WO2025050037A1

WO2025050037A1 - Microrna-15b antagomir and cerium oxide nanoparticles, and uses of same for treatment of intrauterine growth restriction

Info

Publication number: WO2025050037A1
Application number: PCT/US2024/044860
Authority: WO
Inventors: Sarkis DERDERIAN; Kenneth LIECHTY; Sudipta Seal; Carlos ZGHEIB; James BARDILL; Lauren Gallagher
Original assignee: University of Colorado System; University of Colorado Colorado Springs; University of Central Florida Research Foundation Inc
Current assignee: University of Colorado System; University of Colorado Colorado Springs; University of Central Florida Research Foundation Inc
Priority date: 2023-08-31
Filing date: 2024-08-30
Publication date: 2025-03-06
Anticipated expiration: 2026-02-28

Abstract

The compositions, methods, and systems described herein are useful in treating subjects suffering from or at risk of developing Intrauterine Growth Restriction.

Description

MICR0RNA-15B ANTAGOMIR AND CERIUM OXIDE NANOPARTICLES, AND USES OF SAME FOR TREATMENT OF INTRAUTERINE GROWTH RESTRICTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63/535,907, entitled “MICRORNA-15B ANTAGOMIR AND CERIUM OXIDE NANOPARTICLES, AND USES OF SAME FOR TREATMENT OF INTRAUTERINE GROWTH RESTRICTION ", filed on August 31, 2023, the contents of which are hereby incorporated herein in their entirety.

REFERENCE TO A SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 30, 2024, is named MIRNA_ANTAGOMIR_PCT and is 3 kilobytes in size.

TECHNICAL FIELD

[0003] The disclosed compositions processes, methods, and systems are directed to treatment of Intrauterine Growth Restriction with microRNA inhibitors.

BACKGROUND

[0004] Intrauterine Growth Restriction (IUGR) is a fetal disease process accounting for nearly 50% of stillbirths and is defined as fetal weight below the 10th percentile at any gestational age. While many risk factors exist, the pathogenesis of IUGR culminates in aberrant placental angiogenesis and uterine insufficiency. Infants with IUGR are born smaller than normal, and they may have a variety of health problems, including low birth weight, premature birth, difficulty maintaining body temperature, poor feeding, respiratory distress, and an increased risk of infection. They may also be at higher risk for long-term health issues, such as developmental delays, behavioral problems, and chronic diseases such as diabetes and cardiovascular disease. Currently, there are limited to no effective treatments for IUGR. Early detection and management of IUGR can help improve outcomes for both mother and baby. Current approaches, however, rely on regular prenatal care, including ultrasound, to help identity’ IUGR early enough to allow for timely intervention. SUMMARY

[0005] In one aspect, a composition for treating intrauterine growth restriction (IUGR), includes an RNA oligonucleotide, where the RNA oligonucleotide is configured to specifically hybridize to miRNA-15b and suppress an effect of miRNA-15b activity. For example, the RNA oligonucleotide can be an miRNA-15b antagomir. In some embodiments, the RNA oligonucleotide includes at least 18 nucleotides that are complementary to SEQ ID NO: 1. In particular embodiments, the RNA oligonucleotide is fully complementary to SEQ ID NO: 1. In various embodiments, the RNA oligonucleotide can include a chemical modification to at least one nucleotide and/or to a backbone molecule of said RNA oligonucleotide. The composition may also include where the RNA oligonucleotide is conjugated to a nanoparticle. The composition may also include where the RNA oligonucleotide is fully complementary to SEQ ID NO: 1. The composition may also include where the nanoparticle is a lipid nanoparticle. The composition may also include where the nanoparticle is a cerium oxide nanoparticle (CNP). The composition may also include where the miRNA-15b is covalently conjugated to the CNPs via a linker selected from carbonyldiimidazole (CDI) and N,N'-disuccinimidyl carbonate (DSC). The composition may also include where the nanoparticle is coated with one or more biocompatible molecules. The nanoparticle may have a particle size of about 2 nm to about 10 nm, or more particularly about 3 nm to about 5 nm.

[0006] In an aspect, the composition can be included in a pharmaceutical formulation. In some embodiments, the formulation can further include miRNA-146a-conjugated cerium oxide nanoparticles. In various embodiments, miRNA-146a is covalently conjugated to the CNPs via a linker selected from carbonyldiimidazole (CDI) and N,N'-disuccinimidyl carbonate (DSC). The miRNA-146a-conjugated cerium oxide nanoparticles may be coated with one or more biocompatible molecules. The nanoparticles may have a particle size of about 2 nm to about 10 nm. or more particularly about 3 nm to about 5 nm.

[0007] In an aspect, a method of treating intrauterine growth restriction (IUGR) in a pregnant subject can include administering to the subject a therapeutically effective amount of the pharmaceutical formulation. In some embodiments the administering is performed via injection or infusion. The administration route can selected from intrauterine, intraplacental and intraamniotic. The method may also include administering the formulation a plurality of times, such as two to six times. In some embodiments the formulation is administered to the subject in the second trimester of pregnancy, the third trimester of pregnancy, or both. The method may also include where treating includes increasing expression of VEGF in the subject, reducing or preventing oxidative stress in the subject.

[0008] In an aspect, a method of diagnosing intrauterine growth restriction (IUGR) in a subject can comprise steps of determining a level of miRNA-15b in a biological sample from the subject and comparing the level of miRNA-15b in the biological sample to a reference level of miRNA-15b, where the presence of a level of miRNA-15b in the subject that is above the reference level indicates that the subject has IUGR. In some embodiments, the biological sample is selected from placental tissue and amniotic fluid. The method may also further include administering to the subject a therapeutically effective amount of an RNA oligonucleotide that is configured to bind to miRNA-15b and to suppress an effect of miRNA-15b activity.

[0009] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows a diagram showing various risk factors and phenomena associated with intrauterine Growth Restriction (IUGR).

[0011] FIG. 2 shows an illustrative depiction of the action of microRNA ("miR") on gene expression.

[0012] FIG. 3 shows a diagram illustrating post-transcriptional control of angiogenesis by microRNA-15b ("miR-15b").

[0013] FIG. 4 shows a diagram describing a murine calorie-restriction model of IUGR.

[0014] FIG. 5 shows a bar graph of the results of an MTT assay of HTR8 trophoblasts exposed to differing media conditions.

[0015] FIG. 6 shows a bar graph of the results of a BRD-u assay of HTR8 trophoblasts exposed to differing media conditions.

[0016] FIG. 7 shows a bar graph of miRNA-15b expression in HTR8 trophoblasts exposed to differing media conditions.

[0017] FIG. 8 shows a graph of miRNA-15b expression over time in HTR8 trophoblasts exposed to differing media conditions.

[0018] FIG. 9 A through FIG. 9C show box plots of relative placental gene expression of angiogenic mediators associated with IUGR in a murine model of IUGR. Middle line in box represents the median; lower box bounds the first quartile; upper box bounds the 3^rd quartile. Whiskers represent the 95% confidence interval of the mean. Abbreviations: miR, microRNA; IUGR, intrauterine growth restriction; VEGFa, vascular endothelial growth factor alpha; Bcl-2, B-cell lymphoma-2.

[0019] FIG. 10A through FIG. 10D show box plots of relative placental gene expression of inflammatory' mediators associated with IUGR: microRNA-146a (FIG. 10A); Nuclear Factor kappa Bl (FIG. 10B), Interleukin 8 (FIG. 10C) and Interleukin 6 (FIG. 10D. Middle line in box represents the median; lower box bounds the first quartile; upper box bounds the 3^rd quartile. Whiskers represent the 95% confidence interval of the mean. Abbreviations: IUGR, intrauterine growth restriction; miR, microRNA; NFkBl, Nuclear Factor kappa Bl; IL-8, Interleukin 8; IL-6, Interleukin 6.

[0020] FIG. 11 A through FIG. 11C show box plots of relative placental gene expression of hypoxia associated proteins Hy poxia Inducible Factor 1 Subunit alpha (FIG. 11 A), NADPH oxidase 2 (FIG. 1 IB) and Superoxide Dismutase 2 (FIG. 11C). Middle line in box represents the median; lower box bounds the first quartile; upper box bounds the 3^rd quartile. Whiskers represent the 95% confidence interval of the mean. Abbreviations: IUGR, intrauterine growth restriction; HIF-la, Hypoxia Inducible Factor 1 Subunit alpha; SOD2, Superoxide Dismutase 2; Nox2, NADPH oxidase 2.

TERMS AND DEFINITIONS

[0021] The following terms and phrases include the meanings provided below. The provided definitions are intended to aid in describing particular embodiments, and are not intended to limit the claimed compositions, methods, compounds, systems, and therapies. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0022] The term “about'’ or “approximately’’ means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 30%, 25%, 20%, 15%. 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%. 1%, 0.5%. or 0.05% of a given value or range. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series. [0023] ‘‘Nanoparticle’' refers to any particle having a measurable length in one dimension for example a diameter, of less than about 1000 nm. The presently disclosed nanoparticles may have a maximum measured dimension of about 500 nm or less, 100 nm or less, 900 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, and 20 nm or less. In many embodiments, the disclosed nanoparticles may have a greatest dimension in the size range of about 2-10 nm, for example about 3-5 nm.

[0024] A “patient” or “subject” includes a mammal or animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or guinea pig. The animal can be a mammal such as a non-primate or a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent, or adult of any or indeterminant sex.

[0025] “Biological sample” as used herein may refer to any sample removed, extracted, or derived from a patient or subject. In many embodiments a biological sample includes tissues, cells, protein, nucleic acids, etc. A biological sample may be processed for viewing or analysis, such as protein or nucleic acid quantitation. A control sample or reference sample may be a sample obtained from a similar subject, tissue, or cell that does not have, or is not expected to have, a disease or condition that is being assayed. In some embodiments, a reference sample may include samples from two or more subjects.

[0026] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.

[0027] The phrase “therapeutically effective amount” means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

DETAILED DESCRIPTION

[0028] Intrauterine growth restriction is defined as fetal weight below the 10^th percentile at any gestational age. There are multiple risk factors for developing IUGR that are broadly categorized as maternal, fetal and placental but all ultimately culminate in aberrant angiogenesis and placental insufficiency that results in deficient nutrient delivery' to the fetus and a relative hypoxic state (FIG. 1). Diagnosis of IUGR typically involves measuring the fetus's growth and weight during pregnancy, through ultrasound and other tests. Treatment options can depend on the cause and severity of IUGR, but currently include close monitoring of the fetus, medication for the mother, early delivery, or other interventions.

[0029] It is believed that microRNAs may play a role in the development of IUGR. MicroRNAs are short (20-24 nucleotides) non-coding RNAs that regulate gene expression at the post-transcriptional level by binding specific messenger RNAs which promote degradation or suppression of messenger RNA translation (FIG. 2). Therefore, Applicants hypothesize that microRNAs may be promising approaches to target specific aspects of disease pathogenesis.

[0030] Specific microRNAs have previously been implicated in angiogenesis. Particularly, miRNA-15b has been correlated with dysregulation of angiogenesis through the downregulation of VEGFa (FIG. 3). The nucleotide sequence of miRNA-15b is provided herein as SEQ ID NO: 1: UAGCAGCACAUCAUGGUUUACA. Oxidative stress markers including HIF1 -alpha, SOD2 and N0X2 have also been connected to the regulation of angiogenesis through hypoxia.

[0031] Applicants have found that miRNA-15b is upregulated in placentas from an IUGR mouse model. This upregulated miRNA-15b led to a decrease in the expression of vascular endothelial growth factor (VEGF). Therefore, Applicants hypothesize that miRNA-15b upregulation in mouse placentas with IUGR reduces angiogenesis development. This discovery' makes miRNA-15b a target for IUGR treatment.

[0032] Applicants describe herein a treatment to target miRNA-15b in a tissue or organ subject, more particularly in the placenta. In some embodiments, a formulation for treating IUGR can comprise an RNA oligonucleotide that functions to inhibit miRNA-15b. The RNA oligonucleotide therefore promotes an increased angiogenic environment to help IUGR placental development. In some embodiments, the RNA oligonucleotide is an miRNA-15b antagomir.

[0033] Chemically modified single-stranded RNA analogues complementary to a specific miRNA are effective as antagomirs. Accordingly, miRNA- 15b antagomirs of the present disclosure comprise a sequence that is substantially complementary to the nucleic acid sequence specific to miRNA-15b or to a portion thereof. In certain embodiments the miRNA- 15b antagomir comprises one or more sequences that combined are fully complementary' to miRNA-15b. In other embodiments, the miRNA-15b antagomir can comprise at least 18, at least 19, at least 20, or at least 21 bases that are complementary to miRNA-15b. Where the complementary' sequences are less than the total of their respective oligonucleotide, said sequences may be consecutive or non-consecutive.

[0034] In various embodiments, the miRNA-15b antagomir can comprise a sufficient degree of complementarity such that stable and specific binding occurs between miRNA- 15b and the miRNA-15b antagomir. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility or expression, and there is a sufficient degree of complementarity' to avoid non-specific binding of the oligonucleotide to non-target sequences under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. MiRNA-15b antagomirs suitable in the embodiments described herein may be identified by screening an oligonucleotide library/, or a library' of nucleic acid molecules, under hybridization conditions and selecting for those which hybridize to miRNA-15b.

[0035] RNA oligonucleotides of the present disclosure may be modified, either by manipulation of the chemical backbone of the nucleic acids or by covalent or non-covalent attachment of other moieties to the backbone or selected bases. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety; a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine or a polyethylene glycol chain. In each or any⁷ case, such manipulation or attachment may serve to modify the stability, cellular, tissue or organ uptake of the RNA oligonucleotides, or otherwise enhance their efficacy in inhibiting miRNA-15b.

[0036] RNA oligonucleotides as described herein may be configured for any delivery' technique appropriate to deliver them to a target tissue or organ, including genetic engineering techniques, liposome-mediated delivery including lipid nanoparticle delivery, viral vectors or the like. In some embodiments, one or more RNA oligonucleotides may be introduced into placental tissues using nanoparticles. Nanoparticle delivery methods have the potential for reduced immune response compared to viral vectors and also could increase the stability' of the microRNA-15b. In one embodiment, the nanoparticle may be an RNA oligonucleotide, such as a miRNA-15b antagomir, conjugated to a cerium oxide nanoparticle (also referred to as “CeCh nanoparticles.” “nanoceria,” or “CNP”). The CNPs may have a size range of about 2-10 nm. and in particular about 3-5 nm.

[0037] CNPs may be covalently conjugated to, or otherwise incorporate (i.e., non- covalently imbedded in or associated with), an RNA oligonucleotide described herein, such as a miRNA-15b antagomir. Oligonucleotides contain phosphate groups carrying a negative charge along the chain that can electrostatically interact with the positively charged surface of the CNPs. In addition, oligonucleotides have hydroxyl groups of ribose and amino groups available for conjugation with the CNPs. Modifying a terminal end of the oligonucleotide with functional group (amino, thiol, azide) for conjugation is also an option that may be utilized. Providing an appropriate excess of oligonucleotide in reaction medium (e.g. 10-15 molecules per nanoparticle), conjugation can be accomplished via different reactions. For example, amino groups of an oligonucleotide can be coupled with CNP hydroxyl groups or functional groups of a CNP coating after activation with carbonyldiimidazole (CDI), N,N'-disuccinimidyl carbonate (DSC) or other bifunctional activating agents. In particular embodiments, CNP hydroxyl groups are reacted with CDI or DSC to create an intermediate that can react with a primary' amine group. The RNA oligonucleotide can be modified with a terminal 3’ primary amine group that then reacts with the activated nanoparticle intermediate, leaving the final nanoparticle-oligonucleotide conjugate.

[0038] In addition to impaired angiogenesis, an inflammatory milieu has been shown to be associated with IUGR. In fact, a chronic maternal inflammatory state is an independent risk factor for IUGR. In addition, placentas affected by IUGR demonstrate increased inflammatory mediators, and patients who suffered from IUGR in utero are at an increased risk for developing inflammatory disease processes in adolescence and adulthood. Increased or persistent inflammation results in the accumulation of reactive oxygen species (ROS) and increased oxidative stress, a state seen in IUGR. The present disclosure provides compositions comprising miRNA-15b antagomir-CNP conjugates configured to decrease the inflammatory' response, resulting in decreased ROS and oxidative stress and lead to improved outcomes.

[0039] In some embodiments, engineered CNPs of the present technology may possess properties that are able to overcome increased inflammation and oxidative stress associated with IUGR. For example, these CNPs may scavenge excess ROS, similar to the catalytic activity of superoxide dismutase (SOD) and catalase. In some embodiments, the CNPs can be doped to increase the content of the Ce³⁺ oxidation state, thereby enlarging the redox activity and thus the scavenging capacity' of the CNPs. In some embodiments, the CNPs are doped with a lanthanide selected from one or more of Europium (Eu). Lanthanum (La), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Homium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), and Lutetium (Lu).

[0040] Bioavailability and internalization of CNPs can be enhanced by coating the nanoparticles with a biocompatible molecule. In some embodiments, coating with hyaluronic acid may improve the biocompatibility' and behavior of CNPs in a target tissue or organ, changing its stability in the biofluids, preventing aggregation that causes cytotoxicity, changing the biodistribution profile due to acquired bioadhesive and biodegradable properties, as well as retention time within cellular space, mechanism of uptake and exocytosis.

[0041] A well-established regulator of inflammation is miRNA-I46a, and this microRNA has been associated with chronic inflammation in disease processes such as type II diabetes mellitus and rheumatoid arthritis. MiRNA-146a acts as a “molecular brake” on the inflammatory response by targeting and repressing the activation of the NFKB inflammatory pathway. Expression of miRNA-146a is significantly down-regulated in diabetic wounds and mesenchymal stem cell correction of impaired wound healing is associated with increased miRNA-146a expression and down-regulation of inflammatory cytokine production. In some embodiments, a formulation for treating IUGR comprising a miRNA- 15b antagomir may further comprise miRNA-146a. More particularly, such a formulation can comprise nanoparticles conjugated to one or both of these oligonucleotides. For example, CNPs conjugated to miRNA-15b antagomir may be provided with CNPs conjugated to miRNA-146a. In another example, CNPs conjugated to both miRNA-15b antagomir and miRNA-146a may be provided.

[0042] Methods of treating IUGR in a subject are provided by the present disclosure. In some embodiments, such methods can comprise administering to a subject a therapeutically effective amount of one or more oligonucleotides as described above. In some embodiments, the method comprises administering to the subject a formulation comprising nanoparticles conjugated to the oligonucleotides. In some embodiments, the administered formulation can comprise CNPs conjugated to miRNA-15b antagomir. In some embodiments, the administered formulation can further comprise CNPs conjugated to miRNA-146a. In some embodiments, the administered formulation can comprise CNPs conjugated to both miRNA-15b antagomir and miRNA-146a. [0043] The mode and route of administration may selected to accomplish delivery of the oligonucleotide to the placenta of a pregnant subject. In particular embodiments, administration comprises intrauterine, intrapl acental or intraamniotic injection or infusion through e.g., percutaneous or transvaginal routes. Compositions for administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Vehicles include sodium chloride solution, 1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. In various embodiments, administration can be made one or more times during the subject's pregnancy, for example, one time, two times, three times, four times, five times, or six times. In some embodiments, the formulation is administered to the subject in the second trimester of pregnancy, the third trimester of pregnancy, or both.

[0044] In some embodiments, a method of diagnosing IUGR in a subject or identifying a subject as a candidate for a treatment described herein can comprise ascertaining whether an miRNA-15b expression is dysregulated to a degree indicative of IUGR. This method can comprise determining a level of miRNA-15b expression in a biological sample from the subject and comparing said level to a reference level of miRNA-15b, where a level of miRNA-15b above the reference level indicates that the subject has IUGR. The sample may comprise any tissue, cells, or bodily fluid from which miRNA-15b expression level may be ascertained by known protein or nucleic acid quantitation techniques. In some embodiments the sample is selected from placental tissue and amniotic fluid. In certain embodiments, the method can further comprise following positive identification of IUGR with a treatment as described above.

[0045] Any methods disclosed herein comprise one or more steps or actions for performing the described method. The method steps and/or actions may be interchanged with one another. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order and/or use of specific steps and/or actions may be modified.

[0046] References to approximations are made throughout this specification, such as by use of the terms “substantially" and “about." For each such reference, it is to be understood that, in some embodiments, the value, feature, or characteristic may be specified wdthout approximation. For example, where qualifiers such as “about” and “substantially” are used, these terms include within their scope the qualified words in the absence of their qualifiers. All ranges also include both endpoints.

[0047] Similarly, in the above description of embodiments, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure. This method of disclosure, however, is not to be interpreted as reflecting an intention that any claim require more features than those expressly recited in that claim. Rather, as the following claims reflect, inventive aspects lie in a combination of fewer than all features of any single foregoing disclosed embodiment.

[0048] The claims following this written disclosure are hereby expressly incorporated into the present written disclosure, with each claim standing on its own as a separate embodiment. This disclosure includes all permutations of the independent claims with their dependent claims. Moreover, additional embodiments capable of derivation from the independent and dependent claims that follow are also expressly incorporated into the present written description.

[0049] Without further elaboration, it is believed that one skilled in the art can use the preceding description to utilize the invention to its fullest extent. The claims and embodiments disclosed herein are to be construed as merely illustrative and exemplary, and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having ordinary⁷ skill in the art, with the aid of the present disclosure, that changes may be made to the details of the above-described embodiments without departing from the underlying principles of the disclosure herein. In other words, various modifications and improvements of the embodiments specifically disclosed in the description above are within the scope of the appended claims. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified. The scope of the invention is therefore defined by the following claims and their equivalents.

EXAMPLES

[0050] The following examples illustrate various aspects of the disclosure, and should not be considered limiting.

Example 1: Exploring Placental Insufficiency in an In Vitro Model of IUGR

[0051] The Applicants hypothesized that post-transcriptional regulation, specifically through microRNAs, is associated with impaired angiogenesis and that their dysregulation may lead to the development of the placental insufficiency seen in IUGR. To identify placental insufficiency in IUGR, human HTR8 trophoblast cells were employed to assess the effect of nutrient deprivation on potential microRNA expression. This cell line depicts the invasive properties of trophoblastic cells evident during the first trimester of pregnancy. Trophoblast cells were plated in either normal media or media diluted 1: 1 with PBS to mimic a nutrient deficient media.

[0052] To confirm this model recapitulates IUGR. an MTT assay was used to measure cellular metabolic activity as an indicator of cell viabi 1 ity. proliferation and cytotoxicity. As shown in FIG. 5, less proliferation was seen in the nutrient mediums, thus indicating this model mimics the murine caloric restricted model discussed below.

[0053] A BRD-u assay was performed to further validate this model. This assay is used to detect in vitro DNA synthesis. As shown in FIG. 6, DNA synthesis was reduced in a dose responsive manner. These results further supported the utility of this model for evaluating post-transcriptional regulators and downstream targets of IUGR.

[0054] The trophoblast model was employed to assess miRNA- 15b expression in IUGR. As shown in FIG. 7, miRNA-15b gene expression was significantly increased in the IUGR population compared to controls at 48 hours of exposure to selected media.

[0055] To assess whether in vitro nutrient restriction affected miRNA expression in trophoblast cells at different timepoints, RT-qPCR was used to quantify expression of microRNAs of interest in cells under control baseline conditions, and cells collected after 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours of nutrient restriction. As shown in FIG. 8, miRNA- 15b is upregulated in HTR-8 cells that were cultured in media-restricted conditions.

Example 2: Exploring Dvsregulation of Angiogenesis and Sequelae in a Murine Model of IUGR

[0056] It was hypothesized that post-transcriptional regulation, specifically through microRNAs are associated with impaired angiogenesis and that their dysregulation may lead to the development of the placental insufficiency seen in IUGR. To test this hypothesis, a murine model of IUGR was utilized as shown in FIG. 4.

Materials and Methods

[0057] Pregnant mice were provided ad libitum access to food between E1-E8. From E9- E18, dams received either a 50% caloric restricted diet (IUGR) or continued ad libitum access (controls). Placentas from three different dams were harvested at El 8.5 to allow for adequate effects of nutrient deprivation and avoid maternal consumption of placentas. All procedures were approved by the International Animal Care and Use Committee (IACUC) at the University of Colorado (Aurora, CO) and care of the animals was in accord with the National Institutes of Health guidelines for ethical animal treatment.

[0058] RNA was extracted using a column-based extraction technique. Total cellular miR from harvested mouse placentas was purified using the Qiagen® miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) per manufacturer's protocol. The tissue was prepared by adding lysis buffer to 50mg of placental tissue and homogenized using the Omni tissue homogenizer. After extraction of both the miR and mRNA of downstream genes, the Qiagen RNeasy® MinElute Cleanup Kit (Qiagen, Valencia, CA, USA) was used to remove any contaminating DNA and concentrate the samples. RNA concentration was measured using the NanoDrop™ 2000c spectrophotometer (Thermofisher Scientific, Waltham, MA, USA). After RNA extraction, cDNA was synthesized, and relative mRNA and miR levels were evaluated by quantitative real-time PCR using exon spanning primers, and quantification was performed using the cycle threshold (AACt) method. Primers for miR-15b, miR-146a. as well as mRNAs of interest were used to compare levels of expression between IUGR and control placentas. Data were analyzed utilizing Student t-test with a p-value < 0.05 signifying significance. Statistical analyses were performed using Prism GraphPad version 6 (GraphPad Software, Inc., La Jolla, CA).

Results

[0059] Ten placentas in the IUGR group and ten placentas in the control group were harvested at E18.5. Expression of the anti -angiogenic miR-15b was upregulated in the IUGR group (FIG. 9A). VEGFa, which is downregulated in response to increased expression of miR-15b, was suppressed (FIG. 9B). B-cell lymphoma-2 (Bcl-2), a tumor suppressor protein, regulated by VEGFa did not demonstrate a significant difference (FIG. 9C). As many additional miRNAs have been shown to be associated with IUGR including miR-199a, miR-let7f and miR-21, levels of expression of these miRNA were compared. No significant differences in expression levels were seen between IUGR and control placentas for these miRNAs.

[0060] The inflammatory milieu associated with IUGR was explored by analyzing gene expression of the anti-inflammatory miRNA-146a as well as pro-inflammatory mediators. Gene expression of miR-146a was downregulated within placentas affected by IUGR (FIG. 10A). In addition, gene expression of the downstream regulatory target nuclear factor kappa Bl (NFkBl) was upregulated (FIG. 10B) as were two pro-inflammatory cytokines modulated by NFkBl, Interleukin-6 (IL-6, FIG. 10C) and Interleukin-8 (IL-8, FIG. 10D). [0061] These data suggest that an inflammatory state is associated with IUGR. As NFkBl, IL-6 and IL-8 are also elevated in chronic disease processes such as cardiovascular disease and type II diabetes mellitus, our findings suggests that inflammatory pathways associated with FGR may be the same ones associated with sequela of FGR.

[0062] Oxidative stress has been associated with impaired angiogenesis and chronic inflammation. Therefore, the gene expression of mRNAs found to be elevated in the setting of hypoxia was examined. Hypoxia Inducible Factor 1 Subunit alpha (HIF-la, FIG. 11 A), Superoxide Dismutase 2 (SOD2, FIG. 11B), and NADPH oxidase 2 (Nox2, FIG. 11C) were all upregulated in the IUGR mouse model when compared to controls. Despite this hypoxic state, miR-21, a regulator of HIF-la, was not dysregulated in IUGR placentas compared to controls, suggesting that this hypoxic state is a consequence of IUGR rather than a driver of it.

[0063] These data suggest that FGR is associated with impaired angiogenesis and an inflammatory state. Not only is gene expression of key molecules involved in these pathways affected, but miRNAs that regulate them are also dysregulated. Applicants hy pothesize that both the hy poxic and inflammatory state of the placenta are sequela of impaired angiogenesis.

Example 3: In Vitro Transfection of Mice with miRNA-15b Antagomir

[0064] An experiment is conducted to determine the effects of miRNA-15b antagomirs on angiogenesis, autophagy, and the inflammatory response after induction of IUGR using the diet restriction model. The experiment involves injecting microRNAs transplacentally using lentivirus, adeno-associated virus, or nanoparticle delivery systems. For nanoparticle treatments. miRNA-15b antagomirs are chemically conjugated to nanoparticles, or be given as formations with nanoparticles.

[0065] Treatment protocol:

1. Six-to-nine-week-old female C57b6 mice are mated with their male counterparts.

2. Detection of vaginal plugs is performed at El . Plugs are confirmed by lifting the dams by the tail and assessing the vagina for secretions.

3. Pregnant dams are given ad libitum access to standard facility food and water.

4. Beginning on E8, and continuing through the remainder of the pregnancy, the dams receive a calorie-restricted diet (50% of their total required calories) until delivery. This is determined by weight-based measurement of food daily to control daily calories.

5. Dams are weighed daily to ensure they are not losing more than 15% of the bodyweight expected during pregnancy. 6. At selected time(s) from E12 to E20, an exploratory laparotomy is performed and an injection made transplacentally with a sterile micropipette with a dose up to 2 mg of oligonucleotide composition or up to 50-100 microliters of vehicle as applicable.

Treatment groups include: a. PBS b. oligonucleotide alone c. oligonucleotide loaded into lentivirus d. oligonucleotide loaded into AAV e. oligonucleotide-nanoparticle conjugate

7. Forty-eight hours before surgery, acetaminophen (3 mg/mL) is added to their drinking water and continued for 72 hours post-operatively.

8. Pain is assessed daily based on mouse mobility. Mice that appear to have inadequate pain control are euthanized.

[0066] Laparotomy procedure for transplacental injections:

1 . Isoflurane is used as the anesthetic with a box induction of 5% and maintained on a nose cone at 1-3% isoflurane while receiving supplemental oxygen.

2. Before incision, depilatory' cream is applied to the abdomen for 1-2 minutes. The cream is then wiped away with wet gauze to remove the hair and cream.

3. After the removal of the hair, bupivacaine is injected intradermally before the incision for local anesthetic.

4. The abdomen is prepped with three swabs of betadine in a circular motion and three swabs of 70% ethanol in a circular motion to sterilize the area.

5. A sterile surgical drape with a pre-cut hole is placed on the mouse to maintain a sterile field.

6. Previously autoclaved tools are used for the surgery'.

7. The skin is opened separately followed by the muscle.

8. Uterine horns are exposed and kept irrigated with warm sterile saline applied every couple of minutes.

9. Oligonucleotide formulation is injected transplacentally with a sterile micropipette with a dose of up to 2mg (or up to 50-100 microliters).

10. A weight-based dose of buprenorphine ER (1 mg/kg) is injected into the subcutaneous tissue adjacent to the surgical incision at the end of the procedure.

11. Wounds are assessed daily for signs of cellulitis and weights are monitored daily to assess for fetal loss.

12. If there is concern for a surgical site infection, dams are euthanized. 13. At selected time from E12 to E20, the dams are euthanized by anesthetic gas and either thoracotomy or cervical dislocation.

14. The uterine horns are accessed and exposed post-mortem via the previous incision and expose the uterine horns. 15. All placental tissue is either fixed or snap-frozen per routine protocols for micro RNA analysis.

16. The pups are anesthetized by hypothermia (placed on a glove on ice for a few minutes) and euthanized by decapitation with sharp surgical scissors and tissue collected. 17. The tissue samples are processed and assessed for angiogenesis, autophagy, and inflammatory response.

Claims

CLAIMS What is claimed is:

1. A composition for treating intrauterine growth restriction (IUGR), comprising: an RNA oligonucleotide, wherein the RNA oligonucleotide is configured to specifically hybridize to miRNA-15b and suppress an effect of miRNA-15b activity.

2. The composition of claim 1, wherein the RNA oligonucleotide is an miRNA-15b antagomir.

3. The composition of claim 1, wherein the RNA oligonucleotide comprises at least 18 nucleotides that are complementary' to SEQ ID NO: 1.

4. The composition of claim 3, wherein the RNA oligonucleotide comprises at least 20 nucleotides that are complementary to SEQ ID NO: 1.

5. The composition of claim 3, wherein the RNA oligonucleotide is fully complementary to SEQ ID NO: 1.

6. The composition of any one of claims 1 to 5, wherein the RNA oligonucleotide comprises a chemical modification to at least one nucleotide.

7. The composition of any one of claims 1 to 6, wherein the RNA oligonucleotide comprises a chemical modification to a backbone molecule of said RNA oligonucleotide.

8. The composition of claim 6 or 7, wherein the chemical modification is selected from a lipid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine and a polyethylene glycol chain.

9. The composition of any one of claims 1 to 7, wherein the RNA oligonucleotide is conjugated to a nanoparticle.

10. The composition of claim 9, wherein the nanoparticle is a lipid nanoparticle.

11. The composition of claim 9, wherein the nanoparticle is a cerium oxide nanoparticle (CNP).

12. The composition of claim 11, wherein the CNP is doped with a lanthanide selected from one or more of Europium (Eu), Lanthanum (La), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Homium (Ho). Erbium (Er), Thulium (Tm), Yterbium (Yb). and Lutetium (Lu).

13. The composition of claim 11 or 12, wherein the miRNA-15b is covalently conjugated to the CNPs via a linker selected from carbonyldiimidazole (CDI) and N,N'-disuccinimidyl carbonate (DSC).

14. The composition of any one of claims 11 to 13, wherein the nanoparticle is coated with one or more biocompatible molecules selected from is coated with one or more biocompatible molecules.

15. The composition of claim 14, wherein the nanoparticle is coated with hyaluronic acid.

16. The composition of any one of claims 9 to 15, wherein the nanoparticle has a particle size of about 2nm to about 10 nm.

17. The composition of claim 16. wherein the particle size is about 3 nm to about 5 nm.

18. The composition of any one of claims 1 to 17, wherein the effect includes impaired angiogenesis.

19. The composition of any one of claims 1 to 18, wherein the effect includes downregulation of VEGFa.

20. The composition of any one of claims 1 to 19, wherein the effect includes inflammation.

21. The composition of any one of claims 1 to 20, wherein the effect includes oxidative stress.

22. A pharmaceutical formulation comprising: the composition any one of claims 1 to 21; and a pharmaceutically acceptable carrier or diluent.

23. The pharmaceutical formulation of claim 22, further comprising miRNA-146a- conjugated cerium oxide nanoparticles (CNPs).

24. The pharmaceutical formulation of claim 23, wherein the miRNA-146a is covalently conjugated to the CNPs via a linker selected from carbonyldiimidazole (CDI) and N.N'- disuccinimidyl carbonate (DSC).

25. The pharmaceutical formulation of claim 23 or 24, wherein the CNPs are doped with a lanthanide selected from one or more of Europium (Eu), Lanthanum (La). Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Homium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), and Lutetium (Lu).

26. The pharmaceutical formulation of any one of claims 23 to 25, wherein the CNPs have a particle size of about 2nm to about 10 nm.

27. The pharmaceutical formulation of claim 26, wherein the particle size is about 3 nm to about 5 nm.

28. A method of treating intrauterine growth restriction (IUGR) in a pregnant subject, comprising administering to the subject a therapeutically effective amount of the formulation of any one of claims 22 to 27.

29. The method of claim 28, wherein the administering is performed via injection or infusion.

30. The method of claim 29, wherein the administration route is selected from intrauterine, intraplacental and intraamniotic.

31. The method of any one of claims 28 to 30, wherein the formulation is administered a plurality of times.

32. The method of claim 31, wherein the formulation is administered two to six times.

33. The method of any one of claims 28 to 32, wherein the formulation is administered to the subject in the second trimester of pregnancy, the third trimester of pregnancy, or both.

34. The method of any one of claims 28 to 31, wherein treating comprises increasing expression of VEGF in the subject.

35. The method of any one of claims 28 to 34, wherein administering reduces or prevents oxidative stress in the subject.

36. A method of diagnosing intrauterine growth restriction (IUGR) in a subject, the method comprising: determining a level of miRNA-15b in a biological sample from the subject; and comparing the level of miRNA-15b in the biological sample to a reference level of miRNA- 15b, wherein the presence of a level of miRNA-15b in the subject that is above the reference level indicates that the subject has IUGR.

37. The method of claim 36, wherein the biological sample is selected from placental tissue and amniotic fluid.

38. The method of claim 36 or 37, further comprising administering to the subject a therapeutically effective amount of an RNA oligonucleotide that is configured to bind to miRNA-15b and to suppress an effect of miRNA-15b activity.