improved anti-inflammatory function may be determined by a mixed lymphocyte reaction (MLR), in which a reduced inflammation index in the PE-MSCs after healthy EV treatment relative to before treatment indicates improved anti-inflammatory function. Improved anti-inflammatory function may be further or alternatively determined by reduced levels of at least one of CDK4 protein, p53 protein, and TDG protein after healthy EVN treatment relative to before treatment.

[0021] The PE-MSCs may be contacted with about 105to about 1014extracellular vesicles extracted from healthy placental mesenchymal stem cells. In exemplary embodiments, the PE-MSCs are contacted with about 109to about 1014extracellular vesicles. In exemplary embodiments, the PE-MSCs are contacted with about 105to about 108extracellular vesicles. The PE-MSCs may be contacted with the extracellular vesicles for between about 12 hours and about 72 hours. In exemplary embodiments, the PE-MSCs are contacted for between about 24 hours and about 48 hours.

[0022] “ Contacting” a cell or a cell culture refers to adding the healthy EVs to a medium or bufferAtty. Dkt. No. 183161.00029comprising the cell or cell culture. The term “culture” refers to a population of cells grown under controlled conditions in vitro.

[0023] Miscellaneous

[0024] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.”

[0025] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0026] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0027] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values veryAtty. Dkt. No. 183161.00029near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0028] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0029] An “isolated” or “purified” polynucleotide is removed from its natural environment, such as a cell, and is at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which it is naturally associated.

[0030] The terms “protein”, “polypeptide”, and “peptide” refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids.

[0031] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.Atty. Dkt. No. 183161.00029

[0032] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0033] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0034] EXAMPLES

[0035] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0036] Example 1

[0037] Introduction

[0038] Preeclampsia (PE) affects up to 5-8% of pregnancies worldwide with significant effects on pregnancy-related morbidity and mortality. It is the leading cause of fetal growth restriction. Inflammation has been implicated as key condition leading to PE. It is known that the placenta has layers of healthy placenta mesenchymal stem cells (P-MSCs), which are capable of mitigating inflammation. This group has previously published research on the dysfunctional role of PE P-MSCs in inflammation, cell cycle regulation, and the diminished production of immune-suppressive cytokines. Aspirin or acetyl salicylic acid (ASA) treatment remediated these effects, including epigenetic reprogramming. In this innovation, the hypothesis that extracellular vesicles (EVs) from healthy P-MSCs or umbilical cord (UC) could reset PE P-MSCs to mitigate the associated inflammation and to reset cell cycle of P-MSC was tested.

[0039] ResultsAtty. Dkt. No. 183161.00029

[0040] 1. Confirmation ofMSC collection

[0041] We cultured placental MSC from the chorionic membrane. The morphology was spindle-shaped, similar to what was previously described, and consistent regardless of the presence of preeclampsia.

[0042] 2. Mixed Lymphocyte Reaction (MLR) results

[0043] Preeclamptic placental MSC had anti-inflammatory activity and suppressed the MLR reaction, however, not as well as healthy placental MSC. Extracellular vesicle (EV)-treated preeclamptic placental mesenchymal stem cells (PE P-MSCs or PE MSCs) and ASA-treated PE-MSCs suppressed MLR to levels of healthy placental MSC. There was no difference among MSC ability to suppress the MLR reaction between those treated with aspirin and those treated with extracellular vesicles. There was no difference in ability to suppress MLR reaction between MSC treated with extracellular vesicles for 24 versus 48 hours (FIG. 1).

[0044] MSC Western blot results

[0045] PE P-MSCs had increased expression of CDK4, p53 and TDG compared to healthy placental MSCs (P-MSCs). PE P-MSCs had similar expression of p21 compared to P-MSCs. Treatment of preeclamptic P-MSCs with extracellular vesicles decreased the expression of p21, CDK4, p53, and TDG compared to preeclamptic P-MSCs with no treatment. Treated PEC P-MSC, had relatively elevated p53 and TDG compared to healthy P-MSCs, while p21 was decreased, and CKD4 was similar. See Table 1 and FIG. 3.

[0046] Table 1. Western blot results

[0047] Conclusion

[0048] While ASA increased p21, p53, and TDG, and decreased CDK4, EV treatment decreasedAtty. Dkt. No. 183161.00029their expression. Of note, p53 and TDG remained elevated as compared to healthy P-MSCs while p21 was decreased.

[0049] PE P-MSC can be restored towards healthy anti-inflammatory function. The healthy EVs reduced PE-MSC cell cycle with evidence of changes in DNA methylation regulation. These findings are consistent with MSCs exhibiting tissue maintenance during insult. In total, healthy P-MSC EVs may restore the function of PE P-MSCs, as well as other insults during pregnancy. These findings suggest off-the-shelf availability of EVs to treat PE. Combined with prior data on ASA restoring MSC function, this study furthers our understanding of possible methods to reduce PE via anti-inflammatory modality.

[0050] Methods

[0051] Reagents and antibodies

[0052] Phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Medium-high glucose (DMEM), fetal bovine serum (FBS), penicillin / streptomycin (P-S), L-glutamine, bovine serum albumin (BSA), aspirin (acetylsalicylic acid), and protease inhibitor were purchased from Sigma (St Louis, MO) Antibodies used ***

[0053] Sample collection

[0054] Placentas were collected from healthy and preeclamptic singleton pregnancies. UCB were be collected from healthy pregnancies. Preeclampsia was a clinical diagnosis determined by treating team, separate from the research team, as defined by ACOG. Healthy pregnancies were defined as no known placental dysfunction, intraamniotic infection, hypertension, diabetes, HIV or Hepatitis B or Hepatitis C.

[0055] MSC isolation and culture

[0056] After delivery, placentas were collected and examined in the laminar flow hood. Chorionic membrane was separated from the amniotic membrane using sharp dissection and cut into 3-5mm pieces. This tissue was then incubated, filtered, and centrifuged to isolate mesenchymal stem cells as previously described (Romagano). These cells were then plated onto 6-well tissue culture plates and incubated at 37 degrees and 5% CO2 in placental MSC culture media, DMEM with 10% FBS, 1% penicillin / streptomycin and 1% L-glutamine. The cells were examined after 2 to 3 days and noted to be spindle-shaped. Media was replaced as needed, at least weekly, until 80% confluence, at which point the cells were then dis-adhered and split.

[0057] Purification and quantification of extracellular vesiclesAtty. Dkt. No. 183161.00029

[0058] Extracellular vesicles were isolated from healthy P-MSCs via differential ultracentrifugation. Purity and quantity of extracellular vesicles wase confirmed with Nanosight Tracing Analysis for size and particle numbers, and Western blot for CD63, CD9, and Alix.

[0059] MSC extracellular vesicle Western blot

[0060] MSC EV extracts were prepared via snap freeze. After ultracentrifugation, EV pellet was resuspended in 250 microliters of media. 25 microliters of protease inhibitor and 25 microliters of mammalian protein extraction reagent (MPER) were added. The mixture was then submerged in liquid nitrogen for two to three minutes and then transferred to 37 degrees Celsius water bath for two to three minutes. This cycle was repeated 4 times. Total protein was determined using Biorad Bradford protein assay. Laemmli buffer with P-mercaptoethanol (BME) were added to protein samples and the samples were boiled for 10 minutes at 95C to denature protein. Extracts (15 micrograms) were electrophoresed on gradient SDS-PAGE 10-12% at 200V, transferred onto membranes at 4 degrees Celsius using 400 mA for 90 minutes. Precision plus kaleidoscope ladder was used to assess molecular weights. The membranes were washed with PBS 0.1% TWEEN and then incubated in 1-2% milk for 20 minutes. Primary antibody was added at dilution of 1 : 1000 and the Western blot was incubated in the cold room overnight on a shaker. The next day the primary antibody was removed and the membrane washed with PBS-TWEEN four times. Then the membrane was blocked with 1-2% milk and secondary antibodies were added at a dilution of HRP conjugated anti-mouse*** 1:2000 and kept in cold room on shaker for two hours. The membrane was then washed four times after secondary was removed. The Western blood was then developed with Chemiluminescent Test (ECL) and imaged with ChemiDoc XRS+. Band densities were normalized to beta actin. The membranes were then stripped with stripping buffer, washed with PBS 0.1% TWEEN, and re-probed. The following primary antibodies listed in Table 1 were used as primary. The latter was detected with species specific antibodies linked to Horse Radish Peroxidase (HRP).

[0061] MLR

[0062] A mixed lymphocyte reaction (MLR) was used to assess the capacity of the PE-MSC to be licensed into immune suppressor cells following exposure to different types of EVs at different concentrations (e5 to e8) compared to no treatment. Whole blood was collected from two healthy donors in heparinized tubes, blood was added to Ficoll and underwent centrifugation to separate cells based on density. Buffy layer of peripheral blood mononuclear cells (PBMCs) was carefullyAtty. Dkt. No. 183161.00029extracted and re-suspended in PBS and underwent centrifugation again. One donors PBMCs were assigned to be the stimulator cells and were irradiated with 5000 rads from a Cesium (137Cs). The other donor’s PBMCs were assigned to be the responder cells. Cells from both donors were plated at equal concentrations and incubated at 37°C and 5% CO2 for 72 hours with and without the additional MSC treatment described below (Table 2). At 72 h, the cells were pulsed with 1 pCi of tritiated thymidine. After 16 h, the cells were harvested on a Cambridge Technology PHD Cell Harvester. The filters were assessed for tritiated thymidine incorporation on a Beckman Coulter LS6500 Liquid Scintillation Counter.

[0063] Extracellular vesicle collection was as described above with ultracentrifugation. Extracellular vesicles used in the experiment were as follows: EVs from healthy P-MSC, EVs from healthy P-MSC treated with aspirin for 24 hours, healthy p-MSC treated with aspirin for 48 hours, EVs from preeclamptic placental MSC, EVs from preeclamptic placental MSC treated with aspirin for 24 hours, and EVs from preeclamptic placental MSC treated with aspirin for 48 hours. Aspirin dosage was 1 mM in MSC media. This dose was used as it previously has been shown to be a dose tolerated by MSC, allowing them to remain viable and retain their function.

[0064] Table 2. MLR treatment Groups. (PBMC - peripheral blood mononuclear cells; I -irradiated; PEC MSC - preeclamptic mesenchymal stem cells; ASA - aspirinAtty. Dkt. No. 183161.00029

Claims

Atty. Dkt. No. 183161.00029CLAIMSWe claim:

1. A method for treating preeclampsia a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of extracellular vesicles extracted from healthy placental mesenchymal stem cells.

2. The method of claim 1, wherein between about 109and about 1014extracellular vesicles are administered.

3. A method for reducing placental inflammation in a subj ect in need thereof, the method comprising administering to the subject a therapeutically effective amount of extracellular vesicles extracted from healthy placental mesenchymal stem cells.

4. The method of claim 3, wherein between about 109and about 1014extracellular vesicles are administered.

5. The method of claim 3 or 4, wherein the subject has or is suspected of having preeclampsia.

6. A method for improving anti-inflammatory function in preeclampsia placental mesenchymal stem cells (PE-MSCs), the method comprising contacting the PE-MSCs with extracellular vesicles extracted from healthy placental mesenchymal stem cells.

7. The method of claim 6, wherein the PE-MSCs are contacted with about 105to about 1014extracellular vesicles.

8. The method of claim 7, wherein the PE-MSCs are contacted with about 105to about 108extracellular vesicles.

9. The method of any one of claims 6-8, wherein the PE-MSCs are contacted with the extracellular vesicles for between about 12 hours and about 72 hours.

10. The method of claim 9, wherein the PE-MSCs are contacted for between about 24 hours and about 48 hours.

11. The method of any one of claims 6-10, further comprising performing a mixed lymphocyte rection, wherein a reduced inflammation index indicates improved antiinflammatory function.

12. The method of any one of claims 6-11, further comprising measuring levels of CDK4, p53, and TDG, wherein reduced levels of at least one of CDK4, p53, and TDG indicate improved anti-inflammatory function.

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