# Patent

## Title
Compositions and methods related to miRNA modulation of neovascularization or angiogenesis

## Abstract

The present invention concerns methods and compositions for diagnosing and/or treating vascular diseases including cancer, cardiac diseases, vascular diseases of the eye, and inflammatory diseases. The methods involve measuring the levels of one or multiple miRNAs in patient samples and using the test results to diagnose and/or predict an optimal treatment regimen for the patient. Compositions described in the invention include nucleic acids that function as miRNAs or miRNA inhibitors that can be introduced to a patient to reduce or increase vascularization as needed.

## Background
This application claims priority to U.S. Provisional Application Ser. No. 61/051,519 filed May 8, 2008, which is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION
I. Field of the Invention
The present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods and compositions involving microRNA (miRNAs) molecules and disease treatment. Certain aspects of the invention include applications of miRNA therapy for diseases or conditions that involve neovascularization and/or angiogenesis.
II. Background
Pathologic neovascularization refers to the proliferation of blood vessels in tissue not normally containing them, or proliferation of blood vessels of a different kind than usual in a tissue. It includes angiogenesis in tumor growth, diabetic retinopathy, haemangiomas, arthritis, and psoriasis to name a few.
Angiogenesis is the process of forming new blood vessels from pre-existing capillaries. Angiogenesis is tightly regulated and normally does not occur except during development, wound healing, and the formation of the corpus luteum during the female reproductive cycle. This strict regulation is manifested by a balanced production of positive and negative factors, which keep angiogenesis in check. However, this balance becomes abrogated under various pathological conditions, such as cancer, diabetes, and age-related macular degeneration (AMD), resulting in the growth of new blood vessels. It is now well accepted that the progressive growth and metastasis of many solid tumors and loss of vision with diabetes are dependent on the growth of new blood vessels.
Vascular diseases of the eye and tumors of the central nervous system, such as retinoblastoma and primitive neuroectodermal tumors (PNETs), have significant neovascular components. Some tumors of the central nervous system and ocular vascular diseases share similar pathogenesis having a choroidal neovascularization and/or retinal neovascularization component.
Vascular diseases of the eye comprise a major cause of blindness. These diseases include various retinopathies and macular degeneration. Existing treatments include laser ablation of various regions of the retina; vitrectomy or removal of the cloudy vitreous humor and its replacement with a saline solution; and administration of antioxidant vitamins E and C, but none of these methods can cure the disease. Further, existing invasive treatment methods often result in significant loss of vision. Non-invasive methods of treatment are experimental and have not been shown to substantially reduce the risk of blindness or loss of sight.
There is a need for additional compositions and/or methods for treatment of diseases associated with neovascularization and angiogenesis.
SUMMARY OF THE INVENTION
The present invention provides additional diagnostic, prognostic, and/or therapeutic methods by identifying miRNAs that are differentially expressed or mis-regulated in various states of diseased, normal, cancerous, and/or abnormal tissues, including but not limited to diseases characterized by or associated with vascularization and angiogenesis that result in a pathological condition or disease.
A physician may choose to treat a condition associated with neovascularization and/or angiogenesis by surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy and/or other methods. The choice of therapy depends upon the location and severity of the disease, as well as the general state of the patient. Certain aspects of the invention include methods for reducing vascularization in a subject or tissue comprising administering to the subject or tissue in need of such a reduction, in an amount sufficient to reduce vascularization and/or result in a beneficial response, one or more nucleic acid molecule comprising (a) a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more miR-184 (SEQ ID NO:1 and 2), miR-31 (SEQ ID NO:3 and 4), miR-150 (SEQ ID NO:5 and 6), miR-409 (SEQ ID NO:7 and 8), miR-375 (SEQ ID NO:9 and 10), miR-129-5p (SEQ ID NO:11 and 12), miR-142a (SEQ ID NO:13 and 14), miR-29a (SEQ ID NO:15 and 16), miR-129-3p (SEQ ID NO:17 and 18), miR-10b (SEQ ID NO:19 and 20), miR-96 (SEQ ID NO:21 and 22), miR-183 (SEQ ID NO:23 and 24), miR-16 (SEQ ID NO:25 and 26), miR-182 (SEQ ID NO:27 and 28), miR-191 (SEQ ID NO:29 and 30), miR-29c (SEQ ID NO:31 and 32), miR-181c (SEQ ID NO:33 and 34), miR-335 (SEQ ID NO:35 and 36), miR-7026 (SEQ ID NO:37), miR-210 (SEQ ID NO:38 and 39), miR-512-3p (SEQ ID NO:40 and 41), miR-132 (SEQ ID NO:42 and 43), miR-500 (SEQ ID NO:44 and 45), miR-339 (SEQ ID NO:46 and 47), miR-511 (SEQ ID NO:48 and 49), miR-26b (SEQ ID NO:50 and 51), miR-30b (SEQ ID NO:52 and 53), or miR-15a (SEQ ID NO:54 and 55), or complement thereof, and/or (b) an inhibitor of miR-451 (SEQ ID NO:56 and 57), miR-424 (SEQ ID NO:58 and 59), miR-146 (SEQ ID NO:60 and 61), miR-214 (SEQ ID NO:62 and 63), miR-199a (SEQ ID NO:64 and 65), miR-181 (SEQ ID NO:66 and 67), miR-350 (SEQ ID NO:68 and 69), miR-21 (SEQ ID NO:70 and 71), miR-218 (SEQ ID NO:72 and 73), miR-148b (SEQ ID NO:74 and 75), miR-106a (SEQ ID NO:76 and 77), miR-205 (SEQ ID NO:78 and 79), miR-365 (SEQ ID NO:80 and 81), miR-299-5p (SEQ ID NO:82 and 83), ambi-miR-7079 (SEQ ID NO:84), miR-200a (SEQ ID NO:85 and 86), miR-351 (SEQ ID NO:87 and 88), miR-329 (SEQ ID NO:89 and 90), miR-122a (SEQ ID NO:91 and 92), miR-20a (SEQ ID NO:93 and 94), miR-520h (SEQ ID NO:95 and 96), miR-142-5p (SEQ ID NO:97 and 98), miR-203 (SEQ ID NO:99 and 100), miR-211 (SEQ ID NO:101 and 102), miR-145 (SEQ ID NO:103 and 104), let-7b (SEQ ID NO:105 and 106), miR-93 (SEQ ID NO:107 and 108), miR-192 (SEQ ID NO:109 and 110), miR-201 (SEQ ID NO:111 and 112), miR-18a (SEQ ID NO:113 and 114), miR-17-5p (SEQ ID NO:115 and 116), miR-7085 (SEQ ID NO:117), miR-106b (SEQ ID NO:118 and 119), or miR-223 (SEQ ID NO:120 and 121). In certain aspects, a nucleic acid is administered topically, enterally, parenterally or intravitreally. In a further aspect, the nucleic acid molecule is an RNA, DNA, or comprises all or portions of nucleotide analogs or mimetics. An RNA can comprise a complementary RNA region, such as, but not limited to a hairpin structure or multiple (e.g., two) RNA strands. The RNA or DNA molecule can include a nucleotide analog or a modified nucleotide. In still a further aspect, the nucleic acid molecule or miRNA inhibitor is a DNA molecule or is produced from a DNA molecule. An miRNA inhibitor can be an antisense oligonucleotide. The oligonucleotide can comprise all or a portion of nucleotide analogs. In other aspects the DNA is comprised in an expression cassette, such as a plasmid expression vector or a viral expression vector. The nucleic acid molecule can also be comprised in a lipid or viral delivery vehicle.
In certain aspects the subject has, is at risk of developing, or is suspected of having ocular or retinal/choroidal neovascular diseases, cancer, diabetic nephropathy, rheumatoid arthritis, atherosclerotic plaques, endometriosis, Crohn's disease, uterine fibroids, benign prostatic hyperplasia, or psoriasis. The nucleic acids or nucleic acid analogs disclosed herein are useful for treating and/or preventing a variety of angiogenic, microvascular, and macular disorders including macular degeneration (such as wet or neovascular age-related macular degeneration (AMD) and dry or atrophic AMD), macular edema, and secondary indications for inhibiting tumor vascularization, and corneal and iris neovascularization, for example.
A further aspect of the invention includes methods of stimulating vascularization in a subject or tissue comprising administering to a subject in need of such stimulation, in an amount sufficient to stimulate vascularization, one or more nucleic acid molecule comprising (a) a miRNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of miR-451, miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350, miR-21, miR-218, miR-148b, miR-106a, miR-205, miR-365, miR-299-5p, ambi-miR-7079, miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h, miR-142-5p, miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201, miR-18a, miR-17-5p, miR-7085, miR-106b, and/or miR-223; and/or (b) an inhibitor of miR-184, miR-31, miR-150, miR-409, miR-375, miR-129-5p, miR-142a, miR-29a, miR-129-3p, miR-10b, miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c, miR-181c, miR-335, miR-7026, miR-210, miR-512-3p, miR-132, miR-500, miR-339, miR-511, miR-26b, miR-30b, and/or miR-15a. In further aspects of the invention the subject has, is at risk of developing, or is suspected of having coronary artery disease (CAD), cardiac failure, tissue injury, or ischemia.
In yet another aspect the invention includes methods for selecting a vascular therapy for a patient comprising (a) measuring an expression profile of one or more of miR-184, miR-31, miR-150, miR-409, miR-375, miR-129-5p, miR-142a, miR-29a, miR-129-3p, miR-10b, miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c, miR-181c, miR-335, miR-7026, miR-210, miR-512-3p, miR-132, miR-500, miR-339, miR-511, miR-26b, miR-30b, miR-15a, miR-451, miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350, miR-21, miR-218, miR-148b, miR-106a, miR-205, miR-365, miR-299-5p, ambi-miR-7079, miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h, miR-142-5p, miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201, miR-18a, miR-17-5p, miR-7085, miR-106b, and/or miR-223 in a sample; and (b) selecting a therapy based on a comparison of the miRNA expression profile in the patient sample to an expression profile of a normal or non-pathogenic sample, wherein a difference between the expression profiles is indicative of a pathological condition. The altered expression for any of the miRNAs indicates that the patient should be treated with a corresponding therapeutic directed toward the altered miRNA or condition indicated by such altered miRNA.
In some embodiments, it may be useful to know whether a cell expresses a particular miRNA endogenously or whether such expression is affected under particular conditions or when it is in a particular disease state. Thus, in some embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or more miRNA. Consequently, in some embodiments, methods include a step of generating a miRNA profile for a sample. The term “miRNA profile” refers to data regarding the expression pattern of miRNAs in the sample (e.g., one or more miRNA described herein). It is contemplated that the miRNA profile can be obtained using a set of miRNAs, using for example nucleic acid amplification or hybridization techniques well known to one of ordinary skill in the art. In certain embodiments, expression of one or more miRNA described herein is evaluated.
In some embodiments of the invention, an miRNA profile is generated by steps that include one or more of: (a) labeling miRNA in the sample; (b) hybridizing miRNA to a number of probes, or amplifying a number of miRNAs, and/or (c) determining miRNA hybridization to the probes or detecting miRNA amplification products, wherein miRNA expression levels are determined or evaluated. See U.S. Provisional Patent Applications 60/575,743 and 60/649,584, and U.S. patent application Ser. Nos. 11/141,707 and 11/855,792, all of which are hereby incorporated by reference.
Methods of the invention include determining a diagnosis or prognosis for a patient based on miRNA expression or expression levels. In certain embodiments, the elevation or reduction in the level of expression of a particular miRNA or set of miRNAs in a cell is correlated with a disease state as compared to the expression level of that miRNA or set of miRNAs in a normal cell or a reference sample or digital reference. This correlation allows for diagnostic methods to be carried out when the expression level of a miRNA is measured in a biological sample being assessed.
In certain embodiments a method for evaluating a patient includes the steps of determining expression levels of one or more of miR-451, miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350, miR-21, miR-218, miR-148b, miR-106a, miR-205, miR-365, miR-299-5p, ambi-miR-7079, miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h, miR-142-5p, miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201, miR-18a, miR-17-5p, miR-7085, miR-106b, miR-223, miR-184, miR-31, miR-150, miR-409, miR-375, miR-129-5p, miR-142a, miR-29a, miR-129-3p, miR-10b, miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c, miR-181c, miR-335, miR-7026, miR-210, miR-512-3p, miR-132, miR-500, miR-339, miR-511, miR-26b, miR-30b, and/or miR-15a in a biological sample comprising a portion of a tissue or fluid associated with a condition associated with aberrant vascularization, and/or determining a diagnosis or prognosis for the aberrant vascularization condition based on the miRNA expression levels. In a further aspect of the invention, a patient is suspected of having a condition associated with aberrant vascularization. Determining a diagnosis includes, but is not limited to screening for a pathological condition, staging a pathological condition, or assessing response of a pathological condition to therapy. In certain aspects, determining a diagnosis is determining if the patient has a condition associated with aberrant neovascularization or aberrant angiogenesis.
Methods can further comprise normalizing the expression levels of miRNA. Normalizing includes, but is not limited to adjusting expression levels of miRNA relative to expression levels of one or more nucleic acid in the sample.
It is specifically contemplated that miRNA profiles for patients, particularly those suspected of having or at risk of developing a particular disease or condition associated or related to vascularization and/or angiogenesis, can be generated by evaluating any miR or set of miRs discussed in this application. The miRNA profile that is generated from the patient will be one that provides information regarding the particular disease or condition. In certain aspects, a party evaluating miR expression may prepare a recommendation, report and/or summary conveying processed or raw data to a diagnosing physician. In certain aspects, a miRNA profile can be used in conjunction with other diagnostic tests.
Embodiments of the invention include methods for diagnosing, assessing a condition, and/or prognosing a disease or condition associated with or having an accompanying aberrant vascularization in a patient comprising evaluating or determining the expression or expression levels of one or more miRNAs in a sample from the patient. The difference in the expression in the sample from the patient and a reference, such as expression in a normal or non-pathologic sample, is indicative of a pathologic or diseased condition associated with neovascularization and/or angiogenesis. In certain aspects the miRNA expression level is compared to the expression level of a normal cell or a reference sample or a digital reference. Comparing miRNA expression levels includes comparing miRNA expression levels in a sample to miRNA expression levels in a normal tissue sample or reference tissue sample. A normal tissue sample can be taken from the patient being evaluated and can be a normal adjacent tissue to the area being assessed or evaluated.
In certain embodiments the expression of the miRNA is determined by an amplification assay or a hybridization assay. An amplification assay is a quantitative amplification assay, such as, but not limited to quantitative RT-PCR. Hybridization assays include, but are not limited to an array hybridization assay or a solution hybridization assay. An miRNA, amplification product, or probe set can comprise a segment of or be complementary to a corresponding miRNA including all or part of an miRNA sequence described herein. In certain aspects of the invention, a segment can comprise at least or about 5, 6, 7, 8, 9, 10, 11, 12 or more nucleic acid sequences of a miRNA. Other amplification or hybridization sequences may also be included for normalization purposes. The use of an miRNA quantification assay as a clinically relevant diagnostic tool can be enhanced by using an appropriate normalization control. The methods of normalization correct for sample-to-sample variability by comparing a target measurement in a sample to one or more internal controls. Normalization of miRNA quantification assays reduces systematic (non-biological) and non-systematic differences between samples, and can enhance the accurate measurement of differential miRNA expression, for example. The accurate measurement of biologically hardwired differential expression between two groups of samples is the goal of many miRNA qRT-PCR assays. Yet, miRNA levels in qRT-PCR reactions can vary from one sample to the next for reasons that may be technical or biological. Technical reasons may include variability in tissue procurement or storage, inconsistencies in RNA extraction or quantification, or differences in the efficiency of the reverse transcription and/or PCR steps. Biological reasons may include sample-to-sample heterogeneity in cellular populations, differences in bulk transcriptional activity, or alterations in specific miRNA expression that is linked to an aberrant biological program (e.g., a disease state). Given the multiplicity of sources that can contribute to differences in miRNA quantification, results from qRT-PCR assays can be normalized against a relevant endogenous target or targets to minimize controllable variation, and permit definitive interpretations of nominal differences in miRNA expression.
Because conditions related to neovascularization and/or angiogenesis, such as cancer, refer to a class of diseases, it is unlikely that there will be a single treatment and aspects of the invention can be used to determine which treatment will be most effective or most harmful and provide a guide for the physician in evaluating, assessing, and formulating a treatment strategy for a patient. A sample may be taken from a patient having or suspected of having a disease or pathological condition. In certain aspects, the sample can be, but is not limited to tissue (e.g., biopsy, particularly fine needle biopsy), sputum, lavage fluid, blood, serum, plasma, lymph node or other tissue or fluid that may contain cells associated with a neovascular or angiogenic condition. The sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded).
The methods can further comprise or exclude one or more steps including: (a) obtaining a sample from the patient, (b) isolating or obtaining nucleic acids from the sample, (c) reverse transcribing nucleic acids from the sample, (d) labeling the nucleic acids isolated from the sample or an amplification product thereof, (e) hybridizing the labeled nucleic acids to one or more probes or detecting the amplified nucleic acids, (f) analyzing and normalizing data by statistical methods, and/or (g) creating and/or supplying a report of the analysis. Nucleic acids of the invention may include one or more nucleic acids comprising at least one segment having a sequence or complementary sequence of one or more miRNA described herein. In certain aspects, the nucleic acids identify one or more miRNA described herein. Nucleic acids of the invention may be coupled to a support. Such supports are well known to those of ordinary skill in the art and include, but are not limited to glass, plastic, metal, or latex. In particular aspects of the invention, the support can be planar or in the form of a bead or other geometric shapes or configurations.
Embodiments of the invention include kits for analysis of a pathological sample by assessing a miRNA profile for a sample comprising, in suitable container means, one or more miRNA probes and/or amplification primers, wherein the miRNA probes detect or primer amplify one or more miRNA described herein. In certain embodiments the kit contains two or more miRNA hybridization or amplification reagents comprising one or more probe or amplification primer for one or more miRNA selected from miR-451, miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350, miR-21, miR-218, miR-148b, miR-106a, miR-205, miR-365, miR-299-5p, ambi-miR-7079, miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h, miR-142-5p, miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201, miR-18a, miR-17-5p, miR-7085, miR-106b, miR-223, miR-184, miR-31, miR-150, miR-409, miR-375, miR-129-5p, miR-142a, miR-29a, miR-129-3p, miR-10b, miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c, miR-181c, miR-335, miR-7026, miR-210, miR-512-3p, miR-132, miR-500, miR-339, miR-511, miR-26b, miR-30b, and/or miR-15a. The kit can further comprise reagents for labeling miRNA in the sample. The kit may also include labeling reagents comprising at least one amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine-reactive dye.
The present invention also concerns kits containing compositions of the invention or compositions to implement methods of the invention. In some embodiments, kits can be used to evaluate one or more miRNA molecules. In certain embodiments, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more miRNA probes, synthetic miRNA molecules or miRNA inhibitors, or any range and combination derivable therein. In some embodiments, there are kits for evaluating miRNA activity in a cell.
Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.
Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as 1×, 2×, 5×, 10×, or 20× or more, including all values and ranges there between.
Kits for using miRNA probes, synthetic miRNAs, nonsynthetic, and/or miRNA inhibitors of the invention for therapeutic, prognostic, or diagnostic applications are included as part of the invention.
The term “miRNA” or “miR” is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al., 2003, which is hereby incorporated by reference. The term will be used to refer to the single-stranded RNA molecule processed from a precursor. Names of miRNAs and their sequences related to the present invention are provided herein.
Corresponding miRNA sequences that can be used in the context of the invention include, but are not limited to, all or a portion of those sequences in the sequence listing provided herein, as well as the miRNA precursor sequence, or complement of one or more of these miRNAs.
Any embodiment of the invention involving specific miRNAs by name is contemplated also to cover embodiments involving miRNAs whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified miRNA. In other aspects, miRNA of the invention may include additional nucleotides at the 5′, 3′, or both 5′ and 3′ ends of at least, at most or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. It is specifically contemplated that any methods and compositions discussed herein with respect to miRNA molecules or miRNA may be implemented with respect to synthetic miRNAs. Typically, the synthetic miRNA is exposed to the proper conditions to allow it to become or function, at least in part, as a mature miRNA under physiological circumstances.
The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Any embodiment discussed with respect to a particular condition can be applied or implemented with respect to a different condition. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
Throughout this application, the term “about” may be used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIGS. 1A-1B. QRT-PCR quantification of mmu-miR-31, mmu-miR-150, and mmu-miR-184 in mouse retinas undergoing neovascularization. Mean threshold cycle numbers (ΔCT) were determined for RNA from ischemic and control retinas (n=5 each) and the mean difference (ΔΔCT) ranged from about −1.0 (miR-31) to −2.5 (miR-150) (FIG. 1A). Statistical comparisons confirmed that the quantity of each miRNA was significantly reduced in ischemic compared to control retinas (FIG. 1B).
FIG. 2. Putative binding regions for mmu-miR-31, -150, and -184 in 3′-untranslated regions (UTRs) of predicted target mRNAs (SEQ ID NOS:122-134).
FIGS. 3A-3F. Effect of different microRNAs on luciferase activity from reporter genes harboring putative miRNA binding sites. Luciferase reporter assays were performed as described in Example 4. Each bar represents the mean luciferase activity (±standard error of mean) calculated from three experiments. pMIR-Luc-Pdgfb-3′UTR (FIGS. 3A and 3D); pMIR luciferase reporter vector containing 3′-UTR from Pdgfb. pMIR-Luc-HIF1α-3′UTR (FIG. 3C); pMIR luciferase reporter vector containing 3′-UTR from Hif1α. pMIR-Luc-Fz4-3′UTR (FIG. 3B); pMIR luciferase reporter vector containing 3′-UTR from Frizzled4. pGL2-Luc-Vegf-3′UTR (FIG. 3E); pGL2-CMV luciferase reporter vector containing 3′-UTR from Vegf. pMIR-Luc-Notch4-3′UTR (FIG. 3F); pMIR luciferase reporter vector containing 3′-UTR from Notch4.
FIGS. 4A-4B. Effect of microRNAs on target gene product levels in neovascularizing retinas in vivo. (FIG. 4A) VEGF levels in homogenates of experimental miRNA-treated and control miRNA-treated retinas, measured by ELISA. Each bar represents the mean VEGF level (±standard error of mean) calculated from three separate experiments. Asterisks indicate levels of VEGF significantly different from control-treated retinas. (FIG. 4B) HIF-1α, PDGFB, VEGF, and Frizzled4 levels in homogenates of experimental miRNA-treated control miRNA-treated retinas, measured by immunoblot analysis. Each immunoblot was repeated at least once with similar results.
FIGS. 5A-5B. Neovascularization in retinas from mouse eyes following ischemia-induced retinopathy. FIG. 5A. Mouse eyes were injected with negative control miRNA. FIG. 5B. Mouse eyes were injected with a mixture of synthetic mmu-miR-31, -150, and -184. Brightly fluorescent areas are areas of neovascularization.
FIGS. 6A-6D. Effect of microRNAs on ischemia-induced retinal neovascularization. Each bar represents the mean area of neovascularization (±standard deviation) calculated from eight mice. Statistical comparisons were made by paired t-test.
FIGS. 7A-7B. Choroidal neovascularization at Bruch's membrane rupture sites following laser photocoagulation. (FIG. 7A) Mouse eyes were injected with negative control miRNA. (FIG. 7B) Mouse eyes were injected with a mixture of synthetic mmu-miR-31, -150, and -184. Brightly fluorescent areas are areas of neovascularization.
FIGS. 8A-8D. Effect of microRNAs on choroidal neovascularization at Bruch's membrane rupture sites in a mouse model. Each bar represents the mean area of choroidal neovascularization (mm3±standard deviation) calculated from at least twelve experimental values. Statistical comparisons were made by paired t-test.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to compositions and methods relating to preparation and characterization of miRNAs, as well as use of miRNAs for therapeutic, prognostic, and/or diagnostic applications, particularly those methods and compositions related to assessing and/or identifying conditions associated with aberrant or abnormal vascularization and/or angiogenesis.
I. NEOVASCULARIZATION AND ANGIOGENESIS
Neovascularization refers to the proliferation of blood vessels that are atypical for tissues containing them or the proliferation of blood vessels distinct from those that are normally found in the tissue. Neovascularization occurs through a complex process called angiogenesis. Angiogenesis is regulated by a mixture of stimulators and inhibitors that are balanced to ensure the proper development of blood vessels in the body. Mis-regulated angiogenesis is the underlying cause of numerous diseases and a contributor to many more.
Inhibition of angiogenesis can be a useful therapy for diseases such as diabetic nephropathy, rheumatoid arthritis, atherosclerotic plaques, endometriosis, Crohn's disease, uterine fibroids, benign prostatic hyperplasia, psoriasis, and cancer. In 1988, interferon α2a was first used as an antiangiogenic drug to treat children with life-threatening hemangiomas, a nonmalignant vascular tumor (White et al., 1989; Folkman, 1989). Several hundred angiogenesis inhibitors have since been discovered and many of these have been evaluated for therapeutic utility in clinical trials. Many of these compounds have not proved useful for disease therapy. However, some cancer patients have experienced dramatic regression of their tumors from antiangiogenic therapy, and others have experienced stabilization of their disease. The first FDA-approved blood vessel therapy for eye disease was Visudyne (QLT Therapeutics/CibaVision), which has shown effectiveness for treating macular degeneration. Additional angiogenesis inhibitors have since been approved for the treatment of macular degeneration.
In contrast, stimulation of angiogenesis can be a useful therapy for the treatment of coronary artery disease (CAD), cardiac failure, tissue injury, and ischemic diseases such as ischemic CAD, critical limb ischemia with various etiologies, and decubitus. The first FDA-approved device to stimulate growth of new blood vessels in diseased hearts was a laser used in a technique referred to as direct myocardial revascularization (DMR) or transmyocardial revascularization (TMR). The first angiogenesis-stimulating medicine was a prescription gel called Regranex (recombinant human platelet-derived growth factor-BB, Ortho-McNeil Pharmaceuticals) that is approved for treatment of diabetic foot ulcers. Numerous angiogenic growth factors and angiogenic gene therapies are now being developed or tested in humans for growing new blood vessels to heal wounds and for restoring blood flow to the heart, limbs, and brain.
Although differences exist among vascular beds throughout the body, the genes and molecules involved in stimulating or inhibiting neovascularization tend to be common. Animal models for retinal and choroidal neovascularization (CNV) have proved useful in understanding the molecular events leading to blood vessel formation (Campochiaro and Hackett, 2003). Consistent with neovascularization in other tissues, retinal and choroidal neovascularization is stimulated by vascular endothelial growth factor (VEGF) (Aiello et al., 1995; Miller et al., 1994; Ozaki et al., 2000; Kwak et al., 2000). Intraocular injections of ranibizumab, an Fab that binds all isoforms of VEGF-A, resulted in significant visual improvement in 35-40% of patients with CNV caused by age-related macular degeneration (AMD) (Rosenfeld et al., 2006; Brown and Regillo, 2007). While this has benefited many patients with CNV, the remaining 60-65% of CNV patients will need another type of treatment to realize vision improvement.
Transcriptional regulation of many genes is clearly important for determining the balance of stimulators and inhibitors of neovascularization. In addition, mounting evidence now suggests that microRNAs may play a complementary role in regulation of neovascularization. MicroRNAs (miRNAs) are small non-coding RNAs, comprising an evolutionarily conserved class of regulatory nucleic acids ranging in size from 14-35 nucleotides in their mature form. miRNA precursors are processed by cellular proteins, including Drosha and Dicer, to generate a short double-stranded miRNA. One of the miRNA strands is incorporated into a complex of proteins and miRNA called the RNA-induced silencing complex (RISC). The miRNA guides the RISC complex to a target mRNA, which is then cleaved or translationally silenced, depending on the degree of sequence complementarity of the miRNA to its target mRNA (Bagga et al, 2005; Lim et al., 2005). In mice, targeted disruption of Dicer, results in death during the embryonic growth phase, due to disruption of vascular development (Yang et al., 2005). More recently, two miRNAs (miR-221 and miR-222) have been shown to modulate the angiogenic properties of human umbilical vein endothelial cells (Poliseno et al., 2006).
Compositions and methods of the present invention can be used to inhibit neovascularization. Accordingly, the present invention discloses methods of using miRNA or miRNA inhibitor for treating a variety of diseases with a neovascularization component (hereinafter collectively referred to as “neovascularization diseases” or “conditions associated with aberrant vascularization or angiogenesis”) or other similar phrases. In one embodiment, the present invention provides a method of treating pathological conditions resulting from angiogenesis or neovascularization comprising administration of an effective amount of an miRNA or an miRNA inhibitor.
Diseases of the eye treatable using the present invention include, but are not limited to retinopathy of prematurity (ROP), age-related macular degeneration (AMD), diabetic retinopathy, hypertensive retinopathy, central retinal vein occlusion (CRVO), branch vein occlusion (BRVO), neovascular glaucoma, ocular ischemic syndrome, occlusive vasculitis, polypoidal choroidal vasculopathy, myopic choroidal neovascularization, radiation retinopathy, chorioretinitis, central serous choroidopathy, central retinal artery occlusion, uveitic macular edema, idiopathic juxtafoveal telangiectasia, angioid streaks, sickle cell retinopathy, and pseudophakic cystoid macular edema. All of these diseases share the same mechanism of choroidal neovascularization and/or retinal neovascularization, the inhibition of which will result in treatment of the diseases.
Other diseases of the eye treatable by the present invention include, but are not limited to, primary ocular tumors, such as uveal melanomas, melanocytomas, retinocytomas, retinal hamartomas and choristomas, retinal angiomas, retinal gliomas and astrocytomas, choroidal hemangiomas, choroidal neurofibromas, choroidal hamartomas and choristomas, ocular lymphomas and ocular phakomatoses; and metastatic ocular tumors related to choroidal and retinal neovascularization.
In addition, the present invention is suitable for the treatment of cancers such as, but not limited to medulloblastoma, pineoblastoma, non-pineal supratententorial, and Ewings sarcoma.
II. miRNA BASED THERAPY
Embodiments of the invention concern nucleic acids that perform the activities of or inhibit endogenous miRNAs when introduced into cells. In certain aspects, nucleic acids are synthetic or non-synthetic miRNA. Sequence-specific miRNA or miRNA inhibitors can be used to inhibit sequentially or in combination the activities of one or more endogenous miRNAs in cells, as well those genes and associated pathways modulated by the endogenous miRNA.
Methods of the invention include supplying or enhancing the activity of one or more miRNAs in a cell. The present invention also concerns inducing certain cellular characteristics by providing to a cell a particular nucleic acid, such as a specific synthetic miRNA molecule. However, in methods of the invention, the miRNA molecule or miRNA inhibitor need not be synthetic. They may have a sequence that is identical to a naturally occurring miRNA or they may not have any design modifications. In certain embodiments, the miRNA molecule is synthetic, as discussed herein.
The particular nucleic acid molecule provided to the cell is understood to correspond to a particular miRNA in the cell, and thus, the miRNA in the cell is referred to as the “corresponding miRNA.” In situations in which a named miRNA molecule is introduced into a cell, the corresponding miRNA will be understood to be the induced or inhibited miRNA or induced or inhibited miRNA function. It is contemplated, however, that the miRNA molecule introduced into a cell is not a mature miRNA but is capable of becoming or functioning as a mature miRNA under the appropriate physiological conditions. It is contemplated that multiple corresponding miRNAs may be involved. A miRNA may have a minimal adverse effect on a subject or patient while supplying a sufficient therapeutic effect, such as amelioration of a condition, growth inhibition of a cell, death of a targeted cell, alteration of cell phenotype or physiology, slowing of cellular growth, sensitization to a second therapy, sensitization to a particular therapy, and the like. Methods include identifying a cell or patient in need of inducing those cellular characteristics. Also, it will be understood that an amount of a synthetic nucleic acid that is provided to a cell or organism is an “effective amount” or “amount sufficient” for a particular result, which refers to an amount needed (or a sufficient amount) to achieve a desired goal, such as inducing a particular cellular characteristic(s), inhibiting vascularization or stimulating vascularization. In certain embodiments of the methods include providing or introducing to a cell a nucleic acid molecule corresponding to a mature miRNA in the cell in an amount effective to achieve a desired physiological result. Moreover, methods can involve providing synthetic or nonsynthetic miRNA molecules. Furthermore, any method articulated using a list of miRNAs using Markush group language may be articulated without the Markush group language and a disjunctive article (i.e., or) instead, and vice versa.
In some embodiments, there is a method for reducing or inhibiting vascularization comprises introducing into or providing a subject, tissue, or cell an effective amount of a synthetic or nonsynthetic miRNA molecule that corresponds to a miRNA sequence disclosed herein, or a complement or inhibitor thereof.
Certain embodiments of the invention include methods of treating a pathologic condition. In one aspect, the method comprises contacting a target cell with one or more nucleic acid, synthetic miRNA, or miRNA comprising at least one nucleic acid segment having all or a portion of a miRNA sequence. The segment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides or nucleotide analog including all integers there between. In certain aspects, one or more nucleotide of a nucleic acid can be modified. An aspect of the invention includes the modulation of gene expression, miRNA expression or function, or mRNA expression or function within a target subject, tissue, or cell.
Typically, an endogenous gene, miRNA or mRNA is modulated in the cell. In particular embodiments, the nucleic acid sequence comprises at least one segment that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical in nucleic acid sequence to one or more miRNA or gene sequence. Modulation of the expression or processing of an endogenous gene, miRNA, or mRNA can be through modulation of the processing of a mRNA, such processing including transcription, transportation and/or translation in a cell. Modulation may also be effected by the inhibition or enhancement of miRNA activity in a cell, tissue, or organ. Such processing may affect the expression of an encoded product or the stability of the mRNA. In still other embodiments, a nucleic acid sequence can comprise a modified nucleic acid sequence. In certain aspects, one or more miRNA sequence may include or comprise a modified nucleobase or nucleic acid sequence.
It will be understood in methods of the invention that a cell or other biological matter such as an organism (including patients or subjects) can be provided a miRNA or miRNA molecule corresponding to a particular miRNA by administering to the cell or organism a nucleic acid molecule that functions as the corresponding miRNA once inside the cell. In certain embodiments, it is specifically contemplated that the miRNA molecule provided to the biological matter is not a mature miRNA molecule but a nucleic acid molecule that can be processed into the mature miRNA once it is accessible to miRNA processing machinery. The term “nonsynthetic” in the context of miRNA means that the miRNA is not “synthetic,” as defined herein. Furthermore, it is contemplated that in embodiments of the invention that concern the use of synthetic miRNAs, the use of corresponding nonsynthetic miRNAs is also considered an aspect of the invention, and vice versa. It will be understand that the term “providing” an agent is used to include “administering” the agent to a patient.
In certain methods of the invention, there is a further step of administering the selected miRNA modulator to a cell, tissue, organ, or organism (collectively “biological matter”) in need of treatment related to modulation of the targeted miRNA or in need of the physiological or biological results discussed herein (such as with respect to a particular cellular pathway or result like inhibition of vascularization or angiogenesis, or stimulation or promotion of vascularization or angiogenesis). Consequently, in some methods of the invention there is a step of identifying a patient in need of treatment that can be provided by the miRNA modulator(s). It is contemplated that an effective amount of a miRNA modulator can be administered in some embodiments. In particular embodiments, there is a therapeutic benefit conferred on the biological matter, where a “therapeutic benefit” refers to an improvement in the one or more conditions or symptoms associated with a disease or condition or an improvement in the prognosis, duration, or status with respect to the disease. It is contemplated that a therapeutic benefit includes, but is not limited to, a decrease in pain, a decrease in morbidity, a decrease in a symptom. For example, with respect to cancer, it is contemplated that a therapeutic benefit can be inhibition of tumor growth, prevention of metastasis, reduction in number of metastases, inhibition of cancer cell proliferation, induction of cell death in cancer cells, inhibition of angiogenesis near cancer cells, induction of apoptosis of cancer cells, reduction in pain, reduction in risk of recurrence, induction of chemo- or radiosensitivity in cancer cells, prolongation of life, and/or delay of death directly or indirectly related to cancer.
Furthermore, it is contemplated that the miRNA compositions may be provided as part of a therapy to a patient, in conjunction with traditional therapies or preventative agents. Moreover, it is contemplated that any method discussed in the context of therapy may be applied as a preventative measure, particularly in a patient identified to be potentially in need of the therapy or at risk of the condition or disease for which a therapy is needed.
A nucleic acid of the invention can enhance the effect or efficacy of a drug, reduce any side effects or toxicity, modify its bioavailability, and/or decrease the dosage or frequency needed. In certain embodiments, the therapeutic drug is a anti-vascularization, pro-vascularization, and/or a cancer therapeutic. Consequently, in some embodiments, there is a method of treating a patient comprising administering to the patient the therapeutic and an effective amount of a miRNA molecule that improves the efficacy of a second therapeutic or protects non-targeted cells from a detrimental affect of a drug. Therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include but are not limited to, for example, 5-fluorouracil, alemtuzumab, amrubicin, bevacizumab, bleomycin, bortezomib, busulfan, camptothecin, capecitabine, cisplatin (CDDP), carboplatin, cetuximab, chlorambucil, cisplatin (CDDP), EGFR inhibitors (gefitinib and cetuximab), procarbazine, mechlorethamine, cyclophosphamide, camptothecin, COX-2 inhibitors (e.g., celecoxib), cyclophosphamide, cytarabine) ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, dasatinib, daunorubicin, dexamethasone, docetaxel, doxorubicin (adriamycin), EGFR inhibitors (gefitinib and cetuximab), erlotinib, estrogen receptor binding agents, bleomycin, plicomycin, mitomycin, etoposide (VP16), everolimus, tamoxifen, raloxifene, estrogen receptor binding agents, taxol, taxotere, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, gefitinib, gemcitabine, gemtuzumab, ibritumomab, ifosfamide, imatinib mesylate, larotaxel, lapatinib, lonafarnib, mechlorethamine, melphalan, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, mitomycin, navelbine, nitrosurea, nocodazole, oxaliplatin, paclitaxel, plicomycin, procarbazine, raloxifene, rituximab, sirolimus, sorafenib, sunitinib, tamoxifen, taxol, taxotere, temsirolimus, tipifarnib, tositumomab, transplatinum, trastuzumab, vinblastin, vincristin, or vinorelbine or any analog or derivative variant of the foregoing.
III. NUCLEIC ACIDS
The present invention concerns nucleic acids, modified nucleic acids, nucleic acid mimetics, miRNAs, and segments thereof that can be employed in therapeutic applications, particularly those applications related to pathological conditions. The molecules may have been endogenously produced by a cell and isolated, or synthesized or produced chemically or recombinantly. They may be isolated and/or purified. Each of the miRNAs described herein includes the corresponding SEQ ID NO and accession numbers for these miRNA sequences. The name of a miRNA is often abbreviated and referred to without a “hsa-” prefix and will be understood as such, depending on the context. Unless otherwise indicated, miRNAs referred to in the application are human sequences identified as miR-X or let-X, where X is a number and/or letter.
In certain aspects, a miRNA designated by a suffix “5P” or “3P” can be used. “5P” indicates that the mature miRNA derives from the 5′ end of the precursor and a corresponding “3P” indicates that it derives from the 3′ end of the precursor, as described on the world wide web at sanger.ac.uk. Moreover, in some embodiments, a miRNA probe is used that does not correspond to a known human miRNA. It is contemplated that these non-human miRNA probes may be used in embodiments of the invention or that there may exist a human miRNA that is homologous to the non-human miRNA. In other embodiments, any mammalian cell, biological sample, or preparation thereof may be employed.
The present invention concerns, in some embodiments, short nucleic acid molecules that function as miRNAs in a cell. The term “short” refers to a length of a single polynucleotide that is at least, at most, or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 100, or 150 nucleotides, including all integers or ranges derivable there between. The nucleic acid molecules are typically synthetic. The term “synthetic” refers to a nucleic acid molecule that is isolated and not produced naturally in a cell. In certain aspects the sequence (the entire sequence) and/or chemical structure deviates from a naturally-occurring nucleic acid molecule, such as an endogenous precursor miRNA or miRNA molecule or complement thereof. While in some embodiments, nucleic acids of the invention do not have an entire sequence that is identical or complementary to a sequence of a naturally-occurring nucleic acid, such molecules may encompass all or part of a naturally-occurring sequence or a complement thereof. It is contemplated, however, that a synthetic nucleic acid administered to a cell may subsequently be modified or altered in the cell such that its structure or sequence is the same as non-synthetic or naturally occurring nucleic acid, such as a mature miRNA sequence. For example, a synthetic nucleic acid may have a sequence that differs from the sequence of a precursor miRNA, but that sequence may be altered once in a cell to be the same as an endogenous, processed miRNA or an inhibitor thereof.
The term “isolated” means that the nucleic acid molecules of the invention are initially separated from different (in terms of sequence or structure) and unwanted nucleic acid molecules such that a population of isolated nucleic acids is at least about 90% homogenous, and may be at least about 95, 96, 97, 98, 99, or 100% homogenous with respect to other polynucleotide molecules. In many embodiments of the invention, a nucleic acid is isolated by virtue of it having been synthesized in vitro separate from endogenous nucleic acids in a cell. It will be understood, however, that isolated nucleic acids may be subsequently mixed or pooled together. In certain aspects, synthetic miRNA of the invention are RNA or RNA analogs. miRNA inhibitors may be DNA or RNA, or analogs thereof.
In some embodiments, there is a miRNA or a synthetic miRNA having a length of between 10 and 130 residues. The present invention concerns miRNA or synthetic miRNA molecules that are, are at least, or are at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 140, 145, 150, 160, 170, 180, 190, 200 or more residues in length, including any integer or any range there between.
In certain embodiments, synthetic miRNA have (a) a “miRNA region” whose sequence or binding region from 5′ to 3′ is identical or complementary to all or a segment of a mature miRNA sequence, and (b) a “complementary region” whose sequence from 5′ to 3′ is between 60% and 100% complementary to the miRNA sequence in (a). In certain embodiments, these synthetic miRNA are also isolated, as defined above. The term “miRNA region” or complement thereof refers to a region on the synthetic miRNA that is at least 75, 80, 85, 90, 95, or 100% identical, including all integers there between, to the entire sequence of a mature, naturally occurring miRNA sequence or a complement thereof. In certain embodiments, the miRNA region is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% identical to the sequence of a naturally-occurring miRNA or complement thereof. In certain aspects, a double stranded RNA can comprise a miR sequence that is 90 to 100% identical to sequences described herein, as described directly above, and a second nucleic acid that is complementary to the miR sequence and is 60, 65, 70, 75, 80, 85, 90, 95, or 100% identical, including all integers there between, to the miR sequence.
The term “complementary region” or “complement” refers to a region of a nucleic acid or mimetic that is or is at least 60% complementary to the mature, naturally occurring miRNA sequence. The complementary region can be at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary including all values and ranges there between. With single polynucleotide sequences, there may be a hairpin loop structure as a result of chemical bonding between the miRNA region and the complementary region. In other embodiments, the complementary region is on a different nucleic acid molecule than the miRNA region, in which case the complementary region is on the complementary strand and the miRNA region is on the active strand.
In some embodiments of the invention a synthetic miRNA contains one or more design element(s). These design elements include, but are not limited to: (i) a replacement group for the phosphate or hydroxyl of the nucleotide at the 5′ terminus of the complementary region; (ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region; or, (iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3′ end of the complementary region and the corresponding nucleotides of the miRNA region. A variety of design modifications are known in the art, see below.
In certain embodiments, a synthetic miRNA has a nucleotide at its 5′ end of the complementary region in which the phosphate and/or hydroxyl group has been replaced with another chemical group (referred to as the “replacement design”). In some cases, the phosphate group is replaced, while in others, the hydroxyl group has been replaced. In particular embodiments, the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2′O-Me (2′ oxygen-methyl), DMTO (4,4′-dimethoxytrityl with oxygen), fluoroscein, a thiol, or acridine, though other replacement groups are well known to those of skill in the art and can be used as well. This design element can also be used with a miRNA inhibitor.
Additional embodiments concern a synthetic miRNA having one or more sugar modifications in the first or last 1 to 6 residues of the complementary region (referred to as the “sugar replacement design”). In certain cases, there is one or more sugar modifications in the first 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein. In additional cases, there are one or more sugar modifications in the last 1, 2, 3, 4, 5, 6 or more residues of the complementary region, or any range derivable therein, have a sugar modification. It will be understood that the terms “first” and “last” are with respect to the order of residues from the 5′ end to the 3′ end of the region. In particular embodiments, the sugar modification is a 2′O-Me modification, a 2° F. modification, a 2′H modification, a 2′amino modification, a 4′thioribose modification or a phosphorothioate modification on the carboxy group linked to the carbon at position 6′. In further embodiments, there are one or more sugar modifications in the first or last 2 to 4 residues of the complementary region or the first or last 4 to 6 residues of the complementary region. This design element can also be used with a miRNA inhibitor. Thus, a miRNA inhibitor can have this design element and/or a replacement group on the nucleotide at the 5′ terminus, as discussed above.
In other embodiments of the invention, there is a synthetic miRNA in which one or more nucleotides in the last 1 to 5 residues at the 3′ end of the complementary region are not complementary to the corresponding nucleotides of the miRNA region (“noncomplementarity”) (referred to as the “noncomplementarity design”). The noncomplementarity may be in the last 1, 2, 3, 4, and/or 5 residues of the complementary miRNA. In certain embodiments, there is noncomplementarity with at least 2 nucleotides in the complementary region.
It is contemplated that synthetic miRNA of the invention have one or more of the replacement, sugar modification, or noncomplementarity designs. In certain cases, synthetic RNA molecules have two of them, while in others these molecules have all three designs in place.
The miRNA region and the complementary region may be on the same or separate polynucleotides. In cases in which they are contained on or in the same polynucleotide, the miRNA molecule will be considered a single polynucleotide. In embodiments in which the different regions are on separate polynucleotides, the synthetic miRNA will be considered to be comprised of two polynucleotides.
When the RNA molecule is a single polynucleotide, there can be a linker region between the miRNA region and the complementary region. In some embodiments, the single polynucleotide is capable of forming a hairpin loop structure as a result of bonding between the miRNA region and the complementary region. The linker constitutes the hairpin loop. It is contemplated that in some embodiments, the linker region is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues in length, or any range derivable therein. In certain embodiments, the linker is between 3 and 30 residues (inclusive) in length.
In addition to having a miRNA region and a complementary region, there may be flanking sequences as well at either the 5′ or 3′ end of the region. In some embodiments, there is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more, or any range derivable therein, flanking one or both sides of these regions.
In some embodiments of the invention, methods and compositions involving miRNA may concern nucleic acids comprising miRNA nucleotide sequences. Nucleic acids may be, be at least, or be at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides, or any range derivable therein, in length. Such lengths cover the lengths of processed miRNA, precursor miRNA, miRNA containing vectors, and therapeutic miRNA. In many embodiments, miRNA are 14-35 nucleotides in length. miRNA precursors are generally between 62 and 10 nucleotides in humans.
It is understood that some nucleic acids are derived from genomic sequences or a gene. In this respect, the term “gene” is used for simplicity to refer to the genomic sequence encoding the precursor nucleic acid or miRNA for a given miRNA or gene. However, embodiments of the invention may involve genomic sequences of a miRNA that are involved in its expression, such as a promoter or other regulatory sequences.
The term “recombinant” may be used and this generally refers to a molecule that has been manipulated in vitro or that is a replicated or expressed product of such a molecule.
The term “nucleic acid” is well known in the art. A “nucleic acid” as used herein will generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” or a C). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
The term “miRNA” generally refers to a single-stranded molecule, but in specific embodiments, molecules implemented in the invention will also encompass a region or an additional strand that is partially (between 10 and 50% complementary across length of strand), substantially (greater than 50% but less than 100% complementary across length of strand) or fully complementary to another region of the same single-stranded molecule or to another nucleic acid. Thus, miRNA nucleic acids may encompass a molecule that comprises one or more complementary or self-complementary strand(s) or “complement(s)” of a particular sequence. For example, precursor miRNA may have a self-complementary region, which is up to 100% complementary. miRNA probes or nucleic acids of the invention can include, can be or can be at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% complementary to their target.
It is understood that a “synthetic nucleic acid” of the invention means that the nucleic acid does not have all or part of a chemical structure or sequence of a naturally occurring nucleic acid or was made by man and not a biologic cell or organism. Consequently, it will be understood that the term “synthetic miRNA” refers to a “synthetic nucleic acid” that functions in a cell or under physiological conditions as a naturally occurring miRNA.
While embodiments of the invention may involve synthetic miRNAs or synthetic nucleic acids, in some embodiments of the invention, the nucleic acid molecule(s) need not be “synthetic.” In certain embodiments, a non-synthetic nucleic acid or miRNA employed in methods and compositions of the invention may have the entire sequence and structure of a naturally occurring mRNA or miRNA precursor or the mature mRNA or miRNA. For example, non-synthetic miRNAs used in methods and compositions of the invention may not have one or more modified nucleotides or nucleotide analogs. In these embodiments, the non-synthetic miRNA may or may not be recombinantly produced. In particular embodiments, the nucleic acid in methods and/or compositions of the invention is specifically a synthetic miRNA and not a non-synthetic miRNA (that is, not a miRNA that qualifies as “synthetic”); though in other embodiments, the invention specifically involves a non-synthetic miRNA and not a synthetic miRNA. Any embodiments discussed with respect to the use of synthetic miRNAs can be applied with respect to non-synthetic miRNAs, and vice versa.
It will be understood that the term “naturally occurring” refers to something found in an organism without any intervention by a person; it could refer to a naturally-occurring wildtype or mutant molecule. In some embodiments a synthetic miRNA molecule does not have the sequence of a naturally occurring miRNA molecule. In other embodiments, a synthetic miRNA molecule may have the sequence of a naturally occurring miRNA molecule, but the chemical structure of the molecule, particularly in the part unrelated specifically to the precise sequence (non-sequence chemical structure) differs from chemical structure of the naturally occurring miRNA molecule with that sequence. In some cases, the synthetic miRNA has both a sequence and non-sequence chemical structure that are not found in a naturally-occurring miRNA. Moreover, the sequence of the synthetic molecules will identify which miRNA is effectively being provided; the endogenous miRNA will be referred to as the “corresponding miRNA.” Corresponding miRNA sequences that can be used in the context of the invention include, but are not limited to, all or a portion of those sequences in the SEQ IDs provided herein, as well as any other miRNA sequence, miRNA precursor sequence, or any sequence complementary thereof. In some embodiments, the sequence is or is derived from or contains all or part of a sequence identified herein to target a particular miRNA (or set of miRNAs) that can be used with that sequence. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or any number or range of sequences there between may be selected to the exclusion of all non-selected sequences.
1. Nucleobase, Nucleoside, Nucleotide, and Modified Nucleotides
As used herein a “nucleobase” refers to a heterocyclic base, such as for example a naturally occurring nucleobase (i.e., an A, T, G, C or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), and naturally or non-naturally occurring derivative(s) and analogs of such a nucleobase. A nucleobase generally can form one or more hydrogen bonds (“anneal” or “hybridize”) with at least one naturally occurring nucleobase in a manner that may substitute for naturally occurring nucleobase pairing (e.g., the hydrogen bonding between A and T, G and C, and A and U).
“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurring purine and/or pyrimidine nucleobases and also derivative(s) and analog(s) thereof, including but not limited to, those a purine or pyrimidine substituted by one or more of an alkyl, carboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiol moiety. Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.) moieties comprise of from about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms. Other non-limiting examples of a purine or pyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil, a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, a bromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a 8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a 5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a 5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, a methylthioadenine, a N,N-diemethyladenine, an azaadenines, a 8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a 6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like. Other examples are well known to those of skill in the art.
As used herein, a “nucleoside” refers to an individual chemical unit comprising a nucleobase covalently attached to a nucleobase linker moiety. A non-limiting example of a “nucleobase linker moiety” is a sugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), including but not limited to a deoxyribose, a ribose, an arabinose, or a derivative or an analog of a 5-carbon sugar. Non-limiting examples of a derivative or an analog of a 5-carbon sugar include a 2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon is substituted for an oxygen atom in the sugar ring. Different types of covalent attachment(s) of a nucleobase to a nucleobase linker moiety are known in the art (Kornberg and Baker, 1992).
As used herein, a “nucleotide” refers to a nucleoside further comprising a “backbone moiety”. A backbone moiety generally covalently attaches a nucleotide to another molecule comprising a nucleotide, or to another nucleotide to form a nucleic acid. The “backbone moiety” in naturally occurring nucleotides typically comprises a phosphorus moiety, which is covalently attached to a 5-carbon sugar. The attachment of the backbone moiety typically occurs at either the 3′- or 5′-position of the 5-carbon sugar. However, other types of attachments are known in the art, particularly when a nucleotide comprises derivatives or analogs of a naturally occurring 5-carbon sugar or phosphorus moiety.
A nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid. RNA with nucleic acid analogs may also be labeled according to methods of the invention. As used herein a “derivative” refers to a chemically modified or altered form of a naturally occurring molecule, while the terms “mimic” or “analog” refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions. As used herein, a “moiety” generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleobase, nucleoside and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).
Additional non-limiting examples of nucleosides, nucleotides or nucleic acids include those in: U.S. Pat. Nos. 5,681,947, 5,652,099 and 5,763,167, 5,614,617, 5,670,663, 5,872,232, 5,859,221, 5,446,137, 5,886,165, 5,714,606, 5,672,697, 5,466,786, 5,792,847, 5,223,618, 5,470,967, 5,378,825, 5,777,092, 5,623,070, 5,610,289, 5,602,240, 5,858,988, 5,214,136, 5,700,922, 5,708,154, 5,728,525, 5,637,683, 6,251,666, 5,480,980, and 5,728,525, each of which is incorporated herein by reference in its entirety.
Labeling methods and kits of the invention specifically contemplate the use of nucleotides that are both modified for attachment of a label and can be incorporated into a miRNA molecule. Such nucleotides include those that can be labeled with a dye, including a fluorescent dye, or with a molecule such as biotin. Labeled nucleotides are readily available; they can be acquired commercially or they can be synthesized by reactions known to those of skill in the art.
Modified nucleotides for use in the invention are not naturally occurring nucleotides, but instead, refer to prepared nucleotides that have a reactive moiety on them. Specific reactive functionalities of interest include: amino, sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate, isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono- or dihalogen substituted pyridine, mono- or disubstituted diazine, maleimide, epoxide, aziridine, sulfonyl halide, acid halide, alkyl halide, aryl halide, alkylsulfonate, N-hydroxysuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde, iodoacetyl, cyanomethyl ester, p-nitrophenyl ester, o-nitrophenyl ester, hydroxypyridine ester, carbonyl imidazole, and the other such chemical groups. In some embodiments, the reactive functionality may be bonded directly to a nucleotide, or it may be bonded to the nucleotide through a linking group. The functional moiety and any linker cannot substantially impair the ability of the nucleotide to be added to the miRNA or to be labeled. Representative linking groups include carbon containing linking groups, typically ranging from about 2 to 18, usually from about 2 to 8 carbon atoms, where the carbon containing linking groups may or may not include one or more heteroatoms, e.g. S, O, N etc., and may or may not include one or more sites of unsaturation. Of particular interest in many embodiments is alkyl linking groups, typically lower alkyl linking groups of 1 to 16, usually 1 to 4 carbon atoms, where the linking groups may include one or more sites of unsaturation. The functionalized nucleotides (or primers) used in the above methods of functionalized target generation may be fabricated using known protocols or purchased from commercial vendors, e.g., Sigma, Roche, Ambion, Biosearch Technologies and NEN. Functional groups may be prepared according to ways known to those of skill in the art, including the representative information found in U.S. Pat. Nos. 4,404,289; 4,405,711; 4,337,063 and 5,268,486, and U.K. Patent 1,529,202, which are all incorporated by reference.
Amine-modified nucleotides are used in several embodiments of the invention. The amine-modified nucleotide is a nucleotide that has a reactive amine group for attachment of the label. It is contemplated that any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T, or C) can be modified for labeling. Examples include, but are not limited to, the following modified ribo- and deoxyribo-nucleotides: 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP; 8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP; N6-(4-amino)butyl-dATP, N6-(6-amino)butyl-dATP, N4-[2,2-oxy-bis-(ethylamine)]-dCTP; N6-(6-Amino)hexyl-dATP; 8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and 5-propargylamino-dUTP. Such nucleotides can be prepared according to methods known to those of skill in the art. Moreover, a person of ordinary skill in the art could prepare other nucleotide entities with the same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP, dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.
B. Preparation of Nucleic Acids
A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production, or biological production. It is specifically contemplated that miRNA probes of the invention are chemically synthesized.
In some embodiments of the invention, miRNAs are recovered or isolated from a biological sample. The miRNA may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small RNA molecules such as miRNA. U.S. patent application Ser. No. 10/667,126 describes such methods and it is specifically incorporated by reference herein. Generally, methods involve lysing cells with a solution having guanidinium and a detergent.
Alternatively, nucleic acid synthesis is performed according to standard methods. See, for example, Itakura and Riggs (1980) and U.S. Pat. Nos. 4,704,362, 5,221,619, and 5,583,013, each of which is incorporated herein by reference. Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide), include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, each incorporated herein by reference. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
A non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Pat. Nos. 4,683,202 and 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Pat. No. 5,645,897, incorporated herein by reference. See also Sambrook et al., 2001, incorporated herein by reference).
Oligonucleotide synthesis is well known to those of skill in the art. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
Recombinant methods for producing nucleic acids in a cell are well known to those of skill in the art. These include the use of vectors (viral and non-viral), plasmids, cosmids, and other vehicles for delivering a nucleic acid to a cell, which may be the target cell (e.g., a cancer cell) or simply a host cell (to produce large quantities of the desired RNA molecule). Alternatively, such vehicles can be used in the context of a cell free system so long as the reagents for generating the RNA molecule are present. Such methods include those described in Sambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporated by reference.
C. Isolation of Nucleic Acids
Nucleic acids may be isolated using techniques well known to those of skill in the art, though in particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating RNA molecules can be employed. Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography. If miRNA from cells is to be used or evaluated, methods generally involve lysing the cells with a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.
In particular methods for separating miRNA from other nucleic acids, a gel matrix is prepared using polyacrylamide, though agarose can also be used. The gels may be graded by concentration or they may be uniform. Plates or tubing can be used to hold the gel matrix for electrophoresis. Usually one-dimensional electrophoresis is employed for the separation of nucleic acids. Plates are used to prepare a slab gel, while the tubing (glass or rubber, typically) can be used to prepare a tube gel. The phrase “tube electrophoresis” refers to the use of a tube or tubing, instead of plates, to form the gel. Materials for implementing tube electrophoresis can be readily prepared by a person of skill in the art or purchased, such as from C.B.S. Scientific Co., Inc. or Scie-Plas.
Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids, particularly miRNA used in methods and compositions of the invention. Some embodiments are described in U.S. patent application Ser. No. 10/667,126, which is hereby incorporated by reference. Generally, this disclosure provides methods for efficiently isolating small RNA molecules from cells comprising: adding an alcohol solution to a cell lysate and applying the alcohol/lysate mixture to a solid support before eluting the RNA molecules from the solid support. In some embodiments, the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well. A solid support may be any structure, and it includes beads, filters, and columns, which may include a mineral or polymer support with electronegative groups. A glass fiber filter or column has worked particularly well for such isolation procedures.
In specific embodiments, miRNA isolation processes include: (a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced; (b) extracting miRNA molecules from the lysate with an extraction solution comprising phenol; (c) adding to the lysate an alcohol solution for forming a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; (d) applying the lysate/alcohol mixture to a solid support; (e) eluting the miRNA molecules from the solid support with an ionic solution; and, (f) capturing the miRNA molecules. Typically the sample is dried and resuspended in a liquid and volume appropriate for subsequent manipulation.
IV. PHARMACEUTICAL FORMULATIONS AND DELIVERY
Methods of the present invention include the delivery of an effective amount of a miRNA or an expression construct encoding the same. An “effective amount” of the pharmaceutical composition, generally, is defined as that amount sufficient to detectably achieve a desired result, for example, to ameliorate, reduce, minimize or limit the extent of a disease or its symptoms. Other more rigorous definitions may apply, including elimination, eradication or cure of disease.
A. Administration
In certain embodiments, it is desired to inhibit vascularization, stimulate vascularization, kill cells, inhibit cell growth, inhibit metastasis, decrease tumor or tissue size, and/or reverse or reduce the malignant or disease phenotype of cells. The routes of administration will vary, naturally, with the location and nature of the lesion or site to be targeted, and include, e.g., intradermal, subcutaneous, regional, parenteral, intravenous, intramuscular, intranasal, systemic, and oral administration and formulation. Direct injection, local injection, or injection into vasculature at a target site is specifically contemplated for target areas. Local, regional, or systemic administration also may be appropriate.
Multiple injections delivered as a single dose comprise about 0.1 to about 0.5 ml volumes. Compositions of the invention may be administered in multiple injections to a tumor or a targeted site. In certain aspects, injections may be spaced at approximately 1 cm intervals.
In the case of surgical intervention, the present invention may be used preoperatively, to render an inoperable target area subject to resection. Alternatively, the present invention may be used at the time of surgery, and/or thereafter, to treat residual or recurring disease. For example, a resected tumor bed may be injected or perfused with a formulation comprising a miRNA or combinations thereof. Administration may be continued post-resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment also is envisioned. Continuous perfusion of an expression construct or a viral construct also is contemplated.
Continuous administration also may be applied where appropriate, for example, where a tumor or other undesired affected area is excised and the tumor bed or targeted site is treated to eliminate residual, microscopic disease. Delivery via syringe or catherization is contemplated. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.
Treatment regimens may vary as well and often depend on lesion type, tumor type, location, immune condition, target site, disease progression, and health and age of the patient. Certain tumor types will require more aggressive treatment. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.
In certain embodiments, the tumor or affected area being treated may not, at least initially, be resectable or operable. Treatments with compositions of the invention may increase the resectability of the tumor or target site due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional treatments subsequent to resection may serve to eliminate microscopic residual disease at the tumor or targeted site.
Treatments may include various “unit doses.” A unit dose is defined as containing a predetermined quantity of a therapeutic composition(s). The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. With respect to a viral component of the present invention, a unit dose may conveniently be described in terms of μg or mg of miRNA or miRNA mimetic. Alternatively, the amount specified may be the amount administered as the average daily, average weekly, or average monthly dose.
miRNA can be administered to the patient in a dose or doses of about or of at least about 0.005, 0.05, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 μg, ng, or mg, or more, or any range derivable therein. Alternatively, the amount specified may be the amount administered as the average daily, average weekly, or average monthly dose, or it may be expressed in terms of mg/kg, where kg refers to the weight of the patient and the mg is specified above. In other embodiments, the amount specified is any number discussed above but expressed as mg/m2 (with respect to tumor size or patient surface area).
B. Injectable Compositions and Formulations
In some embodiments, the method for the delivery of a miRNA or an expression construct encoding such or combinations thereof is via systemic administration. However, the pharmaceutical compositions disclosed herein may also be administered topically, parenterally, subcutaneously, directly, intratracheally, intravenously, intradermally, intramuscularly, or even intraperitoneally as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety).
Injection of nucleic acids may be delivered by syringe or any other method used for injection of a solution, as long as the nucleic acid and any associated components can pass through the particular gauge of needle required for injection. A syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Pat. No. 5,846,225).
Solutions of the active compounds are typically sterile and must be fluid to the extent that easy syringability exists. It must be sufficiently stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
In certain formulations, a water-based formulation is employed while in others, it may be lipid-based. In particular embodiments of the invention, a composition comprising a tumor suppressor protein or a nucleic acid encoding the same is in a water-based formulation. In other embodiments, the formulation is lipid based.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, intralesional, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.
As used herein, a “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
The nucleic acid(s) are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g., the aggressiveness of the disease or cancer, the size of any tumor(s) or lesions, the previous or other courses of treatment. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and subsequent administration are also variable, but are typified by an initial administration followed by other administrations. Such administration may be systemic, as a single dose, continuous over a period of time spanning 10, 20, 30, 40, 50, 60 minutes, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and/or 1, 2, 3, 4, 5, 6, 7, days or more. Moreover, administration may be through a time release or sustained release mechanism, implemented by formulation and/or mode of administration.
C. Combination Treatments
In certain embodiments, the compositions and methods of the present invention involve a miRNA, or miRNA inhibitor, or expression construct encoding such. These miRNA compositions can be used in combination with a second therapy to enhance the effect of the miRNA therapy, or increase the therapeutic effect of another therapy being employed. These compositions would be provided in a combined amount effective to achieve the desired effect, such as inhibiting or stimulating vascularization, killing of a cancer cell and/or inhibition of cellular hyperproliferation. This process may involve contacting the cells with the miRNA or second therapy at the same or different time. This may be achieved by contacting the cell with one or more compositions or pharmacological formulation that includes or more of the agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition provides (1) miRNA; and/or (2) a second therapy. A second composition or method may be administered that includes an antiangiogenic or pro-angiogenic therapy, chemotherapy, radiotherapy, surgical therapy, immunotherapy, or gene therapy.
It is contemplated that one may provide a patient with the miRNA therapy and the second therapy within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
In certain embodiments, a course of treatment will last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It is contemplated that one agent may be given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof, and another agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no treatment is administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.
Various combinations may be employed, for example miRNA therapy is “A” and a second therapy is “B”:
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Administration of any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the vector or any protein or other agent. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy. Cancer therapeutics can involve the application of multiple targeted therapies that include Tarceva, Iressa, Hierceptin, and other targeted therapeutics along with radiation and chemotherapies.
In specific aspects, it is contemplated that a second therapy, such as chemotherapy, radiotherapy, immunotherapy, surgical therapy or other gene therapy, is employed in combination with the miRNA therapy, as described herein.
1. Antiangiogenic Therapy
Antiangiogenic therapy includes, but is not limited to Macugen, Visudyne, and Photodynamic Therapy (PDT) for macular degeneration and other diseases of the eye.
Angiogenesis inhibitors which inhibit angiogenesis in treated tissues can be used in the compositions and methods of the invention. Angiogenesis inhibitors include αv antagonist, and in particular a αvβ3 antagonist. An angiogenesis inhibiting (anti-angiogenesis) αvβ3 antagonist can be a peptide, a RGD-containing peptide, an anti-αvβ33 antibody, an anti-αvβ3 receptor antibody, or an αvβ3 mimetic. Exemplary antiangiogenic substances are described in the teachings of U.S. Pat. Nos. 5,753,230, 5,766,591, and U.S. Patent publications 20080039384, 20080014196, 20080096795, 20080090750, International Publication No. WO 97/45137, the disclosures of which are specifically incorporated herein by reference.
Other compounds have been identified on the basis of the ability for the compound to inhibit angiogenesis and include but are not limited to prostate specific antigen (PSA); soluble VEGFR-1 and NRP-1; Angiopoietin 2; TSP-1 and TSP-2; angiostatin and related molecules; endostatin; vasostatin; calreticulin; platelet factor-4; TIMP and CDAI; Meth-1 and Meth-2; IFN-α, -β and -γ; CXCL10; IL-4, -12 and -18; prothrombin (kringle domain-2); antithrombin III fragment; prolactin; VEGI; SPARC; osteopontin; maspin; canstatin; proliferin-related protein; restin; bevacizumab; carboxyamidotriazole; TNP-470; CM10; suramin; SU5416; thrombospondin; VEGFR antagonists; angiostatic steroids+heparin; Cartilage-Derived Angiogenesis Inhibitory Factor; matrix metalloproteinase inhibitors; 2-methoxyestradiol; tecogalan; thrombospondin; prolactin; αvβ3 inhibitors; and linomide.
2. Chemotherapy
A wide variety of chemotherapeutic agents may be used in accordance with the present invention. The term “chemotherapy” refers to the use of drugs to treat cancer or other hyperproliferative diseases. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer or other hyperproliferative disease. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
a. Alkylating Agents
Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific. Alkylating agents can be implemented to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary. They include: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan. Troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents.
b. Antimetabolites
Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have been used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.
5-Fluorouracil (5-FU) has the chemical name of 5-fluoro-2,4(1H,3H)-pyrimidinedione. Its mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid. Thus, 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers.
c. Antitumor Antibiotics
Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers. Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), and idarubicin, some of which are discussed in more detail below. Widely used in clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to 35-100 mg/m2 for etoposide intravenously or orally.
d. Mitotic Inhibitors
Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors comprise docetaxel, etoposide (VP16), paclitaxel, taxol, taxotere, vinblastine, vincristine, and vinorelbine.
e. Nitrosureas
Nitrosureas, like alkylating agents, inhibit DNA repair proteins. They are used to treat non-Hodgkin's lymphomas, multiple myeloma, malignant melanoma, in addition to brain tumors. Examples include carmustine and lomustine.
3. Radiotherapy
Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair and function properly. Radiotherapy may be used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, or cervix. It can also be used to treat leukemia and lymphoma (cancers of the blood-forming cells and lymphatic system, respectively).
Radiation therapy used according to the present invention may include, but is not limited to, the use of γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.
4. Immunotherapy
In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (HerceptinTM) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a target cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a target cell target. Various effector cells include cytotoxic T cells and NK cells.
In one aspect of immunotherapy, the tumor or disease cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, and chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.
Examples of immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy, e.g., interferons α, β and γ; IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998) gene therapy e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) and monoclonal antibodies e.g., anti-ganglioside GM2, anti-HER-2, anti-p185; Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). A non-limiting list of several known anti-cancer immunotherapeutic agents and their targets includes, but is not limited to (Generic Name (Target)) Cetuximab (EGFR), Panitumumab (EGFR), Trastuzumab (erbB2 receptor), Bevacizumab (VEGF), Alemtuzumab (CD52), Gemtuzumab ozogamicin (CD33), Rituximab (CD20), Tositumomab (CD20), Matuzumab (EGFR), Ibritumomab tiuxetan (CD20), Tositumomab (CD20), HuPAM4 (MUC1), MORAb-009 (Mesothelin), G250 (carbonic anhydrase IX), mAb 8H9 (8H9 antigen), M195 (CD33), Ipilimumab (CTLA4), HuLuc63 (CS1), Alemtuzumab (CD53), Epratuzumab (CD22), BC8 (CD45), HuJ591 (Prostate specific membrane antigen), hA20 (CD20), Lexatumumab (TRAIL receptor-2), Pertuzumab (HER-2 receptor), Mik-beta-1 (IL-2R), RAV12 (RAAG12), SGN-30 (CD30), AME-133v (CD20), HeFi-1 (CD30), BMS-663513 (CD137), Volociximab (anti-Q5 μl integrin), GC1008 (TGFβ), HCD122 (CD40), Siplizumab (CD2), MORAb-003 (Folate receptor alpha), CNTO 328 (IL-6), MDX-060 (CD30), Ofatumumab (CD20), or SGN-33 (CD33). It is contemplated that one or more of these therapies may be employed with the miRNA therapies described herein.
A number of different approaches for passive immunotherapy of cancer exist. They may be broadly categorized into the following: injection of antibodies alone; injection of antibodies coupled to toxins or chemotherapeutic agents; injection of antibodies coupled to radioactive isotopes; injection of anti-idiotype antibodies; and finally, purging of tumor cells in bone marrow.
5. Gene Therapy
In yet another embodiment, a combination treatment involves gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as one or more therapeutic miRNA. Delivery of a therapeutic polypeptide or encoding nucleic acid in conjunction with a miRNA may have a combined therapeutic effect on target tissues. A variety of proteins are encompassed within the invention, some of which are described below. Various genes that may be targeted for gene therapy of some form in combination with the present invention include, but are not limited to inducers of cellular proliferation, inhibitors of cellular proliferation, regulators of programmed cell death, cytokines and other therapeutic nucleic acids or nucleic acid that encode therapeutic proteins.
The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors (e.g., therapeutic polypeptides) p53, FHIT, p16 and C-CAM can be employed.
Other genes that may be employed according to the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
6. Surgery
Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
Also, as described herein, inventive therapies can be used in conjunction with surgeries to the eye.
7. Other Agents
It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
There have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of chemotherapy is the development/acquisition of drug-resistant phenotypes and the development of multiple drug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors and therefore, there is an obvious need for alternative approaches such as gene therapy.
Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 106° F.). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe, including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.
A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated. Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.
Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
This application incorporates U.S. application Ser. No. 11/349,727 filed on Feb. 8, 2006 claiming priority to U.S. Provisional Application Ser. No. 60/650,807 filed Feb. 8, 2005 herein by references in its entirety.
V. EVALUATION OF MIRNA LEVELS
It is contemplated that a number of assays could be employed to analyze miRNAs, their activities, and their effects. Such assays include, but are not limited to, array hybridization, solution hybridization, nucleic acid amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA) (GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Oligo Ligation Assay (OLA), hybridization, and array analysis. U.S. patent application Ser. Nos. 11/141,707, filed May 31, 2005; 11/857,948, filed Sep. 19, 2007; 11/273,640, filed Nov. 14, 2005 and provisional patent application 60/869,295, filed Dec. 8, 2006 are incorporated by reference in their entirety.
A. Sample Preparation
While endogenous miRNA is contemplated for use with compositions and methods of the invention, recombinant or synthetic miRNA—including nucleic acids that are complementary or identical to endogenous miRNA or precursor miRNA—can also be handled and analyzed as described herein. Samples may be biological samples, in which case, they can be from lavage, biopsy, fine needle aspirates, exfoliates, blood, sputum, tissue, organs, semen, saliva, tears, urine, cerebrospinal fluid, body fluids, hair follicles, skin, or any sample containing or constituting biological cells. In certain embodiments, samples may be, but are not limited to, fresh, frozen, fixed, formalin-fixed, preserved, RNA later-preserved, paraffin-embedded, or formalin-fixed and paraffin-embedded. Alternatively, the sample may not be a biological sample, but be a chemical mixture, such as a cell-free reaction mixture (which may contain one or more biological enzymes).
B. Differential Expression Analyses
Methods of the invention can be used to detect differences in miRNA expression or levels between two samples, or a sample and a reference (e.g., a tissue reference or a digital reference representative of a non-cancerous state). Specifically contemplated applications include identifying and/or quantifying differences between miRNA from a sample that is normal and from a sample that is not normal, between a cancerous condition and a non-cancerous condition, between two differently treated samples (e.g., a pretreatment versus a posttreatment sample) or between samples having differing prognosis. Also, miRNA may be compared between a sample believed to be susceptible to a particular therapy or disease and one believed to be not susceptible or resistant to that therapy or disease. A sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition or one believed to be not normal with respect to that disease or condition. It may be compared to a cell and/or tissue that is normal with respect to that disease or condition. Phenotypic traits include symptoms of a disease or condition of which a component is or may or may not be genetic or caused by a neovascular and/or angiogenic condition. It is specifically contemplated that the invention can be used to evaluate differences between stages of a disease.
Phenotypic traits also include characteristics such as longevity, morbidity, susceptibility or receptivity to particular drugs or therapeutic treatments (drug efficacy), and risk of drug toxicity.
In certain embodiments, miRNA profiles may be generated to evaluate and correlate those profiles with pharmacokinetics. For example, miRNA profiles may be created and evaluated for patient samples prior to the patient's being treated or during treatment to determine if there are miRNAs whose expression correlates with the outcome of treatment. Identification of differential miRNAs can lead to a diagnostic assay that can be used to evaluate samples to determine what drug regimen the patient should be provided. In addition, it can be used to identify or select patients suitable for a particular clinical trial. If a miRNA profile is determined to be correlated with drug efficacy or drug toxicity that may be relevant to whether that patient is an appropriate patient for receiving the drug or for a particular dosage of the drug.
A diagnostic assay can be created based on the profiles that doctors can use to identify individuals with a disease or who are at risk to develop a disease. Alternatively, treatments can be designed based on miRNA profiling.
C. Amplification
Many methods exist for evaluating miRNA levels by amplifying all or part of miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs, and primary miRNAs. Suitable nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. In certain embodiments, more than one amplification method may be used, such as reverse transcription followed by real time PCR (Chen et al., 2005 and/or U.S. patent application Ser. No. 11/567,082, filed Dec. 5, 2006, which are incorporated herein by reference in their entirety).
In PCR and q-PCR methods, for example, a set of primers is used for each target sequence. In certain embodiments, the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified. In certain embodiments, a primer is about 15 to about 35 nucleotides in length. In other embodiments, a primer is equal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length. In additional embodiments, a primer is at least 35 nucleotides in length.
In a further aspect, a forward primer can comprise at least one sequence that anneals to a target miRNA and alternatively can comprise an additional 5′ noncomplementary region. In another aspect, a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA. The reverse primer may be independent of the miRNA sequence, and multiple miRNAs may be amplified using the same reverse primer. Alternatively, a reverse primer may be specific for a miRNA.
In some embodiments, two or more miRNAs or nucleic acids are amplified in a single reaction volume or multiple reaction volumes. In certain aspects, one or more miRNA or nucleic may be used as a normalization control or a reference nucleic acid for normalization. Normalization may be performed in separate or the same reaction volumes as other amplification reactions. One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least one miRNA of interest and at least one reference nucleic acid in one reaction volume by using more than one pair of primers and/or more than one probe. The primer pairs comprise at least one amplification primer that uniquely binds each nucleic acid, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs. Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.
A single combined reaction for q-PCR, may be used to: (1) decrease risk of experimenter error, (2) reduce assay-to-assay variability, (3) decrease risk of target or product contamination, and (4) increase assay speed. The qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA-based thermostable DNA polymerase. When two polymerases are used, a “hot start” approach may be used to maximize assay performance (U.S. Pat. Nos. 5,411,876 and 5,985,619, each incorporated herein by reference in its entirety). For example, the components for a reverse transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Pat. Nos. 5,550,044, 5,413,924, and 6,403,341, each incorporated herein by reference in its entirety).
To assess the expression of microRNAs, real-time RT-PCR detection can be used to screen nucleic acids or RNA isolated from samples of interest and a related reference such as normal adjacent tissue (NAT) samples.
A panel of amplification targets is chosen for real-time RT-PCR quantification. The selection of the panel or targets can be based on the results of microarray expression analyses, such as mirVanaTM miRNA Bioarray V1, Ambion. In one aspect, the panel of targets includes one or more miRNA described herein. One example of a normalization target is 5S rRNA and others can be included. Reverse transcription (RT) reaction components are typically assembled on ice prior to the addition of RNA template. Total RNA template is added and mixed. RT reactions are incubated in an appropriate PCR System at an appropriate temperature (15-70° C., including all values and ranges there between) for an appropriate time, 15 to 30 minutes or longer, then at a temperature of 35 to 42 to 50° C. for 10 to 30 to 60 minutes, and then at 80 to 85 to 95° C. for 5 minutes, then placed on wet ice. Reverse Transcription reaction components typically include nuclease-free water, reverse transcription buffer, dNTP mix, RT Primer, RNase Inhibitor, Reverse Transcriptase, and RNA.
PCR reaction components are typically assembled on ice prior to the addition of the cDNA from the RT reactions. Following assembly of the PCR reaction components a portion of the RT reaction is transferred to the PCR mix. PCR reaction are then typically incubated in an PCR system at an elevated temperature (e.g., 95° C.) for 1 minute or so, then for a number of cycles of denaturing, annealing, and extension (e.g., 40 cycles of 95° C. for 5 seconds and 60° C. for 30 seconds). Results can be analyzed, for example, with SDS V2.3 (Applied Biosystems). Real-time PCR components typically include Nuclease-free water, MgCl2, PCR Buffer, dNTP mix, one or more primers, DNA Polymerase, cDNA from RT reaction and one or more detectable label.
Software tools such as NormFinder (Andersen et al., 2004) are used to determine targets for normalization with the targets of interest and tissue sample set. For normalization of the real-time RT-PCR results, the cycle threshold (Ct) value (a log value) for the microRNA of interest is subtracted from the geometric mean Ct value of normalization targets. Fold change can be determined by subtracting the dCt normal reference (N) from the corresponding dCt sample being evaluated (T), producing a ddCt(T-N) value for each sample. The average ddCt(T-N) value across all samples is converted to fold change by 2ddCt. The representative p-values are determined by a two-tailed paired Student's t-test from the dCt values of sample and normal reference.
D. Nucleic Acid Arrays
Certain aspects of the present invention concern the preparation and use of miRNA arrays or miRNA probe arrays, which are ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules or precursor miRNA molecules and are positioned on a support or support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted. Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
Representative methods and apparatus for preparing a microarray have been described, for example, in U.S. Pat. Nos. 5,143,854; 5,202,231; 5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934; 5,468,613; 5,470,710; 5,472,672; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839; 5,554,501; 5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732; 5,593,839; 5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134; 5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695,940; 5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196; 5,871,928; 5,847,219; 5,876,932; 5,919,626; 6,004,755; 6,087,102; 6,368,799; 6,383,749; 6,617,112; 6,638,717; 6,720,138, as well as WO 93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505; WO 96/31622; WO 97/10365; WO 97/27317; WO 99/35505; WO 09923256; WO 09936760; WO0138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586; WO 03087297; WO 03091426; WO03100012; WO 04020085; WO 04027093; EP 373 203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of which are all herein incorporated by reference. Moreover, a person of ordinary skill in the art could readily analyze data generated using an array. Such protocols are disclosed above, and include information found in WO 9743450; WO 03023058; WO 03022421; WO 03029485; WO 03067217; WO 03066906; WO 03076928; WO 03093810; WO 03100448A1, all of which are specifically incorporated by reference.
E. Hybridization
After an array or a set of miRNA probes is prepared and the miRNA in the sample is labeled, the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed. Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001) and WO 95/21944. Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.
VI. KITS
Any of the compositions or components described herein may be comprised in a kit. In a non-limiting example, reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array, nucleic acid amplification, and/or hybridization can be included in a kit, as well reagents for preparation of samples from A subject. The kit may further include reagents for creating or synthesizing miRNA probes or therapeutics. The kits will thus typically comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. In certain aspects, the kit can include amplification reagents. In other aspects, the kit may include various supports, such as glass, nylon, polymeric beads, magnetic beads, and the like, and/or reagents for coupling any probes and/or target nucleic acids. It may also include one or more buffers, such as pharmaceutical buffer, reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, and components for isolating miRNA. Other kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus, may include, for example, a solid support.
Kits for implementing methods of the invention described herein are specifically contemplated. In some embodiments, there are kits for preparing miRNA for multi-labeling and kits for preparing miRNA probes and/or miRNA arrays. In these embodiments, kit comprise, in suitable container means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of the following: (1) poly(A) polymerase; (2) unmodified nucleotides (G, A, T, C, and/or U); (3) a modified nucleotide (labeled or unlabeled); (4) poly(A) polymerase buffer; and, (5) at least one microfilter; (6) label that can be attached to a nucleotide; (7) at least one miRNA probe; (8) reaction buffer; (9) a miRNA array or components for making such an array; (10) acetic acid; (11) alcohol; (12) solutions for preparing, isolating, enriching, and purifying miRNAs or miRNA probes or arrays. Other reagents include those generally used for manipulating RNA, such as formamide, loading dye, ribonuclease inhibitors, and DNase.
In specific embodiments, kits of the invention include an array containing miRNA probes, as described in the application. An array may have probes corresponding to all known miRNAs of an organism or a particular tissue or organ in particular conditions, or to a subset of such probes. The subset of probes on arrays of the invention may be or include those identified as relevant to a particular diagnostic, therapeutic, or prognostic application. For example, the array may contain one or more probes that is indicative or suggestive of (1) a disease or condition (neovascularization or aberrant angiogenesis), (2) susceptibility or resistance to a particular drug or treatment; (3) susceptibility to toxicity from a drug or substance; (4) the stage of development or severity of a disease or condition (one aspect of prognosis); (5) the likelihood of recurrence (one aspect of prognosis) and (6) genetic predisposition to a disease or condition.
For any kit embodiment, including an array, there can be nucleic acid molecules that contain or can be used to amplify a sequence that is a variant of, identical to or complementary to all or part of any of SEQ ID NOs described herein. Any nucleic acid discussed above may be implemented as part of a kit.
The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquotted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent or buffer. It is envisioned that the solvent may also be provided in another container means. In some embodiments, labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg or at least or at most those amounts of dried dye are provided in kits of the invention. The dye may then be resuspended in any suitable solvent, such as DMSO.
The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
Such kits may also include components that facilitate isolation of the labeled miRNA. It may also include components that preserve or maintain the miRNA or that protect against its degradation. Such components may be RNase-free or protect against RNases. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.
Kits of the invention may also include one or more of the following: Control RNA; nuclease-free water; RNase-free containers, such as 1.5 ml tubes; RNase-free elution tubes; PEG dextran; ethanol; acetic acid; sodium acetate; ammonium acetate; guanidinium; detergent; nucleic acid size marker; RNase-free tube tips; and RNase or DNase inhibitors.
It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.
VII. EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
miRNAS Altered During Ischemic Retinopathy-Induced Neovascularization
To examine miRNA expression during retinal neovascularization, the inventors used a murine model of oxygen-induced, ischemic retinopathy that results in reproducible, proliferative retinal neovascularization (Smith et al., 1994.). C57BL/6J mice were placed in 75% oxygen on postnatal day 7 and returned to room air on postnatal day 12. On postnatal day 15, mice were euthanized, eyes were removed, and retinas dissected. Small RNAs were isolated from retinas using the mirVanaTM miRNA Isolation Kit (cat. no. AM1561; Ambion Inc., Austin, Tex., USA) according to the manufacturer's instructions. Small RNAs (5 μg), pooled from ten ischemic retinas or from ten age-matched control retinas, were identified and quantified by microarray analysis. Microarray hybridizations were performed by LC Sciences (Houston, Tex., USA). Small RNAs from ischemic retinas were labeled with Cy3 and small RNAs from control retinas were labeled with Cy5 and hybridized to an array of murine microRNA (mmu-miR) probes with six redundant complementary sequences for each of the mmu-miR transcripts in the Sanger miRBase Release 7.0 (August 2005) (Griffiths-Jones et al., 2006). Red and green fluorescence intensities were determined for each probe. Statistical comparisons were made between the mean differences in intensity for the probes and the intensity differences for control probes. A p value of <0.01 was used to identify differentially expressed microRNAs (Table 1). Ten miRNAs (mmu-miR-451, -214, -424, -199a, -146, -106a, -350, -21, -218, and -148b) had increased expression during neovascularization of ischemic retinas, and six (mmu-miR-451, -214, -424, -199a, -146, and -106a) were increased more than two-fold. Nine miRNAs (mmu-miR-184, -31, -150, -409, -375, -129-5p, -124a, -29a, and -129-3p) had significantly reduced expression during neovascularization of ischemic retinas, and three (mmu-miR-184, -31, and -150) were decreased more than two-fold.
The miRNAs in Table 1 represent particularly useful therapeutic targets for treating conditions associated with vascularization







TABLE 1







MicroRNAs Differentially Expressed During


Neovascularization of Mouse Retinas.











Log Ratio



miRNA
(ischemia/control)














miR-451
2.31



miR-214
2.08



miR-424
2.01



miR-199a
1.52



miR-146
1.50



miR-106a
1.25



miR-350
0.95



miR-21
0.91



miR-218
0.83



miR-148b
0.80



miR-184
−3.50



miR-31
−2.50



miR-150
−2.01



miR-409
−0.81



miR-375
−0.80



miR-129-5p
−0.75



miR-124a
−0.65



miR-29a
−0.63



miR-129-3p
−0.61







Log ratio, the log(2) normalized signal for miRNA from ischemic mice divided by the log(2) normalized signal for miRNA from control mice. Positive log ratio values indicate miRNAs with increased levels during neovascularization. Negative log ratio values indicate miRNAs with decreased levels during neovascularization.






Example 2
Quantification of miRNAS Altered During Neovascularization by ORT-PCR
The inventors validated microarray data for specific miRNAs identified in Example 1. Using small RNAs isolated from retinas as described in Example 1 above, the inventors measured miRNA levels by qRT-PCR as previously described (Raymond et al., 2005), with minor modifications. Briefly, small RNAs were reverse-transcribed with SuperScriptTM III Reverse Transcriptase (Invitrogen Corp.; Carlsbad, Calif., USA) into cDNAs, using either (1) microRNA-specific primers containing a 36 bp 5′ tail from C. elegans genomic sequence or (2) random hexamer-priming. For miRNA amplifications, a miRNA-specific, 16-nucleotide upstream primer and a 19-nucleotide universal primer complementary to the C. elegans tail sequence were used. For standardization, a fragment of 5S rRNA was amplified and quantified. Amplifications were performed in a LightCycler® (Roche Diagnostics Corp., Indianapolis, Ind., USA) using the LightCycler® FastStart DNA Master SYBR Green I reaction mix (Roche). For negative controls, cDNA generated from random hexamer priming was used. The mean threshold cycle numbers (ΔCT) for mmu-miR-31, -150, and -184 amplified from ischemic and control retinas (n=5 for each) were determined, and the mean differences between the two values (ΔΔCT) were calculated. ΔΔCT values ranged from about −1.0 (miR-31) to −2.5 (miR-150) (FIG. 1A). Statistical comparisons confirmed that each of the miRs were significantly reduced in ischemic compared to control retinas (FIG. 1B).
Example 3
Predicted Gene Targets of MMU-MIR-184, MMU-MIR-31, and MMU-MIR-150
The inventors searched selected mRNA transcripts for sequences that might represent target binding sites for the microRNAs shown in Table 1 above. Searches were performed in the 5′- and 3′-untranslated regions (UTR) from a group of genes encoding angiogenic factors and their receptors (VEGF, HGF, FGF-2, P1GF, IGF-1, TGF-beta, PDGF-A, PDGF-B, VG5q, Delta-like 4, SEMA3a, Sema3F, Notch4, EphA2, Roundabout, HIF-1alpha, Frizzed-4, SDF-1, PEDF, endostatin, vasohibin-1, thrombspodin-1, Ang-1, Ang-2, Flt-1, KDR, neuropilin-1, neuropilin-2, CXCR4, Tie1, Tie2, angiostatin). UTR sequences were obtained from the Mus musculus genome sequence at the Ensembl Project (Birney et al., 2004) (at the World Wide Web address ensembl.org/index.html) and were searched for possible microRNA target sites using several target site prediction systems, including miRBase Targets (at World Wide Web address microrna.sanger.ac.uk/targets/v4/), the miranda algorithm (John et al., 2005; Betel et al., 2008) (at the World Wide Web address microrna.org), RNAhybrid (Rehmsmeier et al., 2004) (at the internet address bibiserv.techfak.uni-bielefeld.de/rnahybrid), and PicTar (Krek et al., 2005) (at internet address pictar.bio.nyu.edu). Previously reported criteria (Lewis et al., 2005; Sethupathy et al., 2006) for predicting microRNA targets were used as a guide and included (1) a conserved seed match, i.e., perfect Watson-Crick complementarity between nucleotides 2-7 of the microRNA and a 6 nucleotide section of the 3′-UTR of the mRNA that occurs at corresponding positions for multiple species, (2) a conserved anchoring A, which is an A nucleotide on the 3′-UTR just downstream of the seed match that also occurs in the UTRs of the genes from multiple species, and (3) a conserved m8-t8 match, which is an A:U or G:C match between the eighth nucleotide of the microRNA and the corresponding position in the 3′-UTR in multiple species. Conservation analyses between the mouse UTRs and corresponding UTRs for humans, rats, and dogs were performed using applications at the University of Santa Cruz Genome Bioinformatics internet site, on the World Wide Web at genome.ucsc.edu.
The inventors identified possible gene targets for mmu-miR-31, -150, and -184 (FIG. 2). Predicted target sequences for mmu-miR-31 were identified in Pdgfb, Hif1α, and Frizzled4. Predicted target sequences for mmu-miR-150 were identified in Vegf, Pdgfb, Pdgfa, and Notch4. Predicted target sequences for mmu-miR-184 were identified in Frizzled4. No predicted target genes were identified for up-regulated miRNAs in Table 1.
The predicted gene targets of mmu-miR-31, -150, and -184 represent particularly useful therapeutic targets for treating conditions associated with vascularization, through manipulation of their expression levels.
Example 4
Verification of miRNA Target Genes
The inventors utilized luciferase gene reporter assays to assess the validity of putative miRNA target regulatory sequences in Pdgfb, Frizzled4, Hif1α, Vegf, and Notch4. Putative miRNA target sequences were amplified by PCR using retinal cDNA as template and amplification primers that included restriction sites for SpeI and HindIII. PCR products were cloned into a TOPO TA Cloning® vector (Invitrogen). Recombinant plasmid inserts (miRNA target sequences) were verified by DNA sequencing. For validation of miRNA target sequences in Pdgfb, Frizzled4, Hif1α, and Notch4, the cloned target sequences were excised and subcloned into a luciferase microRNA expression reporter vector (pMIR-REPORTTM, Ambion). For validation of miRNA target sequences in Vegf, the inventors used a pGL2-CMV vector containing a 1.7 kb mouse Vegf 3′-UTR coupled to a luciferase gene (R. C. Nichols, Department of Microbiology and Immunology, Dartmouth School of Medicine, Hanover, N.H.).
Luciferase reporter assays were conducted in human retinal pigment epithelial cells (ARPE-19; American Type Culture Collection, Manassas, Va., USA) cultured in DMEM/F12 medium (HyClone; Logan, Utah, USA) supplemented with 10% fetal bovine serum. After reaching 70% confluence, cells were co-transfected with a synthetic microRNA (mmu-miR-31, -150, or -184; pre-miRTM miRNA Precursor Molecule; Ambion) (25 mmol), 1 μg of reporter vector (pMIR-REPORTTM or pGL2-CMV) containing one of the possible target 3′-UTRs, and 2.5 ng of Renilla luciferase vector (Promega Corp.; Madison, Wis., USA). For negative controls, ARPE-19 cells were co-transfected with a scrambled synthetic microRNA (25 nmol), 1 μg of reporter vector containing one of the possible target 3′-UTRs, and 2.5 ng of Renilla luciferase vector. After 48 hours, cells were harvested and luciferase expression assays were performed using the Dual-Luciferase® Reporter Assay System (Promega). Luciferase expression assays were performed in triplicate for each miRNA/target 3′-UTR combination. Statistical comparisons with negative control assays were made by paired t-test.
Co-transfection of ARPE-19 cells with synthetic mmu-miR-31 and a pMIR vector with the Pdgfb or Hif1α 3′-UTR, significantly reduced luciferase levels expressed by the reporter (FIG. 3A, 3C), verifying that Pdgfb and Hif1α are targets for regulation by mmu-miR-31. Co-transfection with synthetic mmu-miR-31 and a pMIR vector with the Frizzled4 3′-UTR had no effect on luciferase levels from the reporter (FIG. 3B).
Co-transfection or ARPE-19 cells with synthetic mmu-miR-150 and a pMIR vector with the Pdgfb 3′-UTR or with synthetic mmu-miR-150 and a pGL2-CMV vector with the Vegf 3′-UTR, significantly reduced luciferase levels expressed by the reporter (FIG. 3D, 3E), verifying that Pdgfb and Vegf are targets for regulation by mmu-miR-150. Co-transfection with synthetic mmu-miR-150 and a pMIR vector with the Notch4 3′-UTR had no effect on luciferase levels from the reporter (FIG. 3F).
Co-transfection or ARPE-19 cells with synthetic mmu-miR-184 and a pMIR vector with the Frizzled4 3′-UTR significantly reduced luciferase levels expressed by the reporter (FIG. 3B), verifying that Frizzled4 is a target for regulation by mmu-miR-184.
Example 5
Effect of miRNAS on Target Gene Product Levels in Neovascularizing Retinas In Vivo
The inventors evaluated the effects of mmu-miR-31, -250, and -184 on target gene product levels in vivo, during retinal neovascularization induced by ischemic retinopathy. C57/BL6 mice were placed in 75% oxygen on postnatal day 7. On postnatal day 12, mice were returned to room air and given an intraocular injection of a synthetic experimental microRNA (0.005 μmol in 1 μl) (pre-miRTM miRNA Precursor Molecule; Ambion) in one eye and a synthetic control microRNA (0.005 μmol in 1 μl; scrambled microRNA sequence) in the other eye. Synthetic miRNAs were injected with a Harvard Pump Microinjection System (Harvard Apparatus, Holliston, Mass., USA) and pulled glass micropipettes as previously described (Mori et al., 2001). Mice were euthanized at postnatal day 15, eyes were removed, and retinas were dissected and homogenized. Retinas were placed in 200 μl of lysis buffer (50 μl 1M Tris-HCl (pH 7.4), 50 μl of 10% sodium dodecyl sulfate, 5 μl of 100 nM phenylmethanesulfonyl, and 5 ml of sterilized, de-ionized water), homogenized, sonicated at 4° C. for 5 seconds, and centrifuged at 10,000 g for 5 minutes at 4° C. The protein concentration of the supernatants was measured with a bicinchoninic acid protein assay kit (Pierce; Rockford, Ill., USA). Retinal homogenates were used to assess HIF-1α, PDGF-B, and Frizzled 4 by immunoblot analysis and VEGF by both immunoblot analysis and ELISA. For immunoblots, 20 μg of protein were separated by SDS-PAGE on 8-12% acrylamide gels and transferred to a nitrocellulose membrane. Non-specific binding was blocked by incubation in Tris-buffered saline (TBS) containing 5% skim milk, and membranes were hybridized with 0.5 μg/ml rabbit anti-mouse VEGF polyclonal antibody (Abcam Inc.; Cambridge, Mass., USA), rabbit anti-mouse Frizzled 4 antibody (R&D Systems Inc.; Minneapolis, Minn., USA), rabbit anti-mouse PDGF-B or HIF1-α antibody (Santa Cruz Biotechnology® Inc.; Santa Cruz, Calif., USA), or in TBS containing 0.05% Tween 20 and 2.5% skim milk, at 4° C. overnight. After three washes with TBS-Tween-20, the membrane was incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibody (1:5000) (GE Healthcare Bio-Sciences Corp.; Piscataway, N.J., USA). For signal development, membranes were incubated in ECL PlusTM Western Blotting Detection Reagent (GE Healthcare Bio-Sciences) and exposed to X-ray film. ELISAs were performed using a Quantikine VEGF assay kit (R&D Systems Inc.) according to the manufacturer's instructions. Serial dilutions of recombinant VEGF were assayed to generate a standard curve. The limit of detection was 125 pg/ml of VEGF.
Injection of synthetic mmu-miR-31 or -150, but not synthetic mmu-miR-184, caused a significant reduction in VEGF levels in homogenates of neovascularizing retinas (FIG. 4A, FIG. 4B). Since mmu-miR-31 does not target Vegf directly, it is likely that its effect occurs indirectly through one of its actual targets, probably Hif1α. Injection of 1 μl containing 1.66 pmol each of the three synthetic microRNAs also caused a significant reduction in VEGF (FIG. 4A). Intraocular injection of synthetic mmu-miR-31 caused reductions in retinal HIF1-α and PDGF-B (FIG. 4B). Injection of synthetic mmu-miR-150 caused reductions in PDGF-B and VEGF (FIG. 4B). Injections of synthetic mmu-miR-31 or synthetic mmu-miR-184 failed to cause reductions in Frizzled 4 protein in the retinas (FIG. 4B).
Example 6
miRNA Therapy in Mice with Oxygen-Induced Ischemic Retinopathy
Ischemic retinopathy was generated in mice as described above in Example 1. Mice were returned to room air at postnatal day 12 and given a 1 μl intraocular injection containing 2 μg of experimental synthetic microRNA in one eye or 2 μg of control synthetic microRNA in the other eye. Injections were given as described above in Example 5. On postnatal day 17, areas of neovascularization on the surfaces of the mouse retinas were measured as previously described (Lima e Silva et al., 2007). Briefly, mice were given an intraocular injection of 1 μl of rat anti-mouse PECAM-1 antibody (BD Biosciences; San Jose, Calif., USA) and were euthanized twelve hours after antibody injection. Mouse eyes were dissected and fixed in 10% formalin for 4 hours. Intact retinas were dissected, incubated for 40 minutes in a 1:500 dilution of goat anti-rat IgG conjugated with Alexa488 (Invitrogen), washed, and whole mounted. This technique provides selective staining of retinal neovascularization on the surface of the retina (Shen et al., 2007). An observer, masked with respect to treatment groups, examined the slides by fluorescence microscopy and measured the area of neovascularization per retina using computerized image analysis and Image-Pro® PLUS software (Media Cybernetics, Inc.; Bethesda, Md., USA).
Retinas from mouse eyes injected with negative control miRNA showed extensive neovascularization (FIG. 5A). In contrast, retinas from mouse eyes injected with 2 μg of synthetic mmu-miR-31, -150, or -184 showed very little neovascularization on the surface of the retina (FIG. 5B). Computerized image analysis confirmed a significant reduction in mean area of neovascularization per retina in eyes injected with synthetic mmu-miR-31, -150, or -184 (FIG. 6A, FIG. 6B, FIG. 6C) or with a mixture of all three synthetic miRs (FIG. 6D) as compared to eyes injected with negative control miRNA.
Example 7
miRNA Therapy in Mice with Choroidal Neovascularization
The inventors used a model of choroidal neovascularization to further evaluate miRNA therapy. The choroid, also known as the choroidea or choroid coat, is the vascular layer of the eye lying between the retina and the sclera (the fibrous, protective outer layer of the eye). The choroid provides oxygen and nourishment to the outer layers of the retina.
Choroidal neovascularization was generated by modification of a previously described technique (To be, et al., 1998). Briefly, 4 to 5 week old female C57BL/6J mice were anesthetized and the pupils were dilated with 1% tropicamide. Three burns of 532 nm diode laser photocoagulation (75 μm spot size, 0.1 second duration, 120 mW) were delivered to each retina using the slit lamp delivery system of an OcuLight GL Photocoagulator (IRIDEX Corp., Mountain View, Calif., USA). Burns were performed in the 9-, 12-, and 3-o'clock positions of the posterior pole of the retina. Immediately after laser treatment and again on day 7, mice were given an intraocular injection of 1 μl containing 5 μmol of experimental synthetic microRNA or a mixture of three experimental synthetic microRNAs (1.67 μmol each mmu-miR-31, -150, and -184) in one eye and 5 μmol of a scrambled, synthetic microRNA (control) in the other eye. Fourteen days after laser treatment, mice were perfused with fluorescein-labeled dextran (2×106 average MW) (Sigma-Aldrich, St. Louis, Mo., USA), and choroidal whole mounts were prepared and examined by fluorescence microscopy. An observer, masked with respect to treatment group, used image analysis techniques to measure the area of choroidal neovascularization at each Bruch's membrane rupture site (Shen et al., 2006; Lima e Silva et al., 2007). The three choroidal neovascularization areas within each eye were averaged to give one experimental value per eye. Statistical comparisons were made by paired t-test.
Fluorescence microscopy observation revealed that eyes treated with a mixture of three experimental miRNAs (mmu-miR-31, -150, and -184) (FIG. 7A) had smaller choroidal neovascularization lesions than fellow eyes treated with the scrambled, control miRNA (FIG. 7B). Measurement of the area of choroidal neovascularization by computerized image analysis revealed that eyes treated with the mixture of experimental miRNAs had a significant reduction in the mean area of choroidal neovascularization at Bruch's membrane rupture sites when compared to eyes treated with the scrambled, control miRNA (FIG. 8A). Mouse eyes treated individually with mmu-miR-31 (FIG. 8B) or mmu-miR-150 (FIG. 8C) also exhibited reduced choroidal neovascularization. Mouse eyes treated with mmu-miR-184 displayed similar levels of choroidal neovascularization as those treated with the control miRNA (FIG. 8D).
Example 8
miRNAS Altered in RD1 Mouse Model of Neovascularization
The inventors examined miRNA expression changes in a second model of neovascularization, consisting of a transgenic mouse strain (RD1) that contains a phosphodiesterase 6B mutation (Chang et al., 2002). RD1 mice exhibit early onset retinal degeneration wherein they undergo rod degeneration between postnatal day 10 (P10) and postnatal day 22 (P22) and cone degeneration between postnatal day 22 (P22) and postnatal day 35 (P35).
To track changes in miRNA expression during the progression of neo-vascularization in eyes of RD1 mice, animals were sacrificed at P5, P10, P15, P22, and P35, eyes were removed, and retinas dissected. Small RNAs were isolated from retinas using the mirVanaTM miRNA Isolation Kit (Ambion). To account for changes in miRNA expression during normal mouse development, a set of control mice (strain C57) were sacrificed at P5, P10, P15, P22, and P35, eyes were removed, retinas dissected, and small RNAs were isolated as for the RD1 mice.
miRNAs from all mice were purified using the flashPAGETM Fractionator Apparatus (cat. no. AM13100, Ambion) and labeled using the mirVanaTM miRNA Labeling Kit (cat. no. AM1562, Ambion). Labeled miRNAs were hybridized to mirVanaTM miRNA Bioarrays (Ambion). Signals for each miRNA-specific probe were background-subtracted, normalized, and converted to log(2) scale. The normalized expression data for each miRNA at each time point were compared to the corresponding data for the same animals at P10 (the day of retinal degeneration onset).
The inventors observed differential expression of specific miRNAs during retinal neovascularization in RD1 mice as compared to their expression in the control mice (Table 2). Forty-seven miRNAs were observed to be significantly differentially expressed, at one or both of P22 and P35, in the RD1 mice relative to the C57 control mice—mmu-miR-10b, -96, -183, -184, -16, -182, -191, -29c, -181c, -129-3p, -335, -210, -512-3p, -132, -500, -339, -511, -26b, -30b, and -15a and ambi-miR-7026 were all significantly decreased in mouse retinas undergoing neovascularization (Table 2) and mmu-miR-205, -106a, -365, -299-5p, -200a, -351, -329, -122a, -20a, -350, -520h, -142-5p, -203, -211, -145, -93, -192, -106a, -201, -18a, -17-5p, -106b, and -223, mmu-let-7b, ambi-miR-7079, and ambi-miR-7085, were all significantly increased in mouse retinas undergoing neo-vascularization (Table 2). The miRNAs in Table 2 represent particularly useful therapeutic targets for treating conditions associated with vascularization.







TABLE 2







MicroRNAs Differentially Expressed During


Neovascularization of Retinas in RD1 Mice.












Log Ratio
Log Ratio




RD1-C57
RD1-C57



miRNA
(P22)
(P35)















miR-10b
−2.525
−2.792



miR-96
−2.404
−2.58



miR-183
−1.726
−1.938



miR-184
−2.345
−1.829



miR-16
−1.769
−1.24



miR-182
−1.983
−1.171



miR-191
−1.006
−1.153



miR-29c
−2.195
−1.03



miR-181c
−1.096
−1.017



miR-129-3p
−0.428
−1.015



miR-335
−1.005
−0.967



ambi-miR-7026
0.461
−0.88



miR-210
−0.672
−0.862



miR-512-3p
−0.132
−0.845



miR-132
−0.229
−0.832



miR-500
−0.395
−0.77



miR-339
−0.295
−0.712



miR-511
−0.124
−0.695



miR-26b
−1.375
−0.689



miR-30b
−1.35
−0.685



miR-15a
−1.01
−0.601



miR-205
2.588
2.601



miR-106a
0.957
2.585



miR-365
2.332
2.359



miR-299-5p
2.408
2.318



ambi-miR-7079
2.495
2.302



miR-200a
2.104
2.238



miR-351
1.956
2.083



miR-329
0.111
1.367



miR-122a
0.958
1.35



miR-20a
0.755
1.266



miR-350
0.59
1.258



miR-520h
1.094
1.247



miR-142-5p
0.153
1.157



miR-203
0.637
1.142



miR-211
0.593
1.093



miR-145
0.213
1.072



let-7b
0.364
1.06



miR-93
0.969
1.045



miR-192
0.319
1.001



miR-106a
0.83
0.929



miR-201
0.704
0.916



miR-18a
0.611
0.907



miR-17-5p
0.741
0.864



ambi-miR-7085
0.667
0.848



miR-106b
0.549
0.842



miR-223
0.527
0.819







Positive log ratio values indicate miRNAs with increased levels during neovascularization. Negative log ratio values indicate miRNAs with decreased values during neovascularization. P22; post-natal day 22. P35; post-natal day 35.






Example 9
Predicted MicroRNA Targets
The miRNAs that were observed to be significantly differentially expressed in the retinas of mice undergoing neovascularization were evaluated for their capacity to potentially regulate genes associated with angiogenesis. Gene target predictions were performed by searching within the 3′UTRs of 23 angiogenesis-related genes (LRP6, VEGFA, VEFC, VEGF-R, EFEMP1, ECGF1, ELOVL4, EREG, FGF1, FGF2, IGF1, JAG1, NRP1, NRP2, PGF, RS1, RDS, MMP2, MMP9, TIMP1, TIMP2, TIMP3, TLR4) for sites that are perfectly complementary with the core sequences of the selected miRNAs. Predicted targets for miRNAs are shown in Table 3. Many of the miRNAs were predicted to have multiple potential target sites within a single 3′UTR. The number of predicted target interactions is denoted in parentheses. The average number of angiogenesis-related target sites for the neovascularization-associated miRNAs was 4.64, with a range of 0-12. Based on this analysis, it is apparent that the altered expression of one or several of these miRNAs can profoundly influence the expression levels of genes that are known to inhibit or activate blood vessel growth.







TABLE 3







Predicted target genes of miRNAs altered during neovascularization. Genes with multiple


predicted target interaction sites have the number of such sites indicated in parentheses


following the gene name. Some genes had no targets predicted by the methods


described in Example 9.








miRNA
Predicted Target Genes


















miR-10b
NRP2
RS1








miR-96


miR-183
LRP6
MMP9
TIMP3
NRP1


miR-16/miR-15a
VEGFA
LRP6
NRP2 (3)
FGF2 (4)
RDS (2)
RS1


miR-182
VEGFR


miR-191
TIMP2
ELOVL4
RDS
RS1


miR-29c/miR-29a
VEGFA
MMP2
EREG (2)
IGF1 (2)
ELOVL4
EFEMP1
RDS


miR-181c
TIMP3 (2)
IGF1
RDS


miR-129-3p
NRP1
NRP2


miR-335
FGF2
RDS (2)


miR-210
NRP2


miR-512-3p
TIMP2
FGF1
FGF2


miR-132
RS1


miR-500
VEGFR (2)
TIMP2
NRP1
NRP2
FGF2 (2)
ANPEP


miR-339
VEGFA (2)
MMP2
NRP2
CFB
RS1


miR-511
VEGFA
VEGFC
MMP2
NRP1 (2)
FGF2
TLR4 (3)
VMD2
ELOVL4 (2)


miR-26b
JAG1
EREG
IGF1
TLR4
EFEMP1


miR-30b
TIMP3
NRP1


miR-31
TIMP2
IGF1
TLR4 (2)


miR-150
VEGFA
NRP1
FGF1
FGF2 (2)
EREG
IGF1
RS1


miR-129-5p
APOE
TIMP2
NRP1
JAG1
FGF2
ANGPT1
IGF1 (4)
RDS


miR-124a
MMP2
PGF
NRP1
NRP2
JAG1


miR-451
EREG
FBLN5


miR-214
VEGFR
PGF (3)
TIMP3
NRP1
JAG1
CFB
FBLN5
RDS


miR-424
VEGFA
LRP6
NRP2 (3)
FGF2 (4)
RDS (2)
RS1


miR-199a
JAG1


miR-146
TIMP3
NRP2 (2)
JAG1
FGF2
CFH
TLR4


miR-106a/20a/17-
VEGFA
MMP2 (2)
TIMP2
FGF2 (2)
EREG


5p


miR-21
TIMP3 (2)
JAG1
FGF1
FGF2
CFH
TLR4


miR-218
TIMP2
JAG1
IGF1
RDS


miR-148b
NRP1
FGF2
ANGPT2
IFG1
VMD2


miR-205
VEGFA
FGF1
FGF2


miR-365
EREG
TLR4
EFEMP1


miR-299-5p
VEGFR
FGF1


miR-200a
VEGFR
NRP1
NRP2 (2)
FBLN5


miR-329
TIMP3
NRP2
TLR4


miR-122a
TLR4
RS1


miR-520h
VEGFA
MMP2
PGF
FGF2 (2)
EREG
ANGPT1
TLR4


miR-142-5p
NRP1
JAG1
FGF2
ANGPT2
IGF1 (2)


miR-203
TIMP3
NRP1
NRP2
FGF2
EREG
TLR4 (2)
ABCA4


miR-211
MMP9
NRP2
FGF1 (2)
ANGPT1
ELOVL4
RDS


miR-145
NRP1
NRP2
ANGPT2
IGF1
TLR4
ELOVL4


let-7b
IGF1
ELOVL4


miR-93
VEGFA
MMP2 (2)
TIMP2
FGF2 (2)
EREG


miR-192
EREG
IGF1


miR-18a
IGF1


miR-223
TIMP2
FGF2 (2)
ABCA4
EFEMP1


miR-409


miR-375


miR-184









To determine if the number of angiogenesis target sites for the various miRNAs was significant, the inventors randomly selected twelve miRNAs and used the same list of genes and methodology to select potential angiogenesis related target genes. The average number of target sites for the randomly selected miRNAs was 2.7 or almost two fewer than was observed for the neovascularization-related miRNAs. The Student's t-test was used to calculate a p-value of 0.01, suggesting that the variance in the number of target gene predictions for the neovascularization associated miRNAs and the randomly-selected miRNAs is significant. A further indication of the significant difference between the lists of miRNAs is that only one of the randomly selected miRNAs was predicted to have at least two target sites for any single angiogenesis related gene.







TABLE 4







Predicted target genes of randomly selected miRNAs. Genes with multiple


predicted target interaction sites have the number of such sites indicated


in parentheses following the gene name. Some genes had no targets


predicted by the methods described in Example 9.








miRNA
Predicted Target Genes
















miR-24
VEGFA
TIMP2
NRP1
NRP2
EREG
RDS


miR-26a
JAG1
EREG
IGF1
TLR4
EFEMP1


miR-103
VEGFA
NRP2
FGF2
FGF2
TLR4
RS1


miR-143
NRP2
FGF1


miR-9
EREG
ANGPT2
ELOVL4


miR-124
MMP2
PFG
NRP1
NRP2
JAG1


miR-219-5p
TIMP3
TLR4


miR-377
VEGFA
TIMP3
NRP2 (2)
JAG1
IGF1
ABCA4


miR-384
TIMP2
IGF1


miR-7
FBLN5
TLR4


miR-28-5p
PGF
ECGF1
RS1


miR-601
NRP2


miR-890
TIMP3 (2)


miR-99a


miR-202


miR-323-50


miR-126









REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A method for reducing vascularization in a subject or tissue comprising administering to the subject or tissue in need of such a reduction, in an amount sufficient to reduce vascularization, one or more nucleic acid molecules comprising a nucleic acid sequence 17 to 110 nucleotides in length that is at least 80% identical to miR-31.

2. The method of claim 1, wherein the nucleic acid is administered topically, enterally, parenterally or intravitreally.

3. The method of claim 1, wherein the nucleic acid molecule is an RNA.

4. The method of claim 3, wherein the RNA comprises a complementary RNA region that has noncomplementarity with the miRNA in the last 1, 2, 3, 4, and/or 5 nucleotides of the complementary region.

5. The method of claim 4, wherein the RNA comprises a hairpin structure.

6. The method of claim 1, wherein the RNA is 17 to 35 nucleotides in length and comprises two RNA strands, wherein the complementary strand is 1) between 60 and 90% complementary to the miRNA sequence or 2) has a nucleotide analog or modified nucleotide.

7. The method of claim 6, wherein the RNA molecule includes a nucleotide analog or a modified nucleotide in the complementary strand.

8. The method of claim 1, wherein the nucleic acid molecule is a DNA molecule or is produced from a DNA molecule.

9. A method for reducing vascularization in a subject or tissue comprising administering to the subject or tissue in need of such a reduction, in an amount sufficient to reduce vascularization, a vector comprising a nucleic acid molecule encoding a nucleic acid sequence that comprises a sequence that is at least 80% identical to miR-31.

10. The method of claim 1, wherein the subject has, is at risk of developing, or is suspected of having ocular or retinal/choroidal neovascular diseases, cancer, diabetic nephropathy, rheumatoid arthritis, atherosclerotic plaques, endometriosis, Crohn's disease, uterine fibroids, benign prostatic hyperplasia, or psoriasis.

11. The method of claim 6, wherein the modified nucleotide is a sugar modification.

12. The method of claim 6, wherein the complementary strand comprises a replacement group for the phosphate or hydroxyl group.

13. The method of claim 9, wherein the vector is comprised in a lipid or viral delivery vehicle.

14. The method of claim 9, wherein the vector is a viral vector.

15. The method of claim 9, wherein the vector is administered topically, enterally, parenterally or intravitreally.

16. The method of claim 9, wherein the subject has, is at risk of developing, or is suspected of having ocular or retinal/choroidal neovascular diseases, cancer, diabetic nephropathy, rheumatoid arthritis, atherosclerotic plaques, endometriosis, Crohn's disease, uterine fibroids, benign prostatic hyperplasia, or psoriasis.