# Patent

## Title
Single-stranded RNAi agents containing an internal, non-nucleic acid spacer

## Abstract

Single-stranded RNA molecules comprise one or more internal, non-nucleotide spacers, covalently linked with nucleotide portions of the molecule are provided. The single-stranded RNA molecules function as guide or antisense strands that are capable of inhibiting gene expression via an RNA interference mechanism, and thus represent single-stranded RNAi agents. The single-stranded RNAi molecules can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/818,306, filed Aug. 19, 2011, which is a National Stage Entry of PCT Application No. PCT/US2011/048338, filed Aug. 19, 2011 which claims the benefit of U.S. Provisional Application No. 61/376,471, filed Aug. 24, 2010. Each of these prior applications is incorporated herein by reference in its entirety.


REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The sequence listing of the present application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “SIRMIS00133USPCT-SEQLIST-22FEB2013.TXT”, creation date of Feb. 14, 2013, and a size of 212 KB. This sequence listing submitted by EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
The RNA interference (RNAi) pathway is an evolutionarily conserved mode of gene regulation. The RNAi process is initiated by double-stranded RNA (dsRNA) produced from various exogenous or endogenous sources (e.g., experimental introduction, viral infection). The dsRNA is cleaved by Dicer to generate 20-25 nucleotide small-interfering RNA (siRNA) duplexes. These duplexes are then loaded onto the RNA-induced silencing complex (RISC), and before RISC is activated, the passenger/sense strand of the duplex is removed. The single guide/antisense strand remains associated with RISC and directs cleavage of the target mRNA. Thus, duplexed siRNA have become an important tool for both research and nucleic acid-based therapeutics.
RNAi gene silencing can occur via single-stranded or double-stranded RNA molecules. In the last ten years, it has been reported that single-stranded antisense siRNA are almost as potent as the siRNA duplex (see, e.g., Schwarz et al., 2002, Mol. Cell. 10:537-548; Martinez et al., 2002, Cell 110:563-574; Amarzguioui et al., 2003, Nucleic Acids Res. 31:589-595; and Holen et al., 2003, Nucleic Acids Res. 31:2401-2407). There are benefits in utilizing single-stranded RNA molecules, as opposed to duplexed versions, for gene silencing. Their lower molecule weight may make them easier to cross cell membranes. Single-stranded RNA molecules are also half the mass and volume of duplexed siRNA, implicating a manufacturing cost advantage. Thus, there remains a heightened interest in formulating new and advantageous design features suitable for single-stranded RNAi molecules.
SUMMARY OF THE INVENTION
The instant disclosure provides single-stranded RNA molecules which comprise: (a) a nucleic acid portion comprising two or more nucleotide portions, and (b) an internal (as opposed to “terminal”) spacer portion comprising one or more non-nucleotide spacer portions, wherein a non-nucleotide spacer portion covalently links two nucleotide portions of the molecule. The nucleotide portions of the single-stranded RNA molecules of the invention are not complementary to each other and, thus, said portions do not form base pairs. The single-stranded RNA molecules of the invention function as guide or antisense strands that are capable of inhibiting gene expression via an RNA interference mechanism and, thus, represent single-stranded RNAi agents.
A single-stranded RNAi molecule of the invention has a single-stranded oligonucleotide structure and mediates RNA interference against a target RNA. A single-stranded RNAi molecule comprises: (a) a nucleic acid portion comprising a first nucleotide portion (N1) and a second nucleotide portion (N2), wherein said nucleic acid portion comprises at least 8 nucleotides that can base pair with a target RNA, and wherein the total number of nucleotides within the nucleic acid portion is from 8 to 26 nucleotides; and, (b) an internal spacer portion comprising at least a first non-nucleotide spacer portion (S1) that covalently links the first and second nucleotide portions. The first and second nucleotide portions are not self complementary. The total number of nucleotides of a single-stranded RNAi molecule of the invention (e.g., 8 to 26) is distributed between the nucleotide portions of the molecule, wherein each nucleotide portion contains at least one nucleotide.
In one embodiment, the nucleic acid portion of a single-stranded RNAi molecule of the invention contains two nucleotide portions, referred to as the first nucleotide portion (N1) and the second nucleotide portion (N2). The first and second nucleotide portions of an RNAi molecule of the invention are covalently attached to a non-nucleotide spacer portion of the molecule. In another embodiment, the nucleic acid portion of a single-stranded RNAi molecule of the invention contains more than one nucleotide portion (e.g., 3, 4, or 5, referred to as third (N3), fourth (N4) or fifth (N5) nucleotide portions, respectively).
In one embodiment, the internal spacer portion of a single-stranded RNAi molecule of the invention contains only one non-nucleotide spacer portion, referred to as the first non-nucleotide spacer portion (S1). The first non-nucleotide spacer portion (S1) of an RNAi molecule of the invention is covalently attached to two nucleotides and/or non-nucleotide substitutes, each located within a distinct nucleotide portion of the single-stranded molecule. In another embodiment, the internal spacer portion of a single-stranded RNAi molecule of the invention contains more than one non-nucleotide spacer portion (e.g., 2, 3, or 4, referred to as second (S2), third (S3) or fourth (S4) non-nucleotide spacer portions, respectively).
A single-stranded RNAi molecule of the invention comprises a nucleotide sequence that is partially, substantially or perfectly complementary to an RNA target site in a cell.
In one embodiment, a single-stranded RNAi molecule of the invention comprises a nucleotide sequence that is partially, substantially, or perfectly homologous to the guide strand of a naturally-occurring miRNA and, thus, functions as a miRNA mimetic. A single-stranded miRNA mimetic of the invention is designed based on a corresponding, naturally-occurring miRNA, wherein at least one non-nucleotide spacer portion is either located between two adjacent nucleotides of the naturally-occurring miRNA guide strand sequence or substituted for from one to about 12 internal (i.e., non-terminal) nucleotides of the naturally-occurring miRNA guide strand sequence.
In another embodiment, a single-stranded RNAi molecule of the invention is an analog of either a single-stranded siRNA or the guide/antisense strand of a duplex siRNA, wherein the single-stranded RNAi molecule comprises a sequence that is partially, substantially, or perfectly homologous to the corresponding single-stranded siRNA or the guide strand of the corresponding duplex siRNA. The corresponding single-stranded siRNA or duplex siRNA may be known to inhibit gene expression via an RNAi mechanism. In this embodiment, the single-stranded RNAi molecule represents a single-stranded siRNA mimetic. A single-stranded siRNA mimetic of the invention is designed based on a corresponding siRNA, wherein at least one non-nucleotide spacer portion is either located between two adjacent nucleotides of the siRNA guide strand sequence or substituted for from one to about 4 nucleotides of the corresponding siRNA guide strand sequence.
A single-stranded RNAi molecule of the invention can comprise substitutions, chemically-modified nucleotides, and non-nucleotides, including substitutions or modifications in the backbone, sugars, bases, or nucleosides. In certain embodiments, the use of substituted or modified single-stranded RNAi molecules of this disclosure can enable achievement of a given therapeutic effect at a lower dose since these molecules may be designed to have an increased half-life in a subject or biological samples (e.g., serum). Furthermore, certain substitutions or modifications can be used to improve the bioavailability of single-stranded RNAi molecules by targeting particular cells or tissues or improving cellular uptake of the single-stranded RNAi molecules.
The internal spacer portion of a single-stranded RNAi molecule of the invention can comprise one or more non-nucleotide spacer portions. A non-nucleotide spacer portion can include any aliphatic or aromatic chemical group that can be further substituted, wherein said spacer portion does not contain a nucleotide. The spacer portion can be substituted with a chemical moiety that provides additional functionality to a single-stranded RNAi molecule. For example, a non-nucleotide spacer portion can be substituted with a moiety that binds specifically to a target molecule of interest or facilitates/enhances cellular delivery of the molecule. In one embodiment of the invention, a non-nucleotide spacer portion includes an alkyl, alkenyl or alkynyl chain of preferably 1 to 20 carbons that can be optionally substituted.
The single-stranded RNAi molecules of the invention are useful reagents, which can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications. Thus, the prevent invention further includes compositions comprising a single-stranded RNAi molecule of the disclosure and methods for inhibiting expression of one or more corresponding target mRNAs in a cell or organism. This disclosure provides methods and single-stranded RNAi molecule compositions for treating a subject, including a human cell, tissue or individual.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the degree of inhibition of VAMP3 target expression by single-stranded miR-124 analogs containing a C3 spacer using a RT-qPCR assay. The structure and sequence of the analogs are specifically described in Table 2, infra. In the schematic drawings of the miR-124 analogs, the circles represent nucleotides, with the exception of the black circle located at the 5′ terminus which represents a 5′ phosphate. The open circles represent 2′-deoxy-2′-fluoro nucleotides. The black circles located at the 3′ terminus of the schematics represent 2′-O-methyl nucleotides, and the “3” represents the location of the C3 spacer. In the schematic of the “124(21)-8p-16rrr” analog, the three internal black circles represent unmodified ribonucleotides. The longer bars in the graph indicate greater knockdown, and duplicate bars indicate biological replicates.
FIG. 2 shows the degree of inhibition of VAMP3 target expression by single-stranded miR-124 analogs containing a C6 spacer using a RT-qPCR assay. The structure and sequence of the analogs are specifically described in Table 2, infra. In the schematic drawings of the miR-124 analogs, the circles represent nucleotides, with the exception of the black circle located at the 5′ terminus which represents a 5′ phosphate. The open circles represent 2′-deoxy-2′-fluoro nucleotides. The black circles located at the 3′ terminus of the schematics represent 2′-O-methyl nucleotides, and the “6” represents the location of the C6 spacer. In the schematic of the “124(21)-8p-16rrr” analog, the three internal black circles represent unmodified ribonucleotides. The longer bars in the graph indicate greater knockdown, and duplicate bars indicate biological replicates.
FIG. 3 shows the dose-dependent response of VAMP3 expression for a subset of the analogs tested in FIGS. 1 and 2. VAMP3 expression is depicted along the y-axis. The dose of the miR-124 analog tested (see Table 2, infra, for structure and sequences) is depicted along the x-axis, ranging from the lowest doses on the left to the highest doses on the right.
FIG. 4 shows the degree of inhibition by single-stranded miR-124 analogs containing a C3 spacer using a reporter assay that measures knockdown of a co-transfected luciferase reporter that carries two target sites matching the seed region of miR-124. The structure and sequence of the analogs are specifically described in Table 2, infra. In the schematic drawings of the miR-124 analogs, the circles represent nucleotides, with the exception of the black circle located at the 5′ terminus which represents a 5′ phosphate. The open circles represent 2′-deoxy-2′-fluoro nucleotides. The black circles located at the 3′ terminus of the schematics represent 2′-O-methyl nucleotides, and the “3” represents the location of the C3 spacer. In the schematic of the “124(21)-8p-16rrr” analog, the three internal black circles represent unmodified ribonucleotides. The duplicate bars of the graph indicate biological replicates, and longer bars indicate greater inhibition.
FIG. 5 shows the degree of inhibition by single-stranded miR-124 analogs containing a C6 spacer using a reporter assay that measures knockdown of a co-transfected luciferase reporter that carries two target sites matching the seed region of miR-124. The structure and sequence of the analogs are specifically described in Table 2, infra. In the schematic drawings of the miR-124 analogs, the circles represent nucleotides, with the exception of the black circle located at the 5′ terminus which represents a 5′ phosphate. The open circles represent 2′-deoxy-2′-fluoro nucleotides. The black circles located at the 3′ terminus of the schematics represent 2′-O-methyl nucleotides, and the “6” represents the location of the C6 spacer. In the schematic of the “124(21)-8p-16rrr” analog, the three internal black circles represent unmodified ribonucleotides. The duplicate bars indicate biological replicates, and longer bars indicate greater inhibition.
FIGS. 6A and 6B show the dose-dependent response of target expression inhibition of two different luciferase reporters for a subset of the single-stranded miR-124 analogs tested in FIG. 3. In FIG. 6A, the inhibition activity shown is against a luciferase reporter with two matches to the miR-124 seed region, representing the miRNA activity of the tested analogs. In FIG. 6B, the inhibition activity shown is against a luciferase reporter with two full-length matches to miR-124, representing the siRNA activity of the tested analogs.
FIG. 7 compares the knockdown of ApoB mRNA using ApoB-targeted single stranded (guide strand) oligonucleotides having a C3 spacer incorporated at either position 15 (“485 c3@pos15”), 16 (“485 c3@pos16”), 17 (“485 c3@pos17”), 18 (“485 c3@pos185”), or 19 (“485 c3@pos19”) (relative to the 5′ of the oligo) to the corresponding single stranded oligonucleotide without the spacer (“485”) at two different concentrations (100 nM and 10 nM). All of the single stranded molecules are composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotides, a 5′ phosphate, and two 2′-O-methyl nucleotides at the 3′ terminus.
FIGS. 8 and 9 compares ApoB mRNA knockdown using 30 different single strand sequences targeting ApoB with C3 spacer at either position 18 (FIG. 8) or position 19 (FIG. 9) at two concentrations (100 nM and 10 nM). Single strands are notated on the x-axis by the position within the ApoB mRNA which they target. All of the single stranded molecules are composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotides, a 5′ phosphate, and two 2′-O-methyl nucleotides at the 3′ terminus.
FIG. 10 compares ApoB mRNA knockdown at 100 nM concentration using single stranded molecules targeting each of the 30 different ApoB target sites tested in FIGS. 8 and 9—single strands without a C3 spacer (“all-flu-p”), with a C3 spacer at position 18 (“all-flu-c3-18-p”), and with a C3 spacer at position 19 (“all-flu-c3-19-p”). All of the single stranded molecules are composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotides, a 5′ phosphate, and two 2′-O-methyl nucleotides at the 3′ terminus.



DETAILED DESCRIPTION OF THE INVENTION
A. Terms and Definitions
The following terminology and definitions apply as used in the present application.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range, and when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
“About” or “approximately,” as used herein, in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.
As used herein, the terms “including” (and any form thereof, such as “includes” and “include), “comprising” (and any form thereof, such as “comprise” and “comprises”), “having” (and any form thereof, such as “has” or “have”), or “containing” (and any form thereof, such as “contains” or “contain”) are inclusive and open-ended and do not exclude additional, un-recited elements or method steps.
“Analog” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a compound or molecule that is structurally similar to a parent compound or molecule (e.g., a nucleotide, a naturally-occurring miRNA), but differs slightly in composition (e.g., one atom or functional group is different, added, or removed). The analog may or may not have different chemical or physical properties than the original parent compound or molecule and may or may not have improved biological or chemical activity. For example, the analog may be more hydrophilic or it may have altered activity of the parent compound/molecule. The analog may be a naturally or non-naturally occurring (e.g., chemically-modified or recombinant) variant of the original parent compound/molecule. An example of an RNA analog is an RNA molecule comprising a nucleotide analog. A nucleotide analog is a nucleotide that is chemically-modified at the sugar, base or nucleoside, as is generally recognized in the art.
As used herein, the term “mimetic” refers to its meaning as is generally accepted in the art. The term generally refers to a molecule that is structurally different from a reference molecule. For example, a reference molecule for purposes of certain embodiments of the present invention can be a naturally-occurring miRNA molecule, or a single-stranded siRNA molecule, that does not contain a non-nucleotide internal spacer. The mimetic is capable of performing one or more or all of the biological, physiological, and/or chemical functions that are within the capabilities of the reference molecule. The mimetic and the reference molecule do not have to be functional equivalents, but the mimetic should be able to perform one or more functions and exhibit at least 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the reference molecule, as measured and compared using assays or parameters that are suitable to represent the shared function(s). The terms “analog” and “mimetic,” when describing an RNAi molecule of the disclosure that is structurally different from a reference RNAi molecule, can be used interchangeably.
The term “nucleotide” refers to its meaning as is generally recognized in the art. Nucleotides generally comprise a nucleobase, a sugar, and an internucleoside linkage, e.g., a phosphate. The base can be a natural base (standard), a modified base, or a base analog, as are well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Additionally, the nucleotides can be unmodified or modified at the sugar, internucleoside linkage, and/or base moiety (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, and non-standard nucleotides); see, for example, U.S. application Ser. No. 12/064,014.
The terms “polynucleotide” and “oligonucleotide” as used herein refer to the meaning as is generally accepted in the art. The terms generally refer to a chain of nucleotides. “Nucleic acids” and “nucleic acid molecules” are polymers of nucleotides. Thus, “nucleic acids,” “polynucleotides” and “oligonucleotides” are interchangeable herein. One skilled in the art has the general knowledge that nucleic acids are polynucleotides which can be hydrolyzed into monomeric nucleotides. Monomeric nucleotides can be further hydrolyzed into nucleosides.
By “a contiguous stretch of nucleotides” is meant a continuous series of at least 2 nucleotides. The bonds connecting the nucleotides within the stretch are phosphodiester bonds.
The term “RNA” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a molecule comprising at least one ribofuranoside residue, such as a ribonucleotide. The term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The term refers to a double-stranded RNA, a single-stranded RNA, an isolated RNA such as a partially purified RNA, an essentially pure RNA, a synthetic RNA, a recombinantly-produced RNA, or an altered RNA that differs from a naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides therein. Such alterations can include addition of non-nucleotide material, for example, at one or more non-terminal nucleotides of an RNA molecule. As such, nucleotides in the single-stranded RNA molecules of the invention can comprise non-standard nucleotides, such as non-naturally occurring nucleotides, chemically-synthesized and/or modified nucleotides, or deoxynucleotides. The altered RNA is referred to as a “modified RNA” or a “RNA analog.”
The term “pyrimidine” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to conventional pyrimidine bases, including the standard pyrimidine bases uracil, thymidine, and cytosine. In addition, the term pyrimidine is contemplated to embrace non-standard pyrimidine bases or acids, such as 5-methyluracil, 2-thio-5-methyluracil, 4-thiouracil, pseudouracil, dihydrouracil, orotate, 5-methylcytosine, or the like, as well as a chemically-modified bases or “universal bases,” which can be used to substitute for a standard pyrimidine within the nucleic acid molecules of this disclosure.
The term “purine” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to conventional purine bases, including the standard purine bases adenine and guanine. In addition, the term “purine” is contemplated to embrace non-standard purine bases or acids, such as N2-methylguanine, inosine, diaminopurinc and the like, as well as chemically-modified bases or “universal bases,” which can be used to substitute for standard purines herein.
As described herein, a “base pair” can be formed between two nucleotides, a nucleotide and a modified nucleotide, two modified nucleotides, a nucleotide and a nucleotide analog, two nucleotide analogs, a nucleotide and a non-nucleotide substitute moiety, or two non-nucleotide substitute moieties. In a specific embodiment, a non-nucleotide substitute can comprise any chemical moiety that is capable of associating with a component of the cellular RNAi machinery, such as, for example, the PAZ domain, the PIWI domain, and/or other Argonaute protein domains associated with the RISC. Non-traditional Watson-Crick base pairs are also understood as “non-canonical base pairs,” which is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CU reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.
As used herein, the term “complementary” (or “complementarity”) refers to its meaning as is generally accepted in the art. The term generally refers to the formation or existence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of bonds as described herein. With reference to exemplary nucleic acid molecules of the invention, complementarity can be found between a single-stranded RNAi of the invention and an RNA target sequence. The binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et at, 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785).
As used herein, the term “perfectly complementary” (or “perfect complementarity”) between a first nucleic acid molecule (e.g., a single-stranded RNAi molecule of the present invention) and the second nucleic acid molecule (e.g., a target RNA sequence) means that all the contiguous residues of the first nucleic acid sequence will hydrogen bond with the same number of contiguous residues in the second nucleic acid sequence. For example, two or more perfectly complementary nucleic acid strands can have the same number of nucleotides (i.e., have the same length and form one double-stranded region with or without an overhang), or have a different number of nucleotides (e.g., one strand may be shorter but fully contained within a second strand). As an example, if a single-stranded RNAi molecule of the invention has a first nucleotide portion of only 1 nucleotide and a second nucleotide portion of 10 contiguous nucleotides, wherein all of the 10 nucleotides in the second nucleotide portion of the molecule base pair with the RNA target sequence, the RNAi molecule is perfectly complementary with the RNA target sequence. The single nucleotide included in the first nucleotide portion is not included when determining the degree of complementarity because it is not within a contiguous chain of nucleotides. However, in this example, if the first nucleotide portion contains 2 nucleotides, the RNAi molecule is perfectly complementary to the RNA target sequence if the 2 nucleotides of the first nucleotide portion and the 10 nucleotides of the second nucleotide portion base pair with the RNA target sequence.
Complementary nucleic acid molecules may have wrongly paired bases—that is, bases that cannot form a traditional Watson-Crick base pair (i.e., forming a hydrogen bond) or other non-traditional types of base pair (i.e., “mismatched” bases, formed or held together by non-traditional forces that are not hydrogen bonds). The term “partially complementary” (or “partial complementarity”) between a first nucleic acid molecule (e.g., a single-stranded RNAi molecule of the present invention) and second nucleic acid molecule (e.g., a target RNA sequence) indicates that various mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches or non-based paired nucleotides) occur between the nucleotide sequences, which can result in, for example, in bulges or loops. Such partial complementarity can be represented by a percent (%) complementarity that is determined by the number of base paired nucleotides in relation to the total number of nucleotides involved, e.g., about 50%, 60%, 70%, 80%, 90% etc. For example, a first nucleic acid molecule may have 10 nucleotides and a second nucleic acid molecule may have 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules, which may or may not form a contiguous double-stranded region, represents 50%, 60%, 70%, 80%, 90%, or 100% complementarity, respectively. In relation to the present invention, such partial complementarity is permitted to the extent that a single-stranded RNAi molecule of the invention maintains its function, for example the ability to mediate sequence specific RNAi.
A first nucleic acid molecule can be “substantially complementary” to a second nucleic acid. By “substantially complementary” it is meant that a first nucleic acid sequence (e.g., a single-stranded RNAi molecule of the present invention) is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to a second nucleic acid sequence (e.g., a RNA target sequence). As used herein, a first nucleic acid molecule can be both “partially complementary” and “substantially complementary” to a second nucleic acid molecule.
As used herein, the term “homologous” (or “homology”) refers to its meaning as is generally accepted in the art. The term generally refers to the number of nucleotides of the subject nucleic acid sequence that has been matched to identical nucleotides of a reference nucleic acid sequence, typically as determined by a sequence analysis program (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877) or by visual inspection. The term “perfect homology” (or “perfectly homologous”) as used herein refers to complete (100%) homology or “identity” between a reference sequence and a subject nucleic acid sequence. As used herein, the term “substantially homologous” (or “substantial homology”) is meant that the subject sequence shares at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) homologous nucleotides with the nucleotides of the same nucleotide positions in a reference sequence.
The phrase “chemical modification” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to any modification of the chemical structure of the nucleotides that differs from nucleotides of native RNA. The term “chemical modification” encompasses the addition, substitution, or modification of native RNA at the sugar, base, or internucleotide linkage, as described herein or as is otherwise known in the art. In certain embodiments, the teen “chemical modification” can refer to certain forms of RNA that are naturally-occurring in certain biological systems, for example 2′-O-methyl modifications or inosine modifications.
The phrase “modified nucleotide” as used herein refers to its meaning as is generally accepted in the art. The term generally refers a nucleotide that contains a modification in the chemical structure of the base, sugar and/or phosphate of the unmodified (or natural) nucleotide, as is generally known in the art. Non-limiting examples of modified nucleotides are described herein and in U.S. application Ser. No. 12/064,014.
“Percent modification” refers to its meaning as is generally accepted in the art. As used herein, the term generally refers to the number of nucleotides in a single-stranded RNA molecule of the invention that have been modified. The extent of chemical modifications will depend upon various factors well known to one skilled in the art (e.g., target RNA, off-target silencing, degree of endonuclease degradation).
The term “phosphorothioate” refers to its meaning as is generally accepted in the art. The term generally refers to an internucleotide phosphate linkage comprising one or more sulfur atoms in place of an oxygen atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.
As used herein, the term “locked nucleic acid” (LNA) has the structure of the general Formula I:




X and Y are independently selected from the group consisting of —O—, —S—, —N(H)—, —N(R)—, —CH2—, or —CH— (if part of a double bond), —CH2—O—, CH2—S—, CH2—N(H)—, —CH2—N(R)—, —CH2—CH2—, and CH2—CH— (if part of a double bond), —CH—CH—, where R is selected from hydrogen and C1-4-alkyl; Z and Z* are independently selected from an internucleotide linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleobase; and the asymmetric groups may be found in either orientation.

The four chiral centers of Formula I, as shown, are in a fixed configuration, but their configurations are not necessary fixed. As such, the chiral centers can be found in different configurations, such as those represented in Formula II (below). Thus, each chiral center in Formula 1 can exist in either R or S configuration. The definition of R (rectus) and S (sininster) are described in the IUPAC 1974 Recommendations, Section E, Fundamental Stereochemistry: The rules can be found in Pure Appl. Chem. 45, 13-30 (1976) and In “Nomenclature of Organic Chemistry” Pergamon, N.Y., 1979.
The terminal groups are selected independently among from hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O-, Act-O-, mercapto, Prot-S-, Act-S-, C1-6-alkylthio, amino, Prot-N(RH)—, Act-N(RH)—, mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-6-alkynyl, optionally substituted C2-6-alkynyloxy, monophosphate, monothiophosphate, diphosphate, dithiophosphate triphosphate, trithiophosphate, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH2-, Act-O—CH2—, aminomethyl, Prot-N(RH)—CH2—, Act-N(RH)—CH2—, carboxy methyl, sulphonomethyl, where Prot is a protection group for —OH, —SH, and —NH(RH), respectively, Act is an activation group for —OH, —SH, and —NH(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl.
The protection groups of hydroxy substituents comprises substituted trityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy (MMT), and trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl), optionally substituted methoxytetrahydro-pyranyloxy (mthp), silyloxy such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS)7 tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, and phenyldimethylsilyloxy, tert-butylethers, acetals (including two hydroxy groups), acyloxy such as acetyl or halogen substituted acetyls.
“Act” designates an activation group for —OH, —SH, and —NH(RH), respectively. Such activation groups are, for example, selected from optionally substituted O-phosphoramidite, optionally substituted O-phosphortriester, optionally substituted O-phosphordiester, optionally substituted H-phosphonate, and optionally substituted O-phosphonate.
B constitutes a natural or non-natural nucleobase and selected among adenine, cytosine, 5-methylcytosine, isocytosine, pseudoisocytosine, guanine, thymine, uracil, 5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine, and 2-chloro-6-aminopurine.
Preferably, the locked nucleic acid (LNA) used in a single-stranded RNAi molecule of the invention comprises a LNA structure according to any of the Formulas II:




wherein Y is —O—, —S—, —NH—, or N(RH); Z and Z* are independently selected among an internueleotide linkage, a terminal group or a protecting group; and B constitutes a natural or non-natural nucleobase. These exemplary LNA monomers and others, as well as their preparation are described in WO 99/14226 and subsequent applications, WO 00/56746, WO 00/56748, WO 00/66604, WO 00/125248, WO 02/28875, WO 2002/094250 and WO 2003/006475; the disclosure of all of which are incorporated herein by reference.

The term “universal base” refers to its meaning as is generally accepted in the art. The term generally refers to nucleotide base analogs that form base pairs with each of the standard ANA/RNA bases with little discrimination among them, and is recognized by intracellular enzymes. See, e.g., Loakes et al., 1997, J. Mol. Bio. 270:426-435. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carbozamides, and nitroazole derivatives such as 3′-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art. See, e.g., Loakes, 2001, Nucleic Acids Res. 29:2437.
As used herein, the phrase “RNA interference” (also called “RNAi” herein) refers to its meaning as is generally accepted in the art. The term generally refers to the biological process of inhibiting, decreasing, or down-regulating gene expression in a cell, and which is mediated by short interfering nucleic acid molecules (e.g., siRNAs, miRNAs, shRNAs), see for example Zamore and Haley, 2005, Science 309:1519-1524; Vaughn and Martienssen, 2005, Science 309:1525-1526; Zamore et al., 2000, Cell 101:25-33; Bass, 2001, Nature 411:428-429; Elbashir et al., 2001, Nature 411:494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, international PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237; Hutvagner and Zamore, 2002, Science 297:2056-60; McManus et al., 2002, RNA 8:842-850; Reinhart et al, 2002, Gene & Dev. 16:1616-1626; and Reinhart & Bartel, 2002, Science 297:1831). Additionally, the term “RNA interference” (or “RNAi”) is meant to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, single-stranded RNA molecules of the invention can be used to epigenetically silence genes at either the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by single-stranded RNA molecules of the invention can result from modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science 303:672-676; Pal-Bhadra et al., 2004, Science 303:669-672; Allshire, 2002, Science 297:1818-1819; Volpe et al., 2002, Science 297:1833-1837; Jenuwein, 2002, Science 297:2215-2218; and Hall et al., 2002, Science 297:2232-2237). In another non-limiting example, modulation of gene expression by single-stranded RNA molecules of the invention can result from cleavage of RNA (either coding or non-coding RNA) via RISC, or via translational inhibition, as is known in the art or modulation can result from transcriptional inhibition (see for example Janowski et al, 2005, Nature Chemical Biology 1:216-222).
The terms “inhibit,” “down-regulate,” “reduce” or “knockdown” as used herein refer to their meanings as are generally accepted in the art. With reference to exemplary single-stranded RNAi molecules of the invention, the terms generally refer to the reduction in the (i) expression of a gene or target sequence and/or the level of RNA molecules encoding one or more proteins or protein subunits, and/or (ii) the activity of one or more proteins or protein subunits, below that observed in the absence of the single-stranded RNAi molecules of the invention. Down-regulation can also be associated with post-transcriptional silencing, such as RNAi-mediated cleavage, or by alteration in DNA methylation patterns or DNA chromatin structure. Inhibition, down-regulation, reduction or knockdown with an RNAi agent can be in reference to an inactive molecule, an attenuated molecule, an RNAi agent with a scrambled sequence, or an RNAi agent with mismatches. The phrase “gene silencing” refers to a partial or complete loss-of-function through targeted inhibition of an endogenous target gene in a cell. As such, the term is used interchangeably with RNAi, “knockdown,” “inhibition,” “down-regulation,” or “reduction” of expression of a target gene.
To determine the extent of inhibition, a test sample (e.g., a biological sample from an organism of interest expressing the target gene(s) or target sequence(s) or a sample of cells in culture expressing the target gene/sequence) can be contacted with an RNAi molecule that silences, reduces, or inhibits expression of a target gene or sequence. Expression of the target gene/sequence in the test sample is compared to expression of the target gene/sequence in a control sample (e.g., a biological sample from an organism of interest expressing the target gene/sequence or a sample of cells in culture expressing the target gene/sequence) that is not contacted with the RNAi molecule. Control samples (i.e., samples expressing the target gene/sequence) are assigned a value of 100%. Silencing, inhibition, or reduction of expression of a target gene/sequence is achieved when the value of the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10%. Suitable assays include, e.g., examination of protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELBA, microarray hybridization, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
The phrase “improved RNAi activity” generally refers to an increase in RNAi activity measured in vitro and/or in vivo, where the RNAi activity is a reflection of either or both the ability of the RNAi agent to mediate RNAi and the stability of the RNAi agent.
The term “modulate” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to when the expression of a gene, or level of one or more RNA molecules (coding or non-coding), or activity of one or more RNA molecules or proteins or protein subunits, is up-regulated or down-regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the molecule that effects modulation. For example, the term “modulate” in some embodiments can refer to inhibition and in other embodiments can refer to potentiation or up-regulation, e.g., of gene expression.
The term “RNAi agent” or “RNAi molecule” refers to any nucleic acid molecule capable of inhibiting or down-regulating gene expression or viral replication by mediating RNA interference (“RNAi”) or gene silencing in a sequence-specific manner. The RNAi agent can be a double-stranded nucleic acid molecule comprising self-complementary sense (passenger) and antisense (guide) strands, wherein the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. An RNAi agent can be a single-stranded polynucleotide. While not wishing to be bound by theory, an RNAi agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA, or pre-transcriptional or pre-translational mechanisms.
The term “single-stranded RNAi” or “ssRNAi” agent or molecule is an RNAi agent that is a single-stranded, nucleic acid-derived molecule having a nucleotide sequence that is partially, substantially, or perfectly complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof. A second nucleotide sequence with which the single-stranded RNAi agent forms base-pairs is not present. A single-stranded RNAi molecule can further comprise a terminal phosphate group located at one or both of the terminal ends, such as a 5′-phosphate or a 5′, 3′-diphosphate. An ssRNAi molecule/agent can include a miRNA or a miRNA mimetic. A single-stranded RNAi agent of the invention can be loaded into or otherwise associated with RISC and participate in gene silencing via an RNAi mechanism. A single-stranded RNAi molecule of the invention can comprise substitutions, chemically-modified nucleotides, and non-nucleotides. A single-stranded RNAi molecule of the invention can comprise one or more or all ribonucleotides. Certain embodiments of the invention include single-stranded RNAi molecules that comprise substitutions or modifications in the backbone, sugars, bases, or nucleosides.
The term, “miRNA” or “microRNA” is used herein in accordance with its ordinary meaning in the art and refers to small, non-protein coding RNA molecules that are expressed in a diverse array of eukaryotes, including mammals, and are involved in RNA-based gene regulation. Mature, fully processed miRNAs are about 15 to about 30 nucleotides in length. A representative set of known, endogenous miRNA species is described in the publicly available miRBase sequence database, described in Griffith-Jones et al., Nucleic Acids Research, 2004, 32:D109-D111 and Griffith-Jones et al., Nucleic Acids Research, 2006, 34:D 140-D144, and accessible on the World Wide Web at the Welcome Trust Sanger Institute website. The mature, fully-processed miRNAs that are publicly available on the miRBase sequence database are each incorporated by reference herein. A representative set of miRNAs is also included herein in Table 1, infra. Each mature miRNA is partially complementary to one or more messenger RNA (mRNA) molecules, which are the targets of the miRNA, thereby regulating the expression of genes associated with the targets.
The term “miRNA mimetic,” as used herein, refers to a single-stranded RNA molecule that is a mimetic of a naturally-occurring miRNA in a cell. A miRNA mimetic is typically designed based on a corresponding, endogenous miRNA. A miRNA mimetic is capable of modulating the expression of a target mRNA that is also regulated by a corresponding, naturally-occurring miRNA. A single-stranded RNAi molecule of the present invention that is also a miRNA mimetic can be loaded into or otherwise associated with RISC and participates in gene silencing via an RNAi mechanism. A miRNA mimetic of the invention can comprise substitutions, chemically-modified nucleotides, and non-nucleotides. A miRNA mimetic of the invention can comprise one or more or all ribonucleotides. Certain embodiments of the invention include miRNA mimetics that comprise substitutions or modifications in the backbone, sugars, bases, or nucleosides. A naturally-occurring miRNA in a cell is referred to herein as “the corresponding miRNA,” “the endogenous miRNA,” or the “naturally-occurring miRNA.” A single-stranded miRNA mimetic of the invention that is provided to a cell is also understood to target one or more target mRNAs that are also targeted by a corresponding, naturally-occurring miRNA. It is contemplated that a miRNA mimetic of the present invention introduced to a cell is capable of functioning as a naturally-occurring miRNA under appropriate conditions.
As used herein, the term “seed region” (also referred to herein as a “seed sequence”) refers to its meaning as is generally accepted in the art. The term generally refers to at least 6 consecutive nucleotides within nucleotide positions 1 to 10 of the 5′-end of a naturally-occurring mature miRNA, such as one selected from those listed in the publicly available miRBase sequence database (www.mirbase.org) as of the filing date of the present application and/or one selected from those listed in Table 1. The seed sequence nucleotides of positions 1 to 8 are capitalized in the sequences of Table 1. In a naturally-occurring miRNA, the seed region typically determines the target mRNA sequence to which the miRNA may bind and provide gene regulation. As such, multiple naturally-occurring miRNAs can share a seed region or share substantial homology in the seed regions, and these miRNAs are members of the same miRNA family.
The term “siRNA” (also “short interfering RNA” or “small interfering RNA”) is given its ordinary meaning accepted in the art, generally referring to a duplex (sense and antisense strands) of complementary RNA oligonucleotides which may or may not comprise 3′ overhangs of about 1 to about 4 nucleotides and which mediate RNA interference.
The term “siRNA mimetic” or “single-stranded siRNA mimetic,” as used herein, refers to a single-stranded RNAi molecule that is a mimetic of the guide or antisense strand of a corresponding siRNA (either single or double-stranded). A siRNA mimetic is capable of modulating the expression of a target RNA that is also regulated by the corresponding siRNA and, thus, can be loaded into or otherwise associated with RISC and participates in gene silencing via an RNAi mechanism. A single-stranded siRNA mimetic of the invention can comprise substitutions, chemically-modified nucleotides, and non-nucleotides. A siRNA mimetic of the invention can comprise one or more or all ribonucleotides. Certain embodiments of the invention include siRNA nineties that comprise substitutions or modifications in the backbone, sugars, bases, or nucleosides.
The term “gene” as used herein, especially in the context of “target gene” for an RNAi agent, refers to the meaning as is generally accepted in the art. The term generally refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide. The target gene can also include the UTR (i.e., untranslated region) or non-coding region of the nucleic acid sequence. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by the RNAi agents of the invention. RNAi agents targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). A target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. A cell containing a target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science 300:258-260.
The phrases “target site,” “target sequence,” and “target region,” as used herein, refer to their meanings as generally accepted in the art. The terms generally refer to a sequence within a target nucleic acid molecule (e.g., mRNA) that is “targeted,” e.g., for cleavage mediated by an RNAi molecule that contains a sequence within its guide/antisense region that is partially, substantially, or perfectly complementary to that target sequence. A “target site” for a single-stranded RNAi molecule of the present invention refers to a nucleic acid sequence that is partially, substantially, or perfectly complementary to the single-stranded RNAi agent. The target site may be within a coding or a non-coding (i.e., untranslated) region of a target RNA. The target site may be the target site for an endogenous miRNA for which the single-stranded RNAi molecule is a mimetic, in which case the “target site” can also be referred to as a “miRNA target site” or a “corresponding miRNA target site.”
The phrase “sense region” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleotide sequence of an RNAi molecule having complementarity to an antisense region of the RNAi molecule. In addition, the sense region of an RNAi molecule can comprise a nucleic acid sequence having homology or sequence identity with a target nucleic acid sequence. The sense region of an RNAi molecule is also referred to as the sense strand or the passenger strand.
The phrase “antisense region” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleotide sequence of an RNAi molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of an RNAi molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the RNAi molecule. The antisense region of an RNAi molecule is also referred to as the antisense strand or the guide strand.
As used herein, the term “spacer” refers to any chemical group(s) capable of linking either two nucleotides and/or non-nucleotide substitute moieties. As used in the present invention, the “spacer” can connect two nucleotides and/or non-nucleotide substitute moieties by traditional phosphodiester bonds or non-phosphodiester connectors. The spacer is typically an organic entity that is covalently bound to each nucleotide or non-nucleotide substitute and is other than the internucleotide linkages that form the backbone (i.e., the nucleobases which form complementary hybrids).
As used herein, the term “alkyl” is intended to include a saturated aliphatic hydrocarbon group, both branched and straight-chain, having a specified number of carbon atoms. The term “alkyl” also refers to non-aromatic cycloalkyl groups. Preferably, an alkyl group has from 1 to 20 carbons (i.e., C1-C20). For example, C1-C10, as in “C1-C10 alkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear, branched or cyclic arrangement (i.e., cycloalkyl). The term “cycloalkyl” means a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, “alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on, as well as cycloalkyls, including cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on. An alkyl group may be substituted, if indicated.
As used herein, the term “alkenyl” refers to a non-aromatic hydrocarbon radical, straight or branched, containing 2 or more carbon atoms and at least 1 carbon to carbon double bond. The term “alkenyl” also refers to non-aromatic cycloalkenyl groups. Preferably, the alkenyl group has from 1 to 20 carbons (i.e., C1-C20). Alkenyl groups include, for example, ethenyl, propenyl, butenyl and cyclohexenyl. An alkenyl group may contain double bonds and may be substituted, if indicated.
As used herein, the term “alkynyl” refers to a non-aromatic hydrocarbon radical, straight, or branched, containing 2 or more carbon atoms and at least 1 carbon to carbon triple bond. The term “alkynyl” also refers to non-aromatic cycloalkynyl groups. Up to 3 carbon-carbon triple bonds may be present. Preferably, the alkynyl group has from 1 to 20 carbons (i.e., C1-C20). Alkynyl groups include, for example, ethynyl, propynyl, butyryl and cyclooctynl. An alkynyl group may contain triple bonds and may be substituted, if indicated.
The term “aliphatic” as used herein in reference to a chemical group refers to an organic group composed of carbon and hydrogen which does not contain aromatic rings. Aliphatic structures can be cyclic and/or saturated. The carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings. They can also be joined by single bonds (alkanes), double bonds (alkenes) or triple bonds (alkynes). Besides hydrogen, other elements can be bound to the carbon chain or substituted for a carbon within the chain, the most common being oxygen, nitrogen, sulfur and chlorine.
The term “aromatic” as used herein in reference to a chemical group refers to an organic group containing a set of covalently-bound atoms with the following specific characteristics: (1) a delocalized conjugated π system, most commonly an arrangement of alternating single and double bonds; (2) coplanar structure, with all the contributing atoms in the same plane; (3) contributing atoms arranged in one or more rings; and, (4) a number of delocalized π electrons that is even, but not a multiple of 4. An aromatic structure can be composed solely of hydrocarbons (e.g., aryl). Other elements can be bound to or substituted for a carbon of the aromatic structure, the most common being oxygen, nitrogen, sulfur and chlorine (e.g., heteroaryl, substituted aryl, substituted heteroaryl).
The term “substituted” as used in reference to an aliphatic or aromatic organic structure (e.g., an alkyl, alkenyl, alkynyl, aryl) refers to the presence of additional chemical moieties and/or functional groups bound to the carbon chain. For example, a substituted hydrocarbon chain can include a hydrocarbon chain with a heteroatom (e.g., N, O, or S) bound to it. A substituted hydrocarbon chain can also include a hydrocarbon chain that is interrupted with a heteroatom. When substituted, the substituted group(s) is preferably, hydroxyl, halogen, cyano, C1-C4 alkoxy, —O, —S, NO2, SH, NH2, or NR1R2, where R1 and R2 independently are H or C1-C4 alkyl. A substituted alkyl includes oligomers or polymers of ethylene oxide, including but not limited polyethylene glycol (“PEG”).
The term “non-nucleotide” or “non-nucleic acid” refers to any chemical molecule, moiety, group or compound that is not a nucleotide.
As used herein, the term “substitute non-nucleotide moiety” (or “non-nucleotide substitute moiety”) refers to a chemical moiety that is capable of substituting for one or more nucleotides in a single-stranded RNAi molecule of the invention. Substitute non-nucleotide moieties are typically those that allow for non-traditional base-pairing (i.e., not forming traditional hydrogen bonds). In certain embodiments, substitute non-nucleotide moieties of the instant disclosure are those that are capable of associating or otherwise interacting with one or more components of the cellular RNAi machinery, including, for example, the PAZ domain, the PIWI domain and/or other Argonaute protein domains associated with the RISC.
The term “synthetic,” in certain embodiments herein, refers to nucleic acid molecules that are not produced naturally in a cell. The single-stranded RNAi molecules of the invention are typically synthetic.
In certain embodiments, a single-stranded RNAi molecule of the invention may be isolated. The term “isolated,” as used herein in relation to an oligonucleotide, generally refers to a nucleic acid molecule that exists in a physical form differing from any nucleic acid molecules of identical sequence as found in nature. “Isolated” does not require, although it does not prohibit, that the nucleic acid be physically removed from its native environment. For example, a nucleic acid can be said to be “isolated” when it includes nucleotides and/or internucleoside bonds not found in nature. A nucleic acid can be said to be “isolated” when it exists at a purity not found in nature, where purity can be adjudged with respect to the presence of nucleic acids of other sequences, with respect to the presence of proteins, with respect to the presence of lipids, or with respect to the presence of any other component of a biological cell, or when the nucleic acid lacks sequence that flanks an otherwise identical sequence in an organism's genome, or when the nucleic acid possesses sequence not identically present in nature. A single-stranded RNAi molecule of the present invention can be isolated by virtue of its having been synthesized in vitro. It will be understood, however, that isolated nucleic acids may be subsequently mixed or pooled together.
As used herein, “endogenous” refers its meaning as generally accepted in the art. The term generally refers to any material from or produced inside an organism, cell, tissue or system. As used herein, an “endogenous miRNA” is a naturally-occurring miRNA in a cell, tissue, organism, including a mammal, such as, for example, a human. “Exogenous” generally refers to any material introduced from or produced outside an organism, cell, tissue or system.
The term “expression” as used herein refers to its meaning as is generally accepted in the art. The term generally is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
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 marker genes or mRNA or other analyte indicative of the expression level of a gene of interest. Consequently in some embodiments, methods include a step of generating an RNA profile for a sample. The term “RNA profile” or “gene expression profile” refers to a set of data regarding the expression pattern for one or more gene or genetic marker in the sample (e.g., a plurality of nucleic acid probes that identify one or more markers).
By “capable of” is meant that, when RNAi activity is measured by a suitable in vivo or in vitro assay or method, a single-stranded RNAi molecule of the invention demonstrates at least 5% or more of the knockdown effect against a target sequence as compared to the knockdown effect achieved by the corresponding single-stranded RNAi molecule without the internal, non-nucleotide spacer portion(s). Preferably, a single-stranded RNAi molecule of the invention is capable of achieving 25% or more, 35% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or even 100% or more (i.e., equal or more potent RNAi activity) knockdown of the target than a corresponding RNAi molecule against the same target (e.g., a naturally-occurring miRNA or previously-identified siRNA guide strand).
A “vector” is a replicon, such as a plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), as well as other bacterial, yeast, or viral vectors, to which another nucleic acid segment may be operatively inserted so as to bring about replication or expression of the inserted segment. “Expression vector” refers to a vector comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
The terms “composition” or “formulation” as used herein refer to their generally accepted meaning in the art. These terms generally refer to a composition or formulation, such as in a pharmaceutically acceptable carrier or diluent, in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, inhalation, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect. As used herein, pharmaceutical formulations include formulations for human and veterinary use. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: Lipid Nanoparticles (see for example Semple et al., 2010, Nat Biotechnol. 28(2):172-6.); P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant 8:47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998J. Pharm. Sci. 87:1308-1315; Tyler et aL, 1999, FEBS Lett. 421:280-284; Pardridge et al., 1995, PNAS USA. 92:5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15:73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res. 26:4910-4916; and, Tyler et al., 1999, PNAS 96:7053-7058. A “pharmaceutically acceptable composition” or “pharmaceutically acceptable formulation” can refer to a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention to the physical location most suitable for their desired activity.
The terms “patient,” “subject,” “individual” and the like are used interchangeably herein, and refer to any animal or cells or tissues thereof, whether in vitro or in situ, amendable to the methods described herein. They typically refer to an organism, which is a donor or recipient of the single-stranded RNA molecules of this disclosure. In certain non-limiting embodiments, the patient, subject or individual is a mammal or a mammalian cell. In other non-limiting embodiments, the patient, subject or individual is a human or a human cell.
As used herein, the term “therapeutically effective amount” means an amount of a single-stranded RNAi molecule of the present disclosure that is sufficient to result in a decrease in severity of disease symptoms, an increase in frequency or duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease, in the subject (e.g., a mammal or human) to which it is administered. One of ordinary skill in the art can determine such therapeutically effective amounts based on such factors such as the subject's size, the severity of symptoms, and the particular composition or route of administration selected. For example, a therapeutically effective amount of a single-strand RNAi molecule of the invention, individually, in combination, or in conjunction with other drugs, can be used or administered at a therapeutically effective amount to a subject or by administering to a particular cells under conditions suitable for treatment, to, for example, decrease tumor size, or otherwise ameliorate symptoms associated with a particular disorder in the subject.
The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. The term “treatment” as used herein is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for diseases or disorders. Thus, for example, the term “treatment” includes the administration of an agent prior to or following the onset of a disease or disorder thereby preventing or removing all signs of the disease or disorder. As another example, administration of the agent after clinical manifestation of the disease to combat the symptoms of the diseases is also comprised by the term “treatment.”
The term “parenteral” as used herein refers to its meaning as is generally accepted in the art. The term generally refers methods or techniques of administering a molecule, drug, agent, or compound in a manner other than through the digestive tract, and includes epicutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
The phrase “systemic administration” as used herein refers to its meaning as is generally accepted in the art. The term generally refers in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
Other objects, features and advantages of the present invention will become apparent from the 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 the detailed description.
B. Single-Stranded RNAi Molecules of the Invention
The instant disclosure provides single-stranded RNA molecules comprising at least one internal, non-nucleotide spacer that links together two nucleotide portions of the molecule. Thus, a single-stranded RNA molecule of the present invention is not a continuous stretch of nucleotides but comprises more than one nucleotide portion separated by one or more non-nucleotide spacers, wherein the nucleotide portions contain one or more nucleotides, non-nucleotide substitute moieties, or a combination thereof. A single-stranded RNA molecule of the invention functions as a guide or antisense strand that is capable of inhibiting gene expression via an RNA interference mechanism and, thus, represents an RNAi agent. A single-stranded RNAi molecule of the invention comprises sequence that is partially, substantially or perfectly complementary to one or more RNA target sites in a cell.
A single-stranded RNAi molecule of the invention has a single-stranded oligonucleotide structure comprising (a) a nucleic acid portion separated into two or more nucleotide portions, and (b) an internal (as opposed to “terminal”) spacer portion comprising at least one non-nucleotide spacer portion, wherein the non-nucleotide spacer portion(s) covalently links two nucleotides, each within distinct nucleotide portions of the molecule. The nucleotide portions of a single-stranded RNAi molecule of the invention are separated by the non-nucleotide spacer portions, wherein each nucleotide portion contains at least one nucleotide.
In each embodiment of the invention, the nucleic acid portion of a single-stranded RNAi molecule contains at least two nucleotide portions, a first nucleotide portion (N1) (e.g., a 5′-nucleotide portion) and a second nucleotide portion (N2) (e.g., a 3′-nucleotide portion). The nucleic acid portion of a single-stranded RNAi molecule of the invention can comprise more than two nucleotide portions (e.g., a third nucleotide portion (N3), a fourth nucleotide portion (N4) etc.). Within each nucleotide portion of an RNAi molecule of the invention, the nucleotides and/or non-nucleotide moieties are connected by phosphodiester bonds and/or non-phosphodiester connectors. Importantly, the nucleotide portions of a single-stranded RNAi molecule of the invention are not complementary to each other and, thus, said portions do not form significant base-pairing.
In each embodiment of the invention, the internal spacer portion of a single-stranded RNAi molecule contains at least one non-nucleotide spacer portion (S1), referred to here in as a first non-nucleotide spacer portion. In one embodiment of the present invention, a single-stranded RNAi molecule contains one internal, non-nucleotide spacer portion. The internal spacer portion of a single-stranded RNAi molecule of the invention can comprise more than a first non-nucleotide spacer portion (e.g., a second non-nucleotide spacer portion (S2), a third non-nucleotide spacer portion (S3) etc.). In another embodiment, a single-stranded RNAi molecule contains two internal, non-nucleotide spacer portions.
The number of nucleotide portions within the nucleic acid portion of a single-stranded RNAi molecule of the present invention is dependent on the number of non-nucleotide spacer portions within the molecule, and vice versa. For example, if a single-stranded RNAi molecule contains two non-nucleotide spacer portions, it will generally contain three nucleotide portions, as follows: 5′-(first nucleotide portion)-(first non-nucleotide spacer portion)-(second nucleotide portion)-(second non-nucleotide spacer portion)-(third nucleotide portion)-3′. Each non-nucleotide spacer portion of a single-stranded RNAi molecule of the present invention can contain one or more non-nucleotide spacers.
Single-stranded RNAi molecules of the invention have a single-stranded oligonucleotide structure and mediate RNA interference against a target RNA. Single-stranded RNAi molecule of the invention can comprise: (a) a nucleic acid portion comprising a first nucleotide portion (N1) and a second nucleotide portion (N2), wherein said nucleic acid portion comprises at least 8 nucleotides that can base pair with a target site within a target RNA, and wherein the total number of nucleotides within the nucleic acid portion is from 8 to 26 nucleotides; and, (b) an internal spacer portion comprising at least a first non-nucleotide spacer portion (S1) that covalently links the first and second nucleotide portions. The first and second nucleotide portions are not self complementary. All of nucleotides (e.g., 8 to 26) of a single-stranded RNAi molecule of the invention, all located within the nucleic acid portion, are distributed between the nucleotide portions of the molecule, wherein each nucleotide portion contains at least one nucleotide.
In one embodiment, a single-stranded RNAi molecule of the invention comprises a nucleic acid portion containing a total of from 8 to 26 nucleotides or non-nucleotide substitute moieties (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides or non-nucleotide substitute moieties) distributed between the nucleotide portions of the oligonucleotide, wherein at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) of the nucleotides in the molecule can base pair with a target site within a target RNA. For example, a single-stranded RNAi molecule of the invention may contain 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 total nucleotides, wherein 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 of those nucleotides base pair with a target RNA. In one embodiment, the nucleic acid portion of a single-stranded RNAi molecule of the invention contains a total of from 15 to 21 (e.g., 15, 16, 17, 18, 19, 20, or 21) nucleotides. In another embodiment, the nucleic acid portion of a single-stranded RNAi molecule of the invention contains a total of from 18 to 20 (e.g., 18, 19, or 20) nucleotides. In a further embodiment, the nucleic acid portion of a single-stranded RNAi molecule of the invention contains a total of 19 or 20 nucleotides.
The total number of nucleotides or non-nucleotide moieties, or a combination thereof, that make up the nucleotide portions of a single-stranded RNAi molecule of the invention is distributed between those portions of the molecule in any number of ways. As an example, a single-stranded RNAi molecule comprising only one non-nucleotide spacer portion and two nucleotide portions (i.e., the first nucleotide portion and the second nucleotide portion) may have a total of 12 nucleotides. If the first nucleotide portion of the molecule contains a single nucleotide (e.g., at the 5′-terminus of the molecule), the second nucleotide portion of the molecule will contain 11 contiguous nucleotides. Alternatively, if the first nucleotide portion of the molecule contains 5 contiguous nucleotides, the second nucleotide portion of the molecule will contain 7 contiguous nucleotides. In each example, the total number of nucleotides in the molecule is 12. The nucleotides within the nucleotide portions of a single-stranded RNAi molecule of the invention are not complementary to each other and, thus, said portions can not form substantial base-pairing. Within each of the nucleotide portions of the molecule, the nucleotides and/or non-nucleotide moieties are connected by phosphodiester bonds and/or non-phosphodiester connectors.
At least 8 nucleotides within the nucleic acid portion of a single-stranded RNAi molecule of the invention can base pair with a target sequence within a target RNA. Thus, the single-stranded RNAi molecules of the invention comprise a sequence of contiguous nucleotides that is partially, substantially or perfectly complementary to an RNA target site, including a naturally-occurring RNA target site. In one embodiment, all of the contiguous nucleotides within the nucleic acid portion of a single-stranded RNAi molecule of the invention base pair with a target sequence within a target RNA (i.e., perfectly complementary). In another embodiment, at least 50% of the contiguous nucleotides within the nucleic acid portion of a single-stranded RNAi molecule of the invention base pair with a target sequence within a target RNA (i.e., substantially complementary). In another embodiment, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides within the nucleic acid portion of a single-stranded RNAi molecule of the invention base pair with a target sequence within a target RNA.
In one embodiment, a single-stranded RNAi molecule of the invention has a single-stranded oligonucleotide structure comprising: (a) two nucleotide portions, a first nucleotide portion (N1) and a second nucleotide portion (N2); and, (b) one internal, non-nucleotide spacer portion (S1); wherein the oligonucleotide contains a total of from 8 to 26 nucleotides or non-nucleotide substitute moieties (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides or non-nucleotide substitute moieties); and wherein at least 8 of the nucleotides of the molecule can base pair with a target site within a target RNA. The two nucleotide portions of a single-stranded RNAi molecule of this embodiment comprise, in sum, 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or, 26) nucleotides or non-nucleotide moieties, or a combination thereof, that are distributed between the two nucleotide portions in any number of ways (as described above). In one embodiment, the non-nucleotide spacer portion contains one non-nucleotide spacer. In another embodiment, the non-nucleotide spacer portion contains more than one non-nucleotide spacer (e.g., 2, 3, 4, or more). The spacer portion links the first and second nucleotide portions of the single-stranded RNAi molecule. Thus, the spacer portion is covalently linked to both the 3′-terminal nucleotide or non-nucleotide substitute moiety of the first nucleotide portion of the molecule and the 5′-terminal nucleotide or non-nucleotide substitute moiety of the second nucleotide portion of the molecule. The spacer portion of the molecule can be covalently connected to the phosphate backbone of the nucleotide portions (i.e., through the free phosphate of the two, linked nucleotides) by either traditional phosphodiester bonds or non-phosphodiester connectors.
In one embodiment, a single-stranded RNAi molecule of the invention comprises a contiguous nucleotide sequence that is partially, substantially or perfectly homologous to the guide strand of a naturally-occurring miRNA and, thus, functions as a miRNA mimetic. In another embodiment, a single-stranded RNAi molecule of the invention comprises a contiguous nucleotide sequence that is partially, substantially or perfectly homologous to either a single-stranded siRNA or the guide/antisense strand of a duplex siRNA and, thus, functions as a siRNA mimetic. The single-stranded siRNA or duplex siRNA may be known to inhibit gene expression via an RNAi mechanism.
If a single-stranded RNAi molecule of the present invention is an analog of a naturally-occurring miRNA, the naturally-occurring miRNA is referred to herein as “the corresponding miRNA,” and the single-stranded RNAi molecule represents a mimetic of the corresponding miRNA. A single-stranded miRNA mimetic of the present invention is designed based on a corresponding, naturally-occurring miRNA, wherein at least one non-nucleotide spacer portion is either inserted between two nucleotides of the miRNA guide strand sequence or substituted for one or more nucleotides of the miRNA guide strand sequence. A single-stranded miRNA mimetic of the present invention can be an analog of a mature miRNA sequence publicly available in the miRBase database and/or included within Table 1, infra (SEQ ID NOs: 1-1090).
In one embodiment, a single-stranded RNAi molecule as described herein represents a miRNA mimetic, wherein the RNAi molecule comprises a nucleic acid portion of two or more nucleotide portions and an internal spacer portion comprising at least one non-nucleotide spacer portion. As described above, if the nucleic acid portion of the molecule contains only two nucleotide portions (i.e., a first nucleotide portion and a second nucleotide portion), only one non-nucleotide spacer portion will be present. If the nucleic acid portion of the molecule contains three nucleotide portions, two non-nucleotide spacer portions will be present. Each non-nucleotide spacer portion can comprise more than one non-nucleotide spacer (e.g., 2, 3, 4 or more). In one embodiment, the nucleic acid portion of an miRNA mimetic of the invention consists of from 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides or non-nucleotide moieties, or a combination thereof, wherein at least 8 of the nucleotides can base pair with a naturally-occurring miRNA target site. A contiguous nucleotide sequence within the nucleic acid portion of an miRNA mimetic of the invention is partially, substantially or perfectly homologous to a naturally-occurring miRNA guide strand nucleotide sequence. In one embodiment, a contiguous nucleotide sequence within a nucleic acid portion of a single-stranded RNAi molecule of the invention comprises 5 to 8 (i.e., 5, 6, 7, or 8) contiguous nucleotides that are identical (or perfectly homologous) to the whole or a part of a seed sequence of a naturally-occurring miRNA. For example, in one embodiment, an 8 consecutive nucleotide sequence within a nucleotide portion of a single-stranded RNAi molecule is identical to all or a portion of the seed region of a naturally-occurring miRNA (see Table I, infra).
In one embodiment, a miRNA mimetic of the invention has a non-nucleotide spacer portion and two nucleotide portions, wherein the non-nucleotide spacer portion is inserted between two nucleotides of a corresponding, naturally-occurring miRNA sequence, separating the full-length, naturally-occurring miRNA into two distinct nucleotide portions. In another embodiment, more than one non-nucleotide spacer portion is present in a miRNA mimetic of the invention such that the nucleic acid portion of the miRNA mimetic is separated into more than two nucleotide portions. In such cases, the total nucleotide sequence of the miRNA mimetic is perfectly homologous to the corresponding, naturally-occurring miRNA nucleotide sequence. The difference between the naturally-occurring miRNA and the miRNA mimetic in this embodiment is the presence of a non-nucleotide spacer portion.
In another embodiment, a miRNA mimetic of the invention comprises a non-nucleotide spacer portion that substitutes for one or more nucleotides of a naturally-occurring miRNA guide strand sequence. For example, one or more nucleotides may be first deleted from a naturally-occurring miRNA guide strand sequence, leaving a gap in the sequence and producing at least two distinct nucleotide portions. A non-nucleotide spacer portion is then inserted into the gap, covalently linking the distinct nucleotide portions. Thus, in one embodiment, a single-stranded RNAi molecule of the invention represents a miRNA mimetic wherein one or more internal, non-nucleotide spacer portions takes the place of from one to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides of a corresponding, naturally-occurring miRNA sequence (see SEQ ID NOs: 1-1090). A miRNA mimetic may contain more than one non-nucleotide spacer portion. In one embodiment, a single-stranded miRNA mimetic of the invention comprises at least one non-nucleotide spacer portion in the place of from one to 4 (e.g., 1, 2, 3, or 4) nucleotides of a naturally-occurring miRNA nucleotide sequence. In another embodiment, a single-stranded miRNA mimetic of the invention comprises at least one internal, non-nucleotide spacer portion in the place of one or two nucleotides of a corresponding miRNA nucleotide sequence. The non-nucleotide spacer portion bridges the gap resulting from removal of the one or more nucleotides from a miRNA guide strand sequence, connecting by either traditional phosphodiester bonds or non-phosphodiester connectors to the phosphate backbone of the nucleotide portions of the molecule.
Single-stranded RNAi molecules of the invention can also represent an analog of the guide or antisense strand of a duplex or single-stranded siRNA. The duplex or single-stranded siRNA may be known to inhibit target gene expression, or have the potential of inhibiting target gene expression, via an RNAi mechanism. In such a scenario, the siRNA counterpart, and specifically the guide strand of the siRNA (whether single- or double-stranded), is referred to herein as “the corresponding siRNA” or “the corresponding siRNA guide strand,” and the single-stranded RNAi molecule represents a mimetic of the corresponding siRNA guide strand (i.e., “a single-stranded siRNA mimetic”). A single-stranded siRNA mimetic is designed based on the nucleotide sequence of a corresponding siRNA by either inserting one or more internal, non-nucleotide spacer portions within the nucleotide sequence of the corresponding siRNA nucleotide sequence or substituting one or more nucleotides of the corresponding siRNA nucleotide sequence with one or more non-nucleotide spacer portions.
In one embodiment, a single-stranded RNAi molecule of the invention represents a siRNA mimetic, wherein the nucleic acid portion of the single-stranded RNAi molecule comprises two or more nucleotide portions, and the internal spacer portion comprises at least one non-nucleotide spacer portion. As described above, if the nucleic acid portion of the RNAi molecule contains only two nucleotide portions (i.e., a first nucleotide portion and a second nucleotide portion), only one non-nucleotide spacer portion will be present. If the nucleic acid portion of the RNAi molecule contains three nucleotide portions, two non-nucleotide spacer portions will be present. A non-nucleotide spacer portion may comprise more than one non-nucleotide spacer (e.g., 2, 3, 4 or more). In one embodiment, the nucleic acid portion of a siRNA mimetic consists of from 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides or non-nucleotide moieties, or a combination thereof, wherein at least 8 of the nucleotides can base pair with the an RNA target site. The nucleic acid portion of a siRNA mimetic of the invention comprises a contiguous nucleotide sequence that is partially, substantially or perfectly homologous to a corresponding siRNA guide strand nucleotide sequence.
In one embodiment, a siRNA mimetic of the invention has a non-nucleotide spacer portion and two nucleotide portions, wherein the non-nucleotide spacer portion is inserted between two adjacent nucleotides of the corresponding siRNA nucleotide sequence, separating the corresponding siRNA nucleotide sequence into two distinct nucleotide portions. In another embodiment, a siRNA mimetic of the invention can have more than one non-nucleotide spacer portion such that the corresponding siRNA nucleotide sequence is separated into more than two nucleotide portions. In such cases, the total nucleotide sequence of the siRNA mimetic is perfectly homologous to the corresponding siRNA nucleotide sequence. The difference between the corresponding siRNA and the siRNA mimetic in this embodiment is the presence of a non-nucleotide spacer region(s).
In another embodiment, a siRNA mimetic of the invention comprises one or more non-nucleotide spacer portions that substitutes for one or more nucleotides of a corresponding siRNA guide strand nucleotide sequence. For example, one or more nucleotides may be first deleted from a corresponding siRNA nucleotide sequence, leaving a gap in the sequence and producing at least two distinct nucleotide portions. A non-nucleotide spacer portion is then inserted into the gap to link the distinct nucleotide portions. Thus, in one embodiment, a single-stranded RNAi molecule of the present invention represents a siRNA mimetic comprising at least one internal, non-nucleotide spacer portion, wherein said non-nucleotide spacer portion takes the place of from one to 4 (e.g., 1, 2, 3, or 4) nucleotides of a corresponding siRNA nucleotide sequence. The siRNA mimetic may contain more than one non-nucleotide spacer portion. In another embodiment, a single-stranded RNAi molecule of the present invention represents a siRNA mimetic comprising at least one internal, non-nucleotide spacer portion, wherein said non-nucleotide spacer portion takes the place of one or two nucleotides of a corresponding siRNA nucleotide sequence. The non-nucleotide spacer portion(s) bridges the gap resulting from removal of the one or more nucleotides from the siRNA guide strand sequence, connecting by either traditional phosphodiester bonds or non-phosphodiester connectors to the phosphate backbone of the nucleotide portions of the molecule.
In another embodiment, single-stranded RNAi molecules of the invention can be designed de novo for the purpose of knocking down expression of a particular RNA target, including a naturally-occurring RNA target. In this scenario, a target gene is first selected. One of skill in the art then identifies a portion of said gene (i.e., the target site), generally between about 8 and about 26 nucleotides in length, to target with a single-stranded RNAi molecule for gene silencing. In one embodiment of the invention, a contiguous nucleotide sequence within the nucleic acid portion of a single-stranded RNAi molecule described herein is partially, substantially or perfectly complementary to the identified target site sequence and partially, substantially or perfectly homologous to the complement of the corresponding target site sequence. The counterpart sequence of the single-stranded RNAi molecule in this scenario (i.e., a nucleotide sequence that is the complement of the target site sequence) is referred to herein as “the complement of the corresponding target site sequence.” The single-stranded RNAi molecule comprises two or more nucleotide portions and at least one internal, non-nucleotide spacer portion, as described in one or more of the embodiments above.
A single-stranded RNAi molecule of the present invention is capable of producing an RNA interference result. In the case of a single-stranded miRNA mimetic of the invention, the molecule is capable of modulating the expression of a target mRNA that is also regulated by a corresponding naturally-occurring miRNA.
The single-stranded RNAi molecules of the disclosure can further comprise a terminal phosphate group located at one or both of the terminal ends, such as a 5′-phosphate or a 5′, 3′-diphosphate. In some embodiments, a single-stranded RNAi molecule of the invention can comprise substitutions, chemically-modified nucleotides, and non-nucleotides. In certain other embodiments, a single-stranded RNAi molecule of the invention can comprise one or more or all ribonucleotides. Certain embodiments of the invention include single-stranded RNAi molecules that comprise substitutions or modifications in the backbone, sugars, bases, or nucleosides.
The internal, non-nucleotide spacer portion(s) of the single-stranded RNAi molecules of the disclosure, especially in situations where the total number of nucleotides in the resulting RNAi molecule is reduced compared to a corresponding RNAi agent of which the single-stranded RNAi molecule is an analog (e.g., a naturally-occurring miRNA; the guide strand of a siRNA with gene knockdown capability), reduces the susceptibility of the single-stranded RNAi molecule to endonucleases. The internal, non-nucleotide spacer portion(s) can also limit the damage of exonucleases, ultimately helping to preserve the integrity of the single-stranded RNAi agent. The spacer portion also represents an easily accessible region for connecting one or more moieties of interest to the RNAi molecule (e.g., a chemical moiety that facilitates cellular delivery). Therefore, even if the activity of a single-stranded RNAi molecule of this disclosure is somewhat reduced (e.g., by less than about 20%, or 30%, or even 40%) as compared to a corresponding single-stranded RNAi molecule without the spacer portion (e.g., a naturally-occurring miRNA; the guide strand of a previously identified siRNA with gene knockdown capability), the overall activity of the analog can be greater than that of its counterpart due to improved stability or delivery of the molecule. Additionally, since the yield of synthesis is usually higher for shorter RNA strands, the cost of large-scale synthesis in connection with therapeutic applications may also be substantially reduced using the single-stranded RNAi molecules of the present invention.
In one embodiment, a single-stranded RNAi molecule of the invention can be represented or depicted by Formula III:

5′N1-S1-N2 3′

wherein N1, representing a first nucleotide portion, consists of either one nucleotide or a contiguous stretch of nucleotides; S1, representing a non-nucleotide spacer portion, consists of one or more non-nucleotide spacers; and N2, representing a second nucleotide portion, consists of either one nucleotide or a contiguous stretch of nucleotides. The total number of nucleotides in N1 and N2 is from 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides, and at least 8 nucleotides of the molecule can base pair with a target site within a target RNA. The “nucleotide(s)” within N1 and N2 are either nucleotides, modified nucleotides, nucleotide analogs, or non-nucleotides substitute moieties, or a combination thereof. In one embodiment, individually, N1 and N2 can consist of between one and 25 nucleotides, wherein the sum of N1 and N2 is from 8 to 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides. N1 and N2 are not self complementary and, thus, cannot participate in substantial base-pairing with each other. Within a contiguous stretches of nucleotides of the molecule, the nucleotides are connected by phosphodiester bonds and/or non-phosphodiester connectors. The spacer portion (S1) is covalently attached to the 3′-terminal nucleotide of the first nucleotide portion (N1) and the 5′-terminal nucleotide of the second nucleotide portion of the molecule (N2). For example, the spacer portion can comprise one or more phosphoramidite spacers attached to the free phosphate group of the adjacent nucleotides by a phosphodiester bond. The spacer portion of the molecule (S1) can consist of a single non-nucleotide spacer or more than one non-nucleotide spacers linked together. If there is more than one non-nucleotide spacer within the S1 portion of the molecule, the spacers can be either the same (i.e., having the same structure) or different (i.e., having different structures). In the case where two non-nucleotide spacers are linked within the S1 portion of the molecule, each spacer is covalently attached to one nucleotide within the N1 and N2 portions of the molecule, respectively. If three non-nucleotide spacers are consecutively linked within the S1 portion of the oligonucleotide, the internal (second) spacer does not form a covalent bond with either the N1 or N2 portions of the molecule. Instead, the internal spacer is covalently attached to the first and third spacers, linking them together.

In another embodiment, a single-stranded RNAi molecule of the invention can be represented or depicted by Formula IV:

5′N1-S1-N2-S2-N3 3′

wherein N1, representing a first nucleotide portion, consists of either one nucleotide or a contiguous stretch of nucleotides; S1, representing a first non-nucleotide spacer portion, consists of one or more non-nucleotide spacers; N2, representing a second nucleotide portion, consists of either one nucleotide or a contiguous stretch of nucleotides; S2, representing a second non-nucleotide internal spacer portion, consists of one or more non-nucleotide spacers; and, N3, representing a third nucleotide portion, consists of either one nucleotide or a contiguous stretch of nucleotides. In one embodiment, the total number of nucleotides in N1, N2, and N3 is from 8 to about 26 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides, and at least 8 nucleotides of the molecule can base pair with a target site within a target RNA. The “nucleotide(s)” within N1, N2 and N3 are either nucleotides, modified nucleotides, nucleotide analogs, or non-nucleotides substitute moieties, or a combination thereof. In one embodiment, individually, the nucleotide portions (N1, N2, N3) can consist of between one and 24 nucleotides, wherein the sum of nucleotides within the molecule is from 8 to 26 nucleotides. The nucleotide portions of the RNAi molecule are not self complementary and, thus, cannot participate in substantial base-pairing with each other. Within each of the contiguous stretches of nucleotides, the nucleotides are connected by phosphodiester bonds and/or non-phosphodiester connectors. The spacer portions are covalently attached to terminal nucleotides of the nucleotide portions of the molecule. In one embodiment, a spacer portion comprises one or more phosphoramidite spacers attached to the free phosphate groups of adjacent nucleotides by phosphodiester bonds. Each spacer portion of the molecule can consist of a single non-nucleotide spacer or more than one non-nucleotide spacer linked together. If there is more than one non-nucleotide spacer within a spacer portion of the molecule, the spacers can be either the same (i.e., having the same structure) or different (i.e., having different structures). When two non-nucleotide spacers are linked within a spacer portion of the molecule, each spacer is covalently attached to a terminal nucleotide within the adjacent nucleotide portions of the molecule. If three non-nucleotide spacers are consecutively linked within a spacer portion of the molecule, the internal (second) spacer does not form a covalent bond with a nucleotide portion of the molecule. Instead, the internal spacer is covalently attached to the first and third spacers, linking them together.

In one aspect of the invention, at least one nucleotide portion of a single-stranded RNAi molecule described herein (e.g., N1, N2, or N3, as described in Formulas III and/or IV) is a contiguous stretch of nucleotides that consists of either from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides, from 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides, from 10 to 20 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides, from 13 to 20 (e.g., 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides, from 5 to 15 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides, or from 1 to 14 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides. In another aspect, the length of at least one nucleotide portion of a single-stranded RNAi molecule of the invention is selected from the group consisting of 18 contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguous nucleotides. In a still further aspect, the length of at least one nucleotide portion of a single-stranded RNAi molecule of the invention is selected from the group consisting of 13 contiguous nucleotides, 14 contiguous nucleotides, or 15 contiguous nucleotides. The length of at least one nucleotide portion of a single-stranded RNAi molecule of the invention can be 18 nucleotides. The length of at least one nucleotide portion of a single-stranded RNAi molecule of the invention can be 19 nucleotides. The length of at least one nucleotide portion of a single-stranded RNAi molecule of the invention can be 20 nucleotides. The length of at least one nucleotide portion of a single-stranded RNAi molecule of the invention can be 21 nucleotides.
In one embodiment, a single-stranded RNAi molecule of the invention is represented by Formula III, wherein N1 consists of 18 contiguous nucleotides; S1 consists of a non-nucleotide spacer; and N2 consists of two contiguous nucleotides. In another embodiment, a single-stranded RNAi molecule of the invention is represented by Formula III, wherein N1 consists of 19 contiguous nucleotides; S1 consists of a non-nucleotide spacer; and N2 consists of one nucleotide. In these embodiments, S1 can be a C3- or C6-alkyl spacer.
In another aspect of the invention, a nucleotide portion of a single-stranded RNAi molecule (e.g., N1, N2, or N3, as described by Formulas III and/or IV) is a contiguous stretch of nucleotides that comprises a sequence of at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc.) nucleotides that is substantially or perfectly complementary to an RNA target region. In another aspect, a nucleotide portion of a single-stranded RNAi molecule comprises a sequence of from 5 to 8 contiguous nucleotides that is substantially or perfectly complementary to a RNA target region. In this portion of the invention, said nucleotide portion of the molecule is a contiguous stretch of nucleotides that consists of either from 1 to 20 nucleotides, from 5 to 20 nucleotides, from 10 to 20 nucleotides, from 13 to 20 nucleotides, from 5 to 15 nucleotides, or from 1 to 14 nucleotides. In another aspect, said nucleotide portion is 18, 19, or 20 contiguous nucleotides in length. In a still further aspect, said nucleotide portion is 13, 14 or 15 contiguous nucleotides in length. In another aspect, the length of said nucleotide portion is selected from the group consisting of 18 contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguous nucleotides. In a still further aspect, the length of said nucleotide portion is selected from the group consisting of 13 contiguous nucleotides, 14 contiguous nucleotides, or 15 contiguous nucleotides. The length of said nucleotide portion can be 18 nucleotides. The length of said nucleotide portion can be 19 nucleotides. The length of said nucleotide portion can be 20 nucleotides. The length of said nucleotide portion can be 21 nucleotides.
In one embodiment, a nucleotide portion of a single-stranded RNAi molecule of the invention comprises from 5 to 8 (e.g., 5, 6, 7, or 8) contiguous nucleotides that are identical (or perfectly homologous) to the whole or a part of a seed sequence of a naturally-occurring miRNA sequence. In one embodiment, the naturally-occurring miRNA sequence is a sequence recited in Table 1, infra. For example, in one embodiment, a 6-nucleotide sequence within a nucleotide portion of a single-stranded RNAi molecule is identical to all or a portion of the seed region of a naturally-occurring miRNA sequence, including a naturally-occurring miRNA sequence selected from Table 1.
In one embodiment, a single-stranded RNAi molecule of the invention can be represented or depicted by Formula III or Formula IV. It should be appreciated that Formulas III and IV represent particular examples of single-stranded RNAi molecules of the present invention. Additional examples encompassed by the present invention include, but are not limited to, RNAi molecules having more than three nucleotide portions.
In one aspect of the present invention, a contiguous nucleotide sequence within the nucleic acid portion of a single-stranded RNAi molecule is partially, substantially, or perfectly homologous to a naturally-occurring endogenous miRNA or to a guide strand of a siRNA. In another aspect of the invention, a contiguous nucleotide sequence within the nucleic acid portion of a single-stranded RNAi molecule is partially, substantially, or perfectly complementary to a target site within a RNA target sequence. In another embodiment, at least one nucleotide portion of a single-strand RNAi molecule of the disclosure is partially, substantially or perfectly homologous to a region of a naturally-occurring endogenous miRNA or the guide strand of a siRNA and/or partially, substantially or perfectly complementary to a target site within a RNA target sequence.
The internal spacer portion of single-stranded RNAi molecules of the invention comprises at least a first non-nucleotide spacer portion. Said non-nucleotide spacer portion comprises a chemical group, typically an organic entity, covalently bound to, and thus linking, at least two nucleotides. The two nucleotides are within distinct nucleotide portions of the molecule. There is no particular limitation in the length of a non-nucleotide spacer portion as long as it does not severely impact the ability of the molecule to form traditional or non-traditional Watson-Crick base pairing with an RNA target sequence and/or to mediate RNAi. A non-nucleotide spacer portion can connect two nucleotides and/or non-nucleotide substitute moieties by traditional phosphodiester bonds or non-phosphodiester connectors. Single-stranded RNAi molecules of the invention comprising non-phosphodiester based connectors linking the nucleotides and/or non-nucleotides to a spacer include, for example, a peptide-based connector, such as one linking the units of an oligo peptide nucleic acid (PNA) (see Boffa et al., 2000, Gene Ther. Mol. Biol. 5:47-53).
Various non-nucleotide moieties as are provided herein or otherwise known in the art can be included within the internal spacer portion of the single-stranded RNAi molecules of the invention. The non-nucleotide spacers comprised within the internal spacer portion of a single-stranded RNAi molecule of the invention can include any non-nucleic acid spacer capable of linking either two nucleotides and/or non-nucleotide substitute moieties by either traditional phosphodiester bonds or non-phosphodiester connectors. The spacer is typically an aliphatic or aromatic organic entity and is other than the internucleotide linkages that form the backbone of the oligonucleotide (i.e., the nucleobases which form complementary hybrids).
Non-limiting examples of non-nucleotide spacers include the following: a polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, 1990, Nucleic Acids Res. 18:6353; Seela and Kaiser, 1987, Nucleic Acids Res. 15:3113; Cload and Schepartz, 1991, J. Am. Chem. Soc. 113:6324; Richardson and Schepartz, 1991, J. Am. Chem. Soc. 113:5109; Ma et al., 1993, Nucleic Acids Res. 27:2585; Ma et al., 1993, Biochemistry 32:1751; Durand et al., 1990, Nucleic Acids Res. 18:6353; McCurdy et al., 1991, Nucleosides & Nucleotides 70:287; Jaschke et al., 1993, Tetrahedron Lett. 34:301; Ono et al., 1991, Biochemistry 30:9914; and others.
In one embodiment of the invention, a spacer is an alkyl, alkenyl or alkynyl chain of from one to 20 carbons (i.e., C1 to C20), preferably from 1 to 12 carbons (i.e., C1 to C12), that is optionally substituted. The hydrocarbon chains can be substituted with additional chemical and/or functional groups (e.g., a moiety that binds specifically to a target molecule of interest).
A chemical moiety that provides additional functionality (e.g., specifically binds to a target molecule of interest or facilitates/enhances cellular delivery of the molecule) to a single-stranded RNAi molecule may be a part of the spacer or covalently attached or linked thereto (e.g., substituted). For example, an additional functional group can impart therapeutic activity to a single-stranded RNAi molecule by assisting in transferring the RNAi molecule compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of RNAi molecules of the invention.
Examples of specific conjugate molecules that may be incorporated within a non-nucleotide spacer itself and/or covalently attached thereto and are contemplated by the instant disclosure are small molecules, lipids or lipophiles, terpenes, phospholipids, antibodies, toxins, cholesterol, a protein binding agent (e.g., a ligand for a cellular receptor that can facilitate cellular uptake), a vitamin, negatively charged polymers and other polymers, for example proteins (e.g., human serum albumin), peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, and those described in, for example, U.S. Patent Publication No. 2005/0196781, and U.S. Patent Publication No. 2006/0293271, the disclosures of which are incorporated herein by reference. These compounds are expected to improve delivery and/or localization of single-stranded RNAi molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). For example, a conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. In other embodiments, a conjugate member is a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. In another embodiment, a conjugate molecule is a peptide that functions, when conjugated to a single-stranded RNAi molecule, to facilitate delivery of the molecule into a target cell, or otherwise enhance delivery, stability, or activity of the molecule when contacted with a biological sample. Exemplary peptide conjugate members for use within these aspects of this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509 as described, for example, in U.S. Patent Application Publication Nos, 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application No. 60/939,578, which are all incorporated herein by reference.
In one embodiment, a non-nucleotide spacer comprises a moiety that specifically binds to a target molecule. The target molecule can be any molecule of interest. For example, the target molecule can be a ligand-binding domain of a protein, thereby preventing or competing with the interaction of the naturally-occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al, 1995, Annu. Rev. Biochem. 64:163; Brody and Gold, 2000, 1 Biotechnol. 74:5; Sun, 2000, Curr. Opin. Mol. Ther. 2:100; Kusser, J., 2000, Biotechnol. 74:21; Hermann and Patel, 2000, Science 257:820; and Jayasena, 1999, Clinical Chem. 45:1628). The spacer portion of a single-stranded RNAi molecule of this disclosure can also conveniently be used to introduce functional chemical groups to an RNAi molecule to enhance properties associated with cellular delivery.
In one embodiment, a conjugate molecule or functional chemical moiety attached via a spacer region of a single-stranded RNAi molecule provides the ability to administer said RNAi molecule to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262:4429) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell 22: 611; Connolly et al., 1982, J. Biol. Chem. 257:939). Lee and Lee (1987, Glycoconjugate J. 4:317) obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem. 24: 1388). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of bioconjugates of this disclosure
Conjugate molecules described herein can be attached to a single-stranded RNAi molecule via non-nucleic acid linkers that are biodegradable. The term “biodegradable linker,” as used in this context, refers to a non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, connecting a conjugate molecule to a single-stranded RNAi molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The term “biodegradable,” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
In one embodiment, a single-stranded RNAi molecule of the invention comprises an internal spacer portion comprising one or more non-nucleotide spacer portions, wherein said one or more non-nucleotide spacer portions (e.g., S1 or S2 within Formula III and IV) comprise or consist of a non-nucleotide spacer selected from the group consisting of a C3, a C6, a C9, and a C12 aliphatic spacer. The number after the “C” indicates the number of carbon atoms in the core spacer structure (e.g., if unsubstituted with additional chemical moieties). Said spacers can be alkyl, alkenyl, or alkynyl groups. Said spacers can also contain phosphoramidite moieties to facilitate covalent linkage to the phosphate backbone of the nucleotide portions of the molecule. In one embodiment, the spacer (S) portion is a C3 phosphoramidite spacer. In another embodiment, the spacer is a C6 phosphoramidite spacer. In a further embodiment, the C3, C6, C9, or C12 spacers are optionally substituted (e.g., with a targeting moiety).
One or more or all of the nucleotides within the nucleotide portions of a single-stranded RNAi molecule of the disclosure may be ribonucleotides, modified ribonucleotides, or suitable nucleotide analogs. Incorporation of nucleotide analogs, such as various known sugar, base, and backbone modifications, and LNA monomer units into disrupted strands may significantly enhance serum stability and prolong target knockdown or expression regulatory effects. The single-stranded RNA molecules of the present invention can functionally accommodate and are compatible with various chemical modifications to varying degrees. For example, from 5% to 100% of the ribonucleotides of a single-stranded RNA molecule of the invention may be modified (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the ribonucleotides of the single-stranded RNAi molecules of the invention may be chemically modified or are replaced with nucleotide analog residues). The improved properties conferred by the functionally compatible chemical modifications to the sugar, base and/or backbone, or by including suitable nucleotide analog residues, are of particular importance for application of these single-stranded RNAi molecules in vivo, for example, for use as a therapeutic agent or as a functional genomic tool.
In a further aspect, a single-stranded RNAi molecule of the invention, according to any of the embodiments herein, are capable of participating in RNAi against a RNA target, including an endogenous RNA target. In one embodiment, the endogenous RNA target is the target of a naturally-occurring miRNA. The inhibition of the RNA target may be achieved via the standard RNA-specific interference mechanism, including miRNA-dependent RNA interference. For example, the inhibition of a miRNA target may be by interaction (e.g., base-pairing, binding, etc.) with the untranslated mRNA region, with which a corresponding endogenous miRNA interacts, which effectuates the translational regulation of one or more downstream genes. Alternatively, the inhibition of a miRNA target may be achieved via a siRNA-like interference mechanism wherein the binding of the miRNA target by the single-stranded RNAi molecule of the invention that is a miRNA mimetic results in the cleavage of the untranslated miRNA target. The single-stranded RNAi molecules of the invention may also inhibit mRNA target via a siRNA-like interference mechanism where the binding of the mRNA target in the sequence coding region (rather than in the non-coding untranslated region) by the single-stranded RNAi molecule of the invention results in cleavage of an mRNA target coding sequence.
C. Substituted and/or Modified Single-Stranded RNAi Molecules
The introduction of substituted and modified nucleotides into single-stranded RNAi molecules of the invention provides a tool for overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules (i.e., having standard nucleotides) that are exogenously delivered. In certain embodiments, the use of substituted or modified single-stranded RNAi molecules of this disclosure can enable achievement of a given therapeutic effect at a lower dose since these molecules may be designed to have an increased half-life in a subject or biological samples (e.g., serum). Furthermore, certain substitutions or modifications can be used to improve the bioavailability of single-stranded RNAi molecules by targeting particular cells or tissues or improving cellular uptake of the single-stranded RNAi molecules. Therefore, even if the activity of a single-stranded RNAi molecule of this disclosure is somewhat reduced (e.g., by less than about 20%, or 30%, or even 40%) as compared to an unmodified or unsubstituted RNAi molecule of the same structure, the overall activity of the substituted or modified RNAi molecule can be greater than that of its native counterpart due to improved stability or delivery of the molecule. Substituted and/or modified single-stranded RNAi molecules can also minimize the possibility of activating an interferon response in, for example, humans.
In certain embodiments, single-stranded RNAi molecules of the invention comprise ribonucleotides at about 5% to about 95% of the nucleotide positions. For example, from one to all (e.g., 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, or 27) of the ribonucleotides of the single-stranded RNAi molecules of the invention can be modified.
In related embodiments, a single-stranded RNAi molecule according to the instant disclosure comprises one or more natural or synthetic non-standard nucleoside. In related embodiments, the non-standard nucleoside is one or more deoxyuridine, L- or D-locked nucleic acid (LNA) molecule (e.g., a 5-methyluridine LNA) or substituted LNA (e.g., having a pyrene), or a universal-binding nucleotide, or a G clamp, or any combination thereof. In certain embodiments, the universal-binding nucleotide can be C-phenyl, C-naphthyl, inosine, azole carboxamide, 1-β-D-ribofuranosyl-4-nitro indole, 1-β-D-ribofuranosyl-5-nitroindole, 1-β-D-ribofuranosyl-6-nitroindole, or 1-β-D-ribofuranosyl-3-nitropyrrole.
Substituted or modified nucleotides, which can be present in the single-stranded RNAi molecules of the invention, comprise modified or substituted nucleotides having characteristics similar to natural or standard ribonucleotides. For example, this disclosure features single-stranded RNAi molecules comprising nucleotides having a Northern conformation (see, e.g., Northern pseudorotation cycle, Saenger, Springer-Verlag ed., 1984), which are known to potentially impart resistant to nuclease degradation while maintaining the capacity to mediate RNAi, at least when applied to siRNA molecules. Exemplary nucleotides having a Northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethyl (MOE) nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 5-methyluridines, or 2′-O-methyl nucleotides). In any of these embodiments, one or more substituted or modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy)phenoxazine). See, e.g., Lin and Mateucci, 1998, J. Am. Chem. Soc. 720:8531.
In certain embodiments, the 5′-terminal end of single-stranded RNAi molecules of the invention is phosphorylated. In any of the embodiments of single-stranded RNAi molecules described herein, the molecule can further comprise a terminal phosphate group, such as a 5′-phosphate (see Martinez et al., 2002, Cell 110:563; Schwarz et al., 2002, Mole. Cell 70:537) or a 5′3′-diphosphate.
In another aspect, a single-stranded RNAi molecule of the invention comprises one or more 5′- and/or a 3′-cap structure at the terminal ends of the molecule. By “cap structure” is meant chemical modifications, which have been incorporated into the ends of oligonucleotide (see, for example, Matulic-Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications can protect certain nucleic acid molecules from exonuclease degradation, and can impart certain advantages in delivery and/or cellular localization. In non-limiting examples: a suitable 5′-cap can be one selected from the group comprising inverted abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.
In another non-limiting example, a suitable 3′-cap can be selected from a group comprising, 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties. For more details, see Beaucage and Iyer, 1993, Tetrahedron 49:1925, which is incorporated by reference herein.
In certain embodiments, this disclosure features modified single-stranded RNAi molecules comprising phosphate backbone modifications, including, for example, one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyimide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331; Mesmaeker et al., 1994, ACS 24-39.
In further embodiments, a single-stranded RNAi molecule comprises one or more (e.g., 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, or 26) 2′-sugar substitutions, such as a 2′-deoxy, 2′-O-2-methoxyethyl, 2′-O-methoxyethyl, 2′-O-methyl, 2′-halogen (e.g., 2′-fluoro), 2′-O-allyl, or the like, or any combination thereof. In still further embodiments, a single-stranded RNAi molecule comprises a terminal cap substituent at one or both terminal ends, such as, for example, an alkyl, abasic, deoxy abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety, or any combination thereof. In certain embodiments, at least one 5′-terminal-end ribonucleotide has a 2′-sugar substitution.
In other embodiments, a single-stranded RNAi molecule comprises one or more (e.g., 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, or 26) substitutions in the sugar backbone, including any combination of ribosyl, 2′-deoxyribosyl, a tetrafuranosyl (e.g., L-α-threofuranosyl), a hexopyranosyl (e.g., β-allopyranosyl, β-altropyranosyl and β-glucopyranosyl), a pentopyranosyl (e.g., β-ribopyranosyl, α-lyxopyranosyl, β-xylopyranosyl and α-arabinopyranosyl), a carbocyclic analog, a pyranose, a furanose, a morpholino, or analogs or derivatives thereof.
In yet other embodiments, a single-stranded RNAi molecule comprises at least one (e.g., 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, or 26) modified internucleoside linkage, such as independently a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate, 3′-alkylene phosphonate, 5′-alkylene phosphonate, chiral phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate, phosphoramidate, 3′-amino phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, boranophosphate linkage, or any combination thereof.
A single-stranded RNAi molecule can comprise one or more modified internucleotide linkages at the 3′-terminal end, the 5′-terminal end, or both of the 3′-terminal and 5′-terminal ends of the molecule. In one embodiment, a single-stranded RNAi molecule of the invention has one modified internucleotide linkage at the 3′-terminal end, such as a phosphorothioate linkage. An exemplary single-stranded RNAi molecule comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages. A further exemplary single-stranded RNAi molecule comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive phosphorothioate internucleotide linkages at, for example, the 5′-terminal end of the molecule. In yet another exemplary single-stranded RNAi molecule, there can be one or more pyrimidine phosphorothioate internucleotide linkages. In a further exemplary single-stranded RNAi molecule, there can be one or more purine phosphorothioate internucleotide linkages.
Many exemplary modified nucleotide bases or analogs thereof useful in single-stranded RNAi molecules of the instant disclosure include 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives of adenine and guanine; 8-substituted adenines and guanines (e.g., 8-aza, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the like); 7-methyl, 7-deaza, and 3-deaza adenines and guanines; 2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (e.g., 5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracils and cytosines; and 6-azouracil. Further useful nucleotide bases can be found in Kurreck, 2003, Eur. J. Biochem. 270:1628; Herdewijn, 2000, Guide Nucleic Acid Develop. 10:297; Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; U.S. Pat. No. 3,687,808, and similar references, all of which are incorporated by reference herein.
Certain substituted or modified nucleotide base moieties are also contemplated. These include 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, or 0-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine). Further, for example, 5-methyluridine and 5-methylcytosine substitutions are known to increase nucleic acid duplex stability, which can be combined with 2′-sugar modifications (e.g., 2′-O-methyl or 2′-methoxyethyl) or internucleoside linkages (e.g., phosphorothioate) that provide the desired nuclease resistance to the modified or substituted single-stranded RNAi molecule.
In further embodiments, at least one pyrimidine of a single-stranded RNAi molecule of the invention is a locked nucleic acid (LNA) in the form of a bicyclic sugar. In a related embodiment, the LNA comprises a base substitution, such as a 5-methyluridine LNA or 2-thio-5-methyluridine LNA. In further embodiments, a ribose of the pyrimidine nucleoside or the internucleoside linkage can be optionally modified.
In any of these embodiments, one or more substituted or modified nucleotides can be a G clamp (e.g., a cytosine analog that forms an additional hydrogen bond to guanine, such as 9-(aminoethoxy) phenoxazine). See, e.g., Lin and Mateucci, 1998, Nucleic Acids Res. 19:3111.
In any of the embodiments described herein, a single-stranded RNAi molecule may include multiple types of modifications. For example, a single-stranded RNAi molecule having at least one ribothymidine or 2-thioribothymidine can further comprise at least one LNA, 2′-methoxy, 2′-fluoro, 2′-deoxy, phosphorothioate linkage, an inverted base terminal cap, or any combination thereof. In certain exemplary embodiments, a single-stranded RNAi molecule can comprise one or more or all uridines substituted with ribothymidine and have up to about 75% LNA substitutions. In other exemplary embodiments, a single-stranded RNAi molecule can comprise from one or more or all uridines substituted with ribothymidine and have up to about 25% 2′-methoxy substitutions. In still other exemplary embodiments, a single-stranded RNAi molecule can comprise one or more or all uridines substituted with ribothymidine and have up to about 100% 2′-fluoro substitutions.
Within certain aspects, the present disclosure also provides single-stranded RNAi molecules comprising one or more universal base nucleotides. The term “universal base” as used herein refers to nucleotide base analogs that form base pairs or hydrogen bonded nucleotide pairs with more than one types of nucleotides. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxyamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see, e.g., Loakes, 2001, Nucleic Acids Research 29:2437-2447). In certain aspects, a single-stranded RNAi molecule disclosed herein can include about 1 to about 10 universal base nucleotides, so long as the resulting RNAi molecule remains capable of modulating one or more of its endogenous targets.
D. Synthesis of Single-Stranded RNAi Molecules
Exemplary molecules of the instant disclosure can be obtained using a number of techniques known to those of skill in the art. For example, the RNAi molecules of the invention can be chemically synthesized, recombinantly produced (e.g., encoded by plasmid), or a combination thereof.
Oligonucleotides or individual contiguous stretches of nucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example, as described in Caruthers et al., 1992, Methods in Enzymol. 211:3; Thompson et al, PCT Publication No. WO 99/54459; Wincott et al., 1995, Nucleic Acids Res. 23:2677; Wincott et al., 1997, Methods Mol. Bio. 74:59; Brennan et al., 1998, Biotechnol. Bioeng. 67:33; and Brennan, U.S. Pat. No. 6,001,311. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Synthesis of RNA without modifications, including certain single-stranded RNAi molecules thereof of this disclosure, can be made using the procedure as described in Usman et al., 1987, J Am. Chem. Soc. 109:7845; Scaringe et al., 1990, Nucleic Acids Res. 18:5433; and Wincott et al., 1995, Nucleic Acids Res. 23:2677; and Wincott et al., 1997, Methods Mol. Bio. 74:59. In certain embodiments, the nucleotide portions of the single-stranded RNAi molecules of the present disclosure can be synthesized separately and joined together with the non-nucleotide spacer portions post-synthetically, for example, by ligation (Moore et al., 1992, Science 256:9923; Draper et al., PCT Publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Res. 19:4247; Bellon et al., 1997, Nucleosides & Nucleotides 16:951; Belton et al., 1997, Bioconjugate Chem. 8:204). In a further embodiment, the nucleotide portion of a single-stranded RNAi molecule of this disclosure can be made as single or multiple transcription products expressed by a polynucleotide (DNA or RNA) vector encoding one or more contiguous stretches of RNAs and directing their expression within host cells. The nucleotide portions are then isolated and joined by ligation with a non-nucleotide spacer portion.
In some embodiments, poi III based constructs are used to express nucleic acid molecules of the invention. Transcription of the single-stranded RNAi molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). (see for example, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). (See also, Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Viral., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et at, 1997, Gene Therapy, 4, 45. Transcripts from poi II or poi III promoters are expressed at high levels in all cells; the levels of a given poi II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al, 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et at, 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et at, International PCT Publication No. WO 96/18736. The above transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
Chemically synthesizing nucleic acid molecules with substitutions or modifications (base, sugar, phosphate, or any combination thereof) can impart resistance to degradation by serum ribonucleases, which may lead to increased potency and other pharmacological and therapeutic benefits. See, e.g., Eckstein et al., PCT Publication No. WO 92/07065; Perrault et at, 1990, Nature 344:565; Pieken et al., 1991, Science 253:314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 77:334; Usman et al., 1994, Nucleic Acids Symp. Ser. 31:163; Beigelman et at, 1995, J. Biol Chem. 270:25702; Burlina et at, 1997, Bioorg. Med. Chem. 5:1999; Karpeisky et al., 1998, Tetrahedron Lett. 39:1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences) 48:39; Velma and Eckstein, 1998, Annu. Rev. Biochem. 67:99; Herdewijn, 2000, Guide Nucleic Acid Drug Dev. 10:291; Kurreck, 2003, Eur. J. Biochem. 270:1628; Dorsett and Tuschl, 2004, Nature Rev. Drug Discov. 3:318; Rossi et al., PCT Publication No. WO 91/03162; Usman et al., PCT Publication No. WO 93/15187; Beigelman et al., PCT Publication No. WO 97/26270; Woolf et al., PCT Publication No. WO 98/13526; Sproat, U.S. Pat. No. 5,334,711; Usman et al., U.S. Pat. No. 5,627,053; Beigelman et al., U.S. Pat. No. 5,716,824; Otvos et al., U.S. Pat. No. 5,767,264; Gold et al., U.S. Pat. No. 6,300,074. Each of the above references discloses various substitutions and chemical modifications to the base, phosphate, or sugar moieties of nucleic acid molecules, which can be used in the single-stranded RNAi molecules described herein.
E. Methods for Designing a Single-Stranded RNAi Molecule
As described herein, the single-stranded RNA molecules of the present invention are capable of inhibiting the expression of a target sequence via an RNAi mechanism. In one embodiment, the single-stranded RNAi molecules can be designed based on a previously identified RNAi agent possessing a desired knockdown function (e.g., siRNA, miRNA). For example, if a single-stranded RNAi molecule of the present invention is a miRNA mimetic, it is derived from a corresponding, naturally-occurring miRNA molecule (see Table 1) or an analog thereof (e.g., a chemically modified form). As of the filing date of the present application, over 3000 miRNA molecules endogenous to a variety of species can be found in publically available databases (see, e.g., the publicly available miRBase sequence database as described in Griffith-Jones et al., 2004, Nucleic Acids Research 32:D109-D111 and Griffith-Jones et al., 2006, Nucleic Acids Research 34:D 140-D144, accessible on the World Wide Web at the Wellcome Trust Sanger Institute website). Table 1 herein contains a list of 1090 mature human miRNA sequences (SEQ ID NO: 1-1090). In another example, a single-stranded RNAi molecule of the present invention may be derived from a previously identified siRNA either known to inhibit expression of a target sequence of choice or has the potential of inhibiting expression of a target mRNA sequence. Specifically, a single-stranded RNAi molecule that is derived from a previously identified RNAi molecule (i.e., the reference RNAi molecule) can be designed by introducing one or more internal, non-nucleotide spacers portions within the guide strand of the reference RNAi molecule. In another embodiment, the single-stranded RNAi molecules can be designed de novo (i.e., not based on a known RNAi agent) for the purpose of knocking down expression of a particular target sequence.
The RNAi activity of a given single-stranded RNAi molecule of the invention can be measured using known methods, such as those described generally in Fire et al., PCT Publication No. WO99/32619, and as described in the Examples section infra. In some embodiments, the instant specification provides methods for selecting more efficacious single-stranded RNAi molecule designs by using one or more reporter gene constructs comprising a constitutive promoter, such as a cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of altering the expression of one or more reporter genes, such as a luciferase, chloramphenicol (CAT), or β-galactosidase, which, in turn, is operably fused in-frame to a portion of the target sequence that is whole or partially complementary to the ssRNAi to be tested. These reporter gene expression constructs may be co-transfected with one or more ssRNAi molecules and a control (e.g., corresponding miRNA mimetic that does not contain the internal non-nucleotide spacer). The capacity of a given ssRNAi molecule to mediate RNAi of a target mRNA may be determined by comparing the measured reporter gene activity in cells transfected with the ssRNAi molecule and the activity in cells transfected with a negative control (i.e., in cells not transfected with the ssRNAi molecule) and a positive control (e.g., in cells transfected with the corresponding miRNA mimetic that does not contain the internal non-nucleotide spacer). The ssRNAi molecules having at least 20% or more, preferably at least 40% or more, or 60% or more, or 80% or more, of the activity of their corresponding RNAi molecule, for example, that do not contain internal non-nucleotide spacers, are selected.
A person of skill in the art can screen single-stranded RNAi molecules of this disclosure containing various non-nucleotide spacers to determine which of molecules possess improved properties (e.g., pharmacokinetic profile, bioavailability, stability) while maintaining the ability to mediate RNAi in, for example, an animal model as described herein or generally known in the art. Similarly, a person of skill in the art can also screen single-stranded RNAi molecules of this disclosure having various conjugates to determine which of the RNAi molecule-conjugate complexes possess improved properties while maintaining the ability to mediate RNAi.
F. Compositions and Methods of Use
As set forth herein, single-stranded RNA molecules of the invention are RNAi agents preferably capable of participating in the cellular RNAi pathway or otherwise capable of modulating the same or related pathway(s) and resulting in the inhibition of a target gene associated with a pathological or diseased condition. In the case of a single-stranded RNA molecule that represents a miRNA mimetic, the ssRNAi molecule is designed to supplement or take the place of a corresponding, naturally-occurring miRNA, the reduced or otherwise unsuitably low levels of which have been associated with pathological or diseased conditions. The single-stranded RNAi molecules of the invention thus are useful reagents, which can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
A single-stranded RNA molecule of the invention can be introduced to a cell, tissue, organism, in vitro system, or in vivo system to mediate RNAi against a target sequence. That target sequence may be an endogenous target gene or sequence. In one embodiment, the single-stranded RNAi molecules of the invention can be used for treating organisms having a disease characterized by the undesired production of a protein.
In the case of a single-stranded RNAi molecule of the invention that is a miRNA mimetic, the target sequence is the target of a corresponding, naturally-occurring miRNA. In such a case, the single-stranded miRNA mimetic may regulate a number of genes, for example, downstream from its mRNA target, whose expression levels are associated with or otherwise regulated by the corresponding, naturally-occurring miRNA. Because aberrant expression levels of certain naturally-occurring miRNAs have been implicated in various human ailments, including, but not limited to, hyperproliferative, angiogenic, or inflammatory diseases, states, or adverse conditions, the single-stranded miRNA mimetics of the present invention can offer valuable therapeutic opportunities. In this context, a single-stranded miRNA mimetic of this disclosure can regulate (e.g., knockdown or up-regulate) expression of one or more downstream genes of its corresponding endogenous miRNA, such that prevention, alleviation, or reduction of the severity or recurrence of one or more associated disease symptoms can be achieved. Alternatively, for various distinct disease models in which expression of one or more target mRNAs are not necessarily reduced or at a lower-than-normal level as a consequence of diseases or other adverse conditions, introducing exogenous miRNA mimetics, such as one or more single-stranded miRNA mimetics of the invention, may nonetheless result in a therapeutic result by affecting the expression levels of genes associated with the disease pathway.
A single-stranded RNAi molecule of invention can also act similar to a siRNA molecule in targeting the coding region of a target gene, inhibiting the expression that gene and, thus, reducing protein production. The protein that would have been produced if not for introduction of the single-stranded RNAi molecule may be associated with a pathological or diseased condition (e.g., cancer).
In accordance with this disclosure herein, a single-stranded RNAi molecule of the invention, compositions thereof, and methods for inhibiting expression of one or more corresponding target mRNAs in a cell or organism are provided. This disclosure provides methods and single-stranded RNAi molecule compositions for treating a subject, including a human cell, tissue or individual.
(i) Pharmaceutical Compositions and Formulations
The present disclosure includes single-stranded RNAi molecule compositions prepared for storage or administration that include a pharmaceutically effective amount of a desired RNAi molecule in a pharmaceutically acceptable carrier or diluent. The single-stranded RNAi molecule compositions of the disclosure can be effectively employed as pharmaceutically-acceptable formulations. Pharmaceutically-acceptable formulations prevent, alter the occurrence or severity of, or treat (alleviate one or more symptom(s) to a detectable or measurable extent) a disease state or other adverse condition in a subject. Thus, a pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration into a cell, or a subject such as a human (e.g., systemic administration). The pharmaceutical compositions of the present disclosure are formulated to allow the single-stranded RNAi molecule(s) contained therein to be bioavailable upon administration to a subject.
In certain embodiments, pharmaceutical compositions of this disclosure can optionally include preservatives, antioxidants, stabilizers, dyes, flavoring agents, or any combination thereof. Exemplary preservatives include sodium benzoate, esters of p-hydroxybenzoic acid, and sorbic acid. A pharmaceutically acceptable formulation includes salts of the above compounds, for example, acid addition salts, such as salts of hydrochloric acid, hydrobromic acid, acetic acid, or benzene sulfonic acid. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., A. R. Gennaro edit., 21st Edition, 2005.
In certain embodiments, aqueous suspensions containing one or more single-stranded RNAi molecules of the invention can be prepared in an admixture with suitable excipients, such as suspending agents or dispersing or wetting agents. Exemplary suspending agents include sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia. Representative dispersing or wetting agents include naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). In certain embodiments, the aqueous suspensions can optionally contain one or more preservatives (e.g., ethyl or w-propyl-p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, or one or more sweetening agents (e.g., sucrose, saccharin). In additional embodiments, dispersible powders and granules suitable for preparation of an aqueous suspension comprising one or more single-stranded RNAi molecules of the invention can be prepared by the addition of water with the single-stranded RNAi molecules in admixture with a dispersing or wetting agent, suspending agent and optionally one or more preservative, coloring agent, flavoring agent, or sweetening agent.
In further embodiments, a single-stranded RNAi molecule of this disclosure can be formulated as oily suspensions or emulsions (e.g., oil-in-water) by suspending the ssRNAi in, for example, a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g., liquid paraffin). Suitable emulsifying agents can be naturally-occurring gums (e.g., gum acacia or gum tragacanth), naturally-occurring phosphatides (e.g., soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monooleate), or condensation products of partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). In certain embodiments, the oily suspensions or emulsions can optionally contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. In related embodiments, sweetening agents and flavoring agents can optionally be added to provide palatable oral preparations. In yet other embodiments, these compositions can be preserved by the optionally adding an anti-oxidant, such as ascorbic acid.
In further embodiments, single-stranded RNAi molecules can be formulated as syrups and elixirs with sweetening agents (e.g., glycerol, propylene glycol, sorbitol, glucose or sucrose). Such formulations can also contain a demulcent, preservative, flavoring, coloring agent, or any combination thereof.
In other embodiments, pharmaceutical compositions comprising a single-stranded RNAi molecule of the invention can be in the form of a sterile, injectable aqueous or oleaginous suspension. The sterile, injectable preparation can also be a sterile, injectable solution or suspension in a non-toxic, parenterally-acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Among the exemplary acceptable vehicles and solvents useful in the compositions of this disclosure is water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of parenteral formulations.
The single-stranded RNAi molecules of the invention can be administered directly, or can be complexed, for example, with cationic lipids or packaged within liposomes, or otherwise delivered to target cells or tissues. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2:139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer et al., 1999, Mol. Membr. Biol. 16:129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol, 137:165-192; and Lee et al., 2000, ACS Symp. Ser. 752:184-192. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example, Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT Publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722).
(ii) Carrier/Delivery Systems
In one aspect, the present invention provides carrier systems containing the single-stranded RNAi molecules described herein. In some embodiments, the carrier system is a lipid-based carrier system, cationic lipid, or liposome nucleic acid complexes, a liposome, a micelle, a virosome, a lipid nanoparticle or a mixture thereof. In other embodiments, the carrier system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex. In additional embodiments, the carrier system is a cyclodextrin-based carrier system such as a cyclodextrin polymer-nucleic acid complex. In further embodiments, the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex. Preferably, the carrier system in a lipid nanoparticle (“LNP”) formulation.
In certain embodiments, the single-stranded RNAi molecules of the invention are formulated with a lipid nanoparticle composition such as is described in U.S. patent application Ser. Nos. 11/353,630, 11/586,102, 61/189,295, 61/204,878, 61/235,476, 61/249,807, and 61/298,022. In certain preferred embodiments, the ssRNAi molecules of the invention are formulated with a lipid nanoparticle composition comprising a cationic lipid/Cholesterol/PEG-C-DMA/DSPC in a 40/48/2/10 ratio or a cationic lipid/Cholesterol/PEG-DMG/DSPC in a 40/48/2/10 ratio. In certain other embodiments, the invention features a composition comprising a ssRNAi molecule of the invention formulated with any of the cationic lipid formulations described in U.S. Patent Application Nos. 61/189,295, 61/204,878, 61/235,476, 61/249,807, and 61/298,022.
Within certain embodiments of this disclosure, pharmaceutical compositions and methods are provided that feature the presence or administration of one or more single-stranded RNAi molecule, combined, complexed, or conjugated with functional moiety, optionally formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, or the like. Such conjugates and/or complexes can be used to facilitate delivery of RNAi molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. Non-limiting, examples of such conjugates are described in U.S. Publication Nos. US2008/0152661 A1 and US2004/0162260 A1 (e.g., CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.) and U.S. patent application Ser. Nos. 10/427,160 and 10/201,394; and U.S. Pat. Nos. 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136; and 5,138,045.
A single-stranded RNAi molecule of this disclosure can include a conjugate member on one or more of the nucleotides, at a terminal and/or internal position(s), and/or on the spacer portion of the molecule. The conjugate member can be, for example, a lipophils, a terpene, a protein binding agent, a vitamin, a carbohydrate, or a peptide. For example, the conjugate member can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, or a C5 pyrimidine linker. In other embodiments, the conjugate member is a glyceride lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. In various embodiments, polyethylene glycol (PEG) can be covalently attached to single-stranded RNAi molecules of the present invention. The attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da).
Within certain embodiments of this disclosure, pharmaceutical compositions and methods are provided that feature the presence or administration of one or more single-stranded RNAi molecule, combined, complexed, or conjugated with a polypeptide or peptide, optionally formulated with a pharmaceutically-acceptable carrier, such as a diluent, stabilizer, buffer, or the like. In certain embodiments, when peptide conjugate partners are used to enhance delivery of one or more single-stranded RNAi molecules of this disclosure into a target cell, or otherwise enhance stability or activity of the molecule when contacted with a biological sample. Exemplary peptide conjugate members for use within these aspects of this disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73, PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404, PN453, and PN509 as described, for example, in U.S. Patent Application Publication Nos. 2006/0040882 and 2006/0014289, and U.S. Provisional Patent Application No. 60/939,578, which are all incorporated herein by reference.
In one embodiment, this disclosure provides compositions suitable for administering single-stranded RNAi molecules of this disclosure to specific cell types, such as hepatocytes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262:4429) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR), Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell 22: 611; Connolly et al., 1982, J. Biol. Chem. 257:939). Lee and Lee (1987, Glycoconjugate J. 4:317) obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem. 24: 1388). The use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of bioconjugates of this disclosure.
In still another embodiment, a single-stranded RNAi molecule of the invention may be conjugated to a polypeptide and admixed with one or more non-cationic lipids or a combination of a non-cationic lipid and a cationic lipid to form a composition that enhances intracellular delivery of the RNAi molecule as compared to delivery resulting from contacting the target cells with a naked RNAi molecule without the lipids. In more detailed aspects of this disclosure, the mixture, complex or conjugate comprising a single-stranded RNAi molecule and a polypeptide can be optionally combined with (e.g., admixed or complexed with) a cationic lipid, such as LipofectineTM. To produce these compositions comprised of a polypeptide, a single-stranded RNAi molecule and a cationic lipid, the RNAi molecule and the polypeptide may be mixed together first in a suitable medium such as a cell culture medium, after which the cationic lipid is added to the mixture to form an RNAi molecule/delivery peptide/cationic lipid composition. Optionally, the peptide and cationic lipid can be mixed together first in a suitable medium such as a cell culture medium, followed by the addition of the single-stranded RNAi molecule to form the RNAi molecule/delivery peptide/cationic lipid composition.
This disclosure also features the use of single-stranded RNAi molecule compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations may offer increased accumulation of drugs in target tissues (Lasic et al., 1995, Chem. Rev. 95:2601; Ishiwata et al., 1995, Chem. Pharm. Bull. 43:1005). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., 1995, Science 267:1215; Oku et al., 1995, Biochim. Biophys. Acta 1238:86). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of nucleic acid molecules as compared to conventional cationic liposomes, which are known to accumulate in tissues of the mononuclear phagocytic system (MPS) (Liu et al., 1995, J. Biol. Chem. 42:24864; Choi et al., PCT Publication No. WO 96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland et al., POT Publication No. WO 96/10392). Long-circulating liposomes may also provide additional protection from nuclease degradation as compared to cationic liposomes, in theory due to avoiding accumulation in metabolically aggressive MPS tissues, such as the liver and spleen.
In some embodiments, the RNAi molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof; such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in U.S. Patent Application Publication No. 20030077829.
In other embodiments, single-stranded RNAi molecules of the invention are complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666. In still other embodiments, the membrane disruptive agent or agents and the RNAi molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310.
In certain embodiments, single-stranded RNAi molecules of the invention are complexed with delivery systems as described in U.S. Patent Application Publication Nos. 2003077829; 20050287551; 20050164220; 20050191627; 20050118594; 20050153919; 20050085486; and 20030158133; and International PCT Publication Nos. WO 00/03683 and WO 02/087541.
In some embodiments, a liposomal formulation of the invention comprises a RNAi molecule of the invention formulated or complexed with compounds and compositions described in U.S. Pat. Nos. 6,858,224; 6,534,484; 6,287,591; 6,835,395; 6,586,410; 6,858,225; 6,815,432; 6,586,001; 6,120,798; 6,977,223; 6,998,115; 5,981,501; 5,976,567; 5,705,385; and U.S. Patent Application Publication Nos. 2006/0019912; 2006/0019258; 2006/0008909; 2005/0255153; 2005/0079212; 2005/0008689; 2003/0077829, 2005/0064595, 2005/0175682, 2005/0118253; 2004/0071654; 2005/0244504; 2005/0265961 and 2003/0077829.
The present disclosure also features a method for preparing single-stranded RNAi molecule nanoparticles. A first solution containing melamine derivatives is dissolved in an organic solvent such as dimethyl sulfoxide, or dimethyl formamide to which an acid such as HCl has been added. The concentration of HCl would be about 3.3 moles of HCl for every mole of the melamine derivative. The first solution is then mixed with a second solution, which includes a nucleic acid dissolved or suspended in a polar or hydrophilic solvent (e.g., an aqueous buffer solution containing, for instance, ethylenediaminetraacetic acid (EDTA), or tris(hydroxymethyl) aminomethane (TRIS), or combinations thereof. The mixture forms a first emulsion. The mixing can be done using any standard technique such as, for example, sonication, vortexing, or in a micro fluidizer. The resultant nucleic acid particles can be purified and the organic solvent removed using size-exclusion chromatography or dialysis or both. The complexed nucleic acid nanoparticles can then be mixed with an aqueous solution containing either polyarginine or a Gln-Asn polymer, or both, in an aqueous solution. A preferred molecular weight of each polymer is about 5000 to about 15,000 Daltons. This forms a solution containing nanoparticles of nucleic acid complexed with the melamine derivative and the polyarginine and the Gln-Asn polymers. The mixing steps are carried out in a manner that minimizes shearing of the nucleic acid while producing nanoparticles on average smaller than about 200 nanometers in diameter. It is believed that the polyarginine complexes with the negative charge of the phosphate groups within the minor groove of the nucleic acid, and the polyarginine wraps around the trimeric nucleic acid complex. At either terminus of the polyarginine other moieties, such as the TAT polypeptide, mannose or galactose, can be covalently bound to the polymer to direct binding of the nucleic acid complex to specific tissues, such as to the liver when galactose is used. While not being bound to theory, it is believed that the Gln-Asn polymer complexes with the nucleic acid complex within the major groove of the nucleic acid through hydrogen bonding with the bases of the nucleic acid. The polyarginine and the Gln-Asn polymer should be present at a concentration of 2 moles per every mole of nucleic acid having 20 base pairs. The concentration should be increased proportionally for a nucleic acid having more than 20 base pairs. For example, if the nucleic acid has 25 base pairs, the concentration of the polymers should be 2.5-3 moles per mole of double-stranded nucleic acid. The resultant nanoparticles can be purified by standard means such as size exclusion chromatography followed by dialysis. The purified complexed nanoparticles can then be lyophilized using techniques well known in the art. One embodiment of the present disclosure provides nanoparticles less than 100 nanometers (nm) comprising a single-stranded RNAi molecule.
(iii) Treatment
Subjects (e.g., mammalian, human) amendable for treatment using the single-stranded RNAi molecules of the invention (optionally substituted or modified or conjugated), compositions thereof, and methods of the present disclosure include those suffering from one or more disease or condition mediated, at least in part, by an aberrant expression level of the target gene or sequence, those at risk of developing a disease caused by or associated with the aberrant levels of a target gene/sequence, or those which are amenable to treatment by replenishing or increasing the level of RNAi mediated by the corresponding ssRNAi molecule, including a hyperproliferative (e.g., cancer), angiogenic, metabolic, or inflammatory (e.g., arthritis) disease or disorder or condition.
Compositions and methods disclosed herein are useful in the treatment of a wide variety of target viruses, including retrovirus, such as human immunodeficiency virus (HIV), Hepatitis C Virus, Hepatitis B Virus, Coronavirus, as well as respiratory viruses, including human Respiratory Syncytial Virus, human Metapneumovirus, human Parainfluenza virus, Rhinovirus and Influenza virus.
In other examples, the compositions and methods of this disclosure are useful as therapeutic tools to treat or prevent symptoms of, for example, hyperproliferative disorders. Exemplary hyperproliferative disorders include neoplasms, carcinomas, sarcomas, tumors, or cancer. More exemplary hyperproliferative disorders include oral cancer, throat cancer, laryngeal cancer, esophageal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, gastrointestinal tract cancer, gastrointestinal stromal tumors (GIST), small intestine cancer, colon cancer, rectal cancer, colorectal cancer, anal cancer, pancreatic cancer, breast cancer, cervical cancer, uterine cancer, vulvar cancer, vaginal cancer, urinary tract cancer, bladder cancer, kidney cancer, adrenocortical cancer, islet cell carcinoma, gallbladder cancer, stomach cancer, prostate cancer, ovarian cancer, endometrial cancer, trophoblastic tumor, testicular cancer, penial cancer, bone cancer, osteosarcoma, liver cancer, extrahepatic bile duct cancer, skin cancer, basal cell carcinoma (BCC), lung cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), brain cancer, melanoma, Kaposi's sarcoma, eye cancer, head and neck cancer, squamous cell carcinoma of head and neck, tymoma, thymic carcinoma, thyroid cancer, parathyroid cancer, Hippel-Lindau syndrome, leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, hairy cell leukemia, lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-cell lymphoma, multiple myeloma, malignant pleural mesothelioma, Barrett's adenocarcinoma, Wilm's tumor, or the like. In other examples, the compositions and methods of this disclosure are useful as therapeutic tools to regulate expression of one or more target gene to treat or prevent symptoms of, for example, inflammatory disorders. Exemplary inflammatory disorders include diabetes mellitus, rheumatoid arthritis, pannus growth in inflamed synovial lining, collagen-induced arthritis, spondylarthritis, ankylosing spondylitis, multiple sclerosis, encephalomyelitis, inflammatory bowel disease, Chron's disease, psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupus erythematosis, graft-versus-host disease, atherosclerosis, and allergies.
Other exemplary disorders that can be treated with single-stranded RNAi molecules, compositions and methods of the instant disclosure include metabolic disorders, cardiac disease, pulmonary disease, neovascularization, ischemic disorders, age-related macular degeneration, diabetic retinopathy, glomerulonephritis, diabetes, asthma, chronic obstructive pulmonary disease, chronic bronchitis, lymphangiogenesis, and atherosclerosis.
Within additional aspects, combination formulations and methods are provided comprising an effective amount of one or more single-stranded RNAi molecules in combination with one or more secondary or adjunctive active agents that are formulated together or administered coordinately with the single-stranded RNAi molecules of the invention to control one or more target gene-associated disease or condition as described herein. Useful adjunctive therapeutic agents in these combinatorial formulations and coordinate treatment methods include, for example, enzymatic nucleic acid molecules, allosteric nucleic acid molecules, guide, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules and other organic or inorganic compounds including metals, salts and ions, and other drugs and active agents indicated for treating one or more target gene-associated disease or condition, including chemotherapeutic agents used to treat cancer, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), or the like. Exemplary chemotherapeutic agents include alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogen mustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, mytomycin, actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin, topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab, rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide, prednisone, leucovorin, oxaliplatin.
To practice the coordinate administration methods of this disclosure, a single-stranded RNAi molecule is administered simultaneously or sequentially in a coordinated treatment protocol with one or more secondary or adjunctive therapeutic agents described herein or known in the art. The coordinate administration may be done in either order, and there may be a time period while only one or both (or all) active therapeutic agents, individually or collectively, exert their biological activities. A distinguishing aspect of all such coordinate treatment methods is that the single-stranded RNAi molecule(s) present in a composition elicits some favorable clinical response, which may or may not be in conjunction with a secondary clinical response provided by the secondary therapeutic agent. For example, the coordinate administration of a single-stranded RNAi molecule with a secondary therapeutic agent as contemplated herein can yield an enhanced (e.g., synergistic) therapeutic response beyond the therapeutic response elicited by either or both the purified single-stranded RNAi molecule and the secondary therapeutic agent alone.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, the physical characteristics of the specific subject under consideration for treatment (e.g., age, body weight, general health, sex, diet), concurrent medication, rate of excretion, drug combination, the severity of the particular disease undergoing therapy, and other factors that those skilled in the medical arts will recognize. For example, an amount between about 0.1 mg/kg and about 140 mg/kg body weight/day of active ingredients may be administered depending on the potency of a single-stranded RNAi molecule of this disclosure (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
Nucleic acid molecules can be administered to cells or organisms by a variety of methods known to those of skill in the art, including administration of formulations that comprise a single-stranded RNAi molecule, or formulations that further comprise one or more additional components, such as a pharmaceutically acceptable carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, or the like. In certain embodiments, a single-stranded RNAi molecule of the invention, and/or the polypeptide can be encapsulated in liposomes, administered by iontophoresis, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (see, e.g., PCT Publication No. WO 00/53722). Alternatively, a nucleic acid/peptide/vehicle combination can be locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of this disclosure, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies, such as those described in Conroy et al, (1999, Clin. Cancer Res. 5:2330) and PCT Publication No. WO 99/31262.
The formulations of the present disclosure, having an amount of a single-stranded RNAi molecule sufficient to treat or prevent a disorder associated with target gene expression are, for example, suitable for topical (e.g., creams, ointments, skin patches, eye drops, ear drops) application or administration. Other routes of administration include oral, parenteral, sublingual, bladder washout, vaginal, rectal, enteric, suppository, nasal, and inhalation. The term “parenteral,” as used herein, includes subcutaneous, intravenous, intramuscular, intraarterial, intraabdominal, intraperitoneal, intraarticular, intraocular or retrobulbar, intraaural, intrathecal, intracavitary, intracelial, intraspinal, intrapulmonary or transpulmonary, intrasynovial, and intraurethral injection or infusion techniques. The compositions of the present disclosure may also be formulated and used as a tablet, capsule or elixir for oral administration, suppository for rectal administration, sterile solution, or suspension for injectable administration, either with or without other compounds known in the art. For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
Further methods for delivery of nucleic acid molecules, such as single-stranded RNAi molecules of this invention, have been described in, for example, Boado et al., 1998, J Pharm. Sci 87:1308; Tyler et al., 1999, FEBS Lett. 421:2m; Pardridge et al., 1995, Proc. Nat'l Acad. Sci. USA 92:5592; Boado, 1995, Adv. Drug Delivery Rev. 15:73; Aldrian-Herrada et al. 1998, Nucleic Acids Res. 26:4910; Tyler et al., 1999, Proc. Nat'l Acad. Sci. USA 96:7053; Akhtar et al., 1992, Trends Cell Bio. 2:139; “Delivery Strategies for Guide Oligonucleotide Therapeutics,” ed. Akhtar, 1995, Maurer et al., 1999 Mol. Membr. Biol. 16:129; Lee et al., 2000, ACS Symp. Ser. 752:184. In addition to in vivo and therapeutic applications, a skilled person in the art will appreciate that the single-stranded RNAi molecules of the present disclosure are useful in a wide variety of in vitro applications, such as in scientific and commercial research (e.g., elucidation of physiological pathways, drug discovery and development), and medical and veterinary diagnostics.
All U.S. patents, U.S. patent publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, figures, and websites referred to in this specification are expressly incorporated herein by reference, in their entirety.
Table 1 lists certain endogenous human miRNA sequences, wherein the seed sequences, confirmed or projected, are capitalized. All miRNA sequences in Table 1 are shown in 5′ to 3′ orientation. Other miRNA sequences of the present invention may be found in the miRBase database, the content of which is incorporated by reference herein.










TABLE 1








SEQ ID


miRNA name
miRBase number
Sequence
NO


















hsa-let-7a
MIMAT0000062
UGAGGUAGuagguuguauaguu
1


 


hsa-let-7a*
MIMAT0004481
CUAUACAAucuacugucuuuc
2


 


hsa-let-7a-2*
MIMAT0010195
CUGUACAGccuccuagcuuucc
3


 


hsa-let-7b
MIMAT0000063
UGAGGUAGuagguugugugguu
4


 


hsa-let-7b*
MIMAT0004482
CUAUACAAccuacugccuuccc
5


 


hsa-let-7c
MIMAT0000064
UGAGGUAGuagguuguaugguu
6


 


hsa-let-7c*
MIMAT0004483
UAGAGUUAcacccugggaguua
7


 


hsa-let-7d
MIMAT0000065
AGAGGUAGuagguugcauaguu
8


 


hsa-let-7d*
MIMAT0004484
CUAUACGAccugcugccuuucu
9


 


hsa-let-7e
MIMAT0000066
UGAGGUAGgagguuguauaguu
10


 


hsa-let-7e*
MIMAT0004485
CUAUACGGccuccuagcuuucc
11


 


hsa-let-7f
MIMAT0000067
UGAGGUAGuagauuguauaguu
12


 


hsa-let-7f-1*
MIMAT0004486
CUAUACAAucuauugccuuccc
13


 


hsa-let-7f-2*
MIMAT0004487
CUAUACAGucuacugucuuucc
14


 


hsa-miR-15a
MIMAT0000068
UAGCAGCAcauaaugguuugug
15


 


hsa-miR-15a*
MIMAT0004488
CAGGCCAUauugugcugccuca
16


 


hsa-miR-16
MIMAT0000069
UAGCAGCAcguaaauauuggcg
17


 


hsa-miR-16-1*
MIMAT0004489
CCAGUAUUaacugugcugcuga
18


 


hsa-miR-17
MIMAT0000070
CAAAGUGCuuacagugcagguag
19


 


hsa-miR-17*
MIMAT0000071
ACUGCAGUgaaggcacuuguag
20


 


hsa-miR-18a
MIMAT0000072
UAAGGUGCaucuagugcagauag
21


 


hsa-miR-18a*
MIMAT0002891
ACUGCCCUaagugcuccuucugg
22


 


hsa-miR-19a*
MIMAT0004490
AGUUUUGCauaguugcacuaca
23


 


hsa-miR-19a
MIMAT0000073
UGUGCAAAucuaugcaaaacuga
24


 


hsa-miR-19b-1*
MIMAT0004491
AGUUUUGCagguuugcauccagc
25


 


hsa-miR-19b
MIMAT0000074
UGUGCAAAuccaugcaaaacuga
26


 


hsa-miR-19b-2*
MIMAT0004492
AGUUUUGCagguuugcauuuca
27


 


hsa-miR-20a
MIMAT0000075
UAAAGUGCuuauagugcagguag
28


 


hsa-miR-20a*
MIMAT0004493
ACUGCAUUaugagcacuuaaag
29


 


hsa-miR-21
MIMAT0000076
UAGCUUAUcagacugauguuga
30


 


hsa-miR-21*
MIMAT0004494
CAACACCAgucgaugggcugu
31


 


hsa-miR-22*
MIMAT0004495
AGUUCUUCaguggcaagcuuua
32


 


hsa-miR-22
MIMAT0000077
AAGCUGCCaguugaagaacugu
33


 


hsa-miR-23a*
MIMAT0004496
GGGGUUCCuggggaugggauuu
34


 


hsa-miR-23a
MIMAT0000078
AUCACAUUgccagggauuucc
35


 


hsa-miR-24-1*
MIMAT0000079
UGCCUACUgagcugauaucagu
36


 


hsa-miR-24
MIMAT0000080
UGGCUCAGuucagcaggaacag
37


 


hsa-miR-24-2*
MIMAT0004497
UGCCUACUgagcugaaacacag
38


 


hsa-miR-25*
MIMAT0004498
AGGCGGAGacuugggcaauug
39


 


hsa-miR-25
MIMAT0000081
CAUUGCACuugucucggucuga
40


 


hsa-miR-26a
MIMAT0000082
UUCAAGUAauccaggauaggcu
41


 


hsa-miR-26a-1*
MIMAT0004499
CCUAUUCUugguuacuugcacg
42


 


hsa-miR-26b
MIMAT0000083
UUCAAGUAauucaggauaggu
43


 


hsa-miR-26b*
MIMAT0004500
CCUGUUCUccauuacuuggcuc
44


 


hsa-miR-27a*
MIMAT0004501
AGGGCUUAgcugcuugugagca
45


 


hsa-miR-27a
MIMAT0000084
UUCACAGUggcuaaguuccgc
46


 


hsa-miR-28-5p
MIMAT0000085
AAGGAGCUcacagucuauugag
47


 


hsa-miR-28-3p
MIMAT0004502
CACUAGAUugugagcuccugga
48


 


hsa-miR-29a*
MIMAT0004503
ACUGAUUUcuuuugguguucag
49


 


hsa-miR-29a
MIMAT0000086
UAGCACCAucugaaaucgguua
50


 


hsa-miR-30a
MIMAT0000087
UGUAAACAuccucgacuggaag
51


 


hsa-miR-30a*
MIMAT0000088
CUUUCAGUcggauguuugcagc
52


 


hsa-miR-31
MIMAT0000089
AGGCAAGAugcuggcauagcu
53


 


hsa-miR-31*
MIMAT0004504
UGCUAUGCcaacauauugccau
54


 


hsa-miR-32
MIMAT0000090
UAUUGCACauuacuaaguugca
55


 


hsa-miR-32*
MIMAT0004505
CAAUUUAGugugugugauauuu
56


 


hsa-miR-33a
MIMAT0000091
GUGCAUUGuaguugcauugca
57


 


hsa-miR-33a*
MIMAT0004506
CAAUGUUUccacagugcaucac
58


 


hsa-miR-92a-1*
MIMAT0004507
AGGUUGGGaucgguugcaaugcu
59


 


hsa-miR-92a
MIMAT0000092
UAUUGCACuugucccggccugu
60


 


hsa-miR-92a-2*
MIMAT0004508
GGGUGGGGauuuguugcauuac
61


 


hsa-miR-93
MIMAT0000093
CAAAGUGCuguucgugcagguag
62


 


hsa-miR-93*
MIMAT0004509
ACUGCUGAgcuagcacuucccg
63


 


hsa-miR-95
MIMAT0000094
UUCAACGGguauuuauugagca
64


 


hsa-miR-96
MIMAT0000095
UUUGGCACuagcacauuuuugcu
65


 


hsa-miR-96*
MIMAT0004510
AAUCAUGUgcagugccaauaug
66


 


hsa-miR-98
MIMAT0000096
UGAGGUAGuaaguuguauuguu
67


 


hsa-miR-99a
MIMAT0000097
AACCCGUAgauccgaucuugug
68


 


hsa-miR-99a*
MIMAT0004511
CAAGCUCGcuucuaugggucug
69


 


hsa-miR-100
MIMAT0000098
AACCCGUAgauccgaacuugug
70


 


hsa-miR-100*
MIMAT0004512
CAAGCUUGuaucuauagguaug
71


 


hsa-miR-101*
MIMAT0004513
CAGUUAUCacagugcugaugcu
72


 


hsa-miR-101
MIMAT0000099
UACAGUACugugauaacugaa
73


 


hsa-miR-29b-1*
MIMAT0004514
GCUGGUUUcauauggugguuuaga
74


 


hsa-miR-29b
MIMAT0000100
UAGCACCAuuugaaaucaguguu
75


 


hsa-miR-29b-2*
MIMAT0004515
CUGGUUUCacaugguggcuuag
76


 


hsa-miR-103-2*
MIMAT0009196
AGCUUCUUuacagugcugccuug
77


 


hsa-miR-103
MIMAT0000101
AGCAGCAUuguacagggcuauga
78


 


hsa-miR-105
MIMAT0000102
UCAAAUGCucagacuccuguggu
79


 


hsa-miR-105*
MIMAT0004516
ACGGAUGUuugagcaugugcua
80


 


hsa-miR-106a
MIMAT0000103
AAAAGUGCuuacagugcagguag
81


 


hsa-miR-106a*
MIMAT0004517
CUGCAAUGuaagcacuucuuac
82


 


hsa-miR-107
MIMAT0000104
AGCAGCAUuguacagggcuauca
83


 


hsa-miR-16-2*
MIMAT0004518
CCAAUAUUacugugcugcuuua
84


 


hsa-miR-192
MIMAT0000222
CUGACCUAugaauugacagcc
85


 


hsa-miR-192*
MIMAT0004543
CUGCCAAUuccauaggucacag
86


 


hsa-miR-196a
MIMAT0000226
UAGGUAGUuucauguuguuggg
87


 


hsa-miR-197
MIMAT0000227
UUCACCACcuucuccacccagc
88


 


hsa-miR-198
MIMAT0000228
GGUCCAGAggggagaugguuc
89


 


hsa-miR-199a-5p
MIMAT0000231
CCCAGUGUucagacuaccuguuc
90


 


hsa-miR-199a-3p
MIMAT0000232
ACAGUAGUcugcacauugguua
91


 


hsa-miR-208a
MIMAT0000241
AUAAGACGagcaaaaagcuugu
92


 


hsa-miR-129-5p
MIMAT0000242
CUUUUUGCggucugggcuugc
93


 


hsa-miR-129*
MIMAT0004548
AAGCCCUUaccccaaaaaguau
94


 


hsa-miR-148a*
MIMAT0004549
AAAGUUCUgagacacuccgacu
95


 


hsa-miR-148a
MIMAT0000243
UCAGUGCAcuacagaacuuugu
96


 


hsa-miR-30c
MIMAT0000244
UGUAAACAuccuacacucucagc
97


 


hsa-miR-30c-2*
MIMAT0004550
CUGGGAGAaggcuguuuacucu
98


 


hsa-miR-30d
MIMAT0000245
UGUAAACAuccccgacuggaag
99


 


hsa-miR-30d*
MIMAT0004551
CUUUCAGUcagauguuuugcugc
100


 


hsa-miR-139-5p
MIMAT0000250
UCUACAGUgcacgugucuccag
101


 


hsa-miR-139-3p
MIMAT0004552
GGAGACGCggcccuguuggagu
102


 


hsa-miR-147
MIMAT0000251
GUGUGUGGaaaugcuucugc
103


 


hsa-miR-7
MIMAT0000252
UGGAAGACuagugauuuuguugu
104


 


hsa-miR-7-1*
MIMAT0004553
CAACAAAUcacagucugccaua
105


 


hsa-miR-7-2*
MIMAT0004554
CAACAAAUcccagucuaccuaa
106


 


hsa-miR-10a
MIMAT0000253
UACCCUGUagauccgaauuugug
107


 


hsa-miR-10a*
MIMAT0004555
CAAAUUCGuaucuaggggaaua
108


 


hsa-miR-10b
MIMAT0000254
UACCCUGUagaaccgaauuugug
109


 


hsa-miR-10b*
MIMAT0004556
ACAGAUUCgauucuaggggaau
110


 


hsa-miR-34a
MIMAT0000255
UGGCAGUGucuuagcugguugu
111


 


hsa-miR-34a*
MIMAT0004557
CAAUCAGCaaguauacugcccu
112


 


hsa-miR-181a
MIMAT0000256
AACAUUCAacgcugucggugagu
113


 


hsa-miR-181a-2*
MIMAT0004558
ACCACUGAccguugacuguacc
114


 


hsa-miR-181b
MIMAT0000257
AACAUUCAuugcugucggugggu
115


 


hsa-miR-181c
MIMAT0000258
AACAUUCAaccugucggugagu
116


 


hsa-miR-181c*
MIMAT0004559
AACCAUCGaccguugaguggac
117


 


hsa-miR-182
MIMAT0000259
UUUGGCAAugguagaacucacacu
118


 


hsa-miR-182*
MIMAT0000260
UGGUUCUAgacuugccaacua
119


 


hsa-miR-183
MIMAT0000261
UAUGGCACugguagaauucacu
120


 


hsa-miR-183*
MIMAT0004560
GUGAAUUAccgaagggccauaa
121


 


hsa-miR-187*
MIMAT0004561
GGCUACAAcacaggacccgggc
122


 


hsa-miR-187
MIMAT0000262
UCGUGUCUuguguugcagccgg
123


 


hsa-miR-196a*
MIMAT0004562
CGGCAACAagaaacugccugag
124


 


hsa-miR-199b-5p
MIMAT0000263
CCCAGUGUuuagacuaucuguuc
125


 


hsa-miR-199b-3p
MIMAT0004563
ACAGUAGUcugcacauugguua
91


 


hsa-miR-203
MIMAT0000264
GUGAAAUGuuuaggaccacuag
126


 


hsa-miR-204
MIMAT0000265
UUCCCUUUgucauccuaugccu
127


 


hsa-miR-205
MIMAT0000266
UCCUUCAUuccaccggagucug
128


 


hsa-miR-205*
MIMAT0009197
GAUUUCAGuggagugaaguuc
129


 


hsa-miR-210
MIMAT0000267
CUGUGCGUgugacagcggcuga
130


 


hsa-miR-211
MIMAT0000268
UUCCCUUUgucauccuucgccu
131


 


hsa-miR-212
MIMAT0000269
UAACAGUCuccagucacggcc
132


 


hsa-miR-181a*
MIMAT0000270
ACCAUCGAccguugauuguacc
133


 


hsa-miR-214*
MIMAT0004564
UGCCUGUCuacacuugcugugc
134


 


hsa-miR-214
MIMAT0000271
ACAGCAGGcacagacaggcagu
135


 


hsa-miR-215
MIMAT0000272
AUGACCUAugaauugacagac
136


 


hsa-miR-216a
MIMAT0000273
UAAUCUCAgcuggcaacuguga
137


 


hsa-miR-217
MIMAT0000274
UACUGCAUcaggaacugauugga
138


 


hsa-miR-218
MLMAT0000275
UUGUGCUUgaucuaaccaugu
139


 


hsa-miR-218-1*
MIMAT0004565
AUGGUUCCgucaagcaccaugg
140


 


hsa-miR-218-2*
MIMAT0004566
CAUGGUUCugucaagcaccgcg
141


 


hsa-miR-219-5p
MIMAT0000276
UGAUUGUCcaaacgcaauucu
142


 


hsa-miR-219-1-3p
MIMAT0004567
AGAGUUGAgucuggacgucccg
143


 


hsa-miR-220a
MIMAT0000277
CCACACCGuaucugacacuuu
144


 


hsa-miR-221*
MIMAT0004568
ACCUGGCAuacaauguagauuu
145


 


hsa-miR-221
MIMAT0000278
AGCUACAUugucugcuggguuuc
146


 


hsa-miR-222*
MIMAT0004569
CUCAGUAGccaguguagauccu
147


 


hsa-miR-222
MIMAT0000279
AGCUACAUcuggcuacugggu
148


 


hsa-miR-223*
MIMAT0004570
CGUGUAUUugacaagcugaguu
149


 


hsa-miR-223
MIMAT0000280
UGUCAGUUugucaaauacccca
150


 


hsa-miR-224
MIMAT0000281
CAAGUCACuagugguuccguu
151


 


hsa-miR-224*
MIMAT0009198
AAAAUGGUgcccuagugacuaca
152


 


hsa-miR-200b*
MIMAT0004571
CAUCUUACgggcagcauugga
153


 


hsa-miR-200b
MIMAT0000318
UAAUACUGccugguaaugauga
154


 


hsa-let-7g
MIMAT0000414
UGAGGUAGuaguuuguacaguu
155


 


hsa-let-7g*
MIMAT0004584
CUGUACAGgccacugccuugc
156


 


hsa-let-7i
MIMAT0000415
UGAGGUAGuaguuugugcuguu
157


 


hsa-let-7i*
MIMAT0004585
CUGCGCAAgcuacugccuugcu
158


 


hsa-miR-1
MIMAT0000416
UGGAAUGUaaagaaguauguau
159


 


hsa-miR-15b
MIMAT0000417
UAGCAGCAcaucaugguuuaca
160


 


hsa-miR-15b*
MIMAT0004586
CGAAUCAUuauuugcugcucua
161


 


hsa-miR-23b*
MIMAT0004587
UGGGUUCCuggcaugcugauuu
162


 


hsa-miR-23b
MIMAT0000418
AUCACAUUgccagggauuacc
163


 


hsa-miR-27b*
MIMAT0004588
AGAGCUUAgcugauuggugaac
164


 


hsa-miR-27b
MIMAT0000419
UUCACAGUggcuaaguucugc
165


 


hsa-miR-30b
MIMAT0000420
UGUAAACAuccuacacucagcu
166


 


hsa-miR-30b*
MIMAT0004589
CUGGGAGGuggauguuuacuuc
167


 


hsa-miR-122
MIMAT0000421
UGGAGUGUgacaaugguguuug
168


 


hsa-miR-122*
MIMAT0004590
AACGCCAUuaucacacuaaaua
169


 


hsa-miR-124*
MIMAT0004591
CGUGUUCAcagcggaccuugau
170


 


hsa-miR-124
MIMAT0000422
UAAGGCACgcggugaaugcc
171


 


hsa-miR-125b
MIMAT0000423
UCCCUGAGacccuaacuuguga
172


 


hsa-miR-125b-1*
MIMAT0004592
ACGGGUUAggcucuugggagcu
173


 


hsa-miR-128
MIMAT0000424
UCACAGUGaaccggucucuuu
174


 


hsa-miR-130a*
MIMAT0004593
UUCACAUUgugcuacugucugc
175


 


hsa-miR-130a
MIMAT0000425
CAGUGCAAuguuaaaagggcau
176


 


hsa-miR-132*
MIMAT0004594
ACCGUGGCuuucgauuguuacu
177


 


hsa-miR-132
MIMAT0000426
UAACAGUCuacagccauggucg
178


 


hsa-miR-133a
MIMAT0000427
UUUGGUCCccuucaaccagcug
179


 


hsa-miR-135a
MIMAT0000428
UAUGGCUUuuuauuccuauguga
180


 


hsa-miR-135a*
MIMAT0004595
UAUAGGGAuuggagccguggcg
181


 


hsa-miR-137
MIMAT0000429
UUAUUGCUuaagaauacgcguag
182


 


hsa-miR-138
MIMAT0000430
AGCUGGUGuugugaaucaggccg
183


 


hsa-miR-138-2*
MIMAT0004596
GCUAUUUCacgacaccaggguu
184


 


hsa-miR-140-5p
MIMAT0000431
CAGUGGUUuuacccuaugguag
185


 


hsa-miR-140-3p
MIMAT0004597
UACCACAGgguagaaccacgg
186


 


hsa-miR-141*
MIMAT0004598
CAUCUUCCaguacaguguugga
187


 


hsa-miR-141
MIMAT0000432
UAACACUGucugguaaagaugg
188


 


hsa-miR-142-5p
MIMAT0000433
CAUAAAGUagaaagcacuacu
189


 


hsa-miR-142-3p
MIMAT0000434
UGUAGUGUuuccuacuuuaugga
190


 


hsa-miR-143*
MIMAT0004599
GGUGCAGUgcugcaucucuggu
191


 


hsa-miR-143
MIMAT0000435
UGAGAUGAagcacuguagcuc
192


 


hsa-miR-144*
MIMAT0004600
GGAUAUCAucauauacuguaag
193


 


hsa-miR-144
MIMAT0000436
UACAGUAUagaugauguacu
194


 


hsa-miR-145
MIMAT0000437
GUCCAGUUuucccaggaaucccu
195


 


hsa-miR-145*
MIMAT0004601
GGAUUCCUggaaauacuguucu
196


 


hsa-miR-152
MIMAT0000438
UCAGUGCAugacagaacuugg
197


 


hsa-miR-153
MIMAT0000439
UUGCAUAGucacaaaagugauc
198


 


hsa-miR-191
MIMAT0000440
CAACGGAAucccaaaagcagcug
199


 


hsa-miR-191*
MIMAT0001618
GCUGCGCUuggauuucgucccc
200


 


hsa-miR-9
MIMAT0000441
UCUUUGGUuaucuagcuguauga
201


 


hsa-miR-9*
MIMAT0000442
AUAAAGCUagauaaccgaaagu
202


 


hsa-miR-125a-5p
MIMAT0000443
UCCCUGAGacccuuuaaccuguga
203


 


hsa-miR-125a-3p
MIMAT0004602
ACAGGUGAgguucuugggagcc
204


 


hsa-miR-125b-2*
MIMAT0004603
UCACAAGUcaggcucuugggac
205


 


hsa-miR-126*
MIMAT0000444
CAUUAUUAcuuuugguacgcg
206


 


hsa-miR-126
MIMAT0000445
UCGUACCGugaguaauaaugcg
207


 


hsa-miR-127-5p
MIMAT0004604
CUGAAGCUcagagggcucugau
208


 


hsa-miR-127-3p
MIMAT0000446
UCGGAUCCgucugagcuuggcu
209


 


hsa-miR-129-3p
MIMAT0004605
AAGCCCUUaccccaaaaagcau
210


 


hsa-miR-134
MIMAT0000447
UGUGACUGguugaccagagggg
211


 


hsa-miR-136
MIMAT0000448
ACUCCAUUuguuuugaugaugga
212


 


hsa-miR-136*
MIMAT0004606
CAUCAUCGucucaaaugagucu
213


 


hsa-miR-138-1*
MIMAT0004607
GCUACUUCacaacaccagggcc
214


 


hsa-miR-146a
MIMAT0000449
UGAGAACUgaauuccauggguu
215


 


hsa-miR-146a*
MIMAT0004608
CCUCUGAAauucaguucuucag
216


 


hsa-miR-149
MIMAT0000450
UCUGGCUCcgugucuucacuccc
217


 


hsa-miR-149*
MIMAT0004609
AGGGAGGGacgggggcugugc
218


 


hsa-miR-150
MIMAT0000451
UCUCCCAAcccuuguaccagug
219


 


hsa-miR-150*
MIMAT0004610
CUGGUACAggccugggggacag
220


 


hsa-miR-154
MIMAT0000452
UAGGUUAUccguguugccuucg
221


 


hsa-miR-154*
MIMAT0000453
AAUCAUACacgguugaccuauu
222


 


hsa-miR-184
MIMAT0000454
UGGACGGAgaacugauaagggu
223


 


hsa-miR-185
MIMAT0000455
UGGAGAGAaaggcaguuccuga
224


 


hsa-miR-185*
MIMAT0004611
AGGGGCUGgcuuuccucugguc
225


 


hsa-miR-186
MIMAT0000456
CAAAGAAUucuccuuuugggcu
226


 


hsa-miR-186*
MIMAT0004612
GCCCAAAGgugaauuuuuuggg
227


 


hsa-miR-188-5p
MIMAT0000457
CAUCCCUUgcaugguggaggg
228


 


hsa-miR-188-3p
MIMAT0004613
CUCCCACAugcaggguuugca
229


 


hsa-miR-190
MIMAT0000458
UGAUAUGUuugauauauuaggu
230


 


hsa-miR-193a-5p
MIMAT0004614
UGGGUCUUugcgggcgagauga
231


 


hsa-miR-193a-3p
MIMAT0000459
AACUGGCCuacaaagucccagu
232


 


hsa-miR-194
MIMAT0000460
UGUAACAGcaacuccaugugga
233


 


hsa-miR-195
MIMAT0000461
UAGCAGCAcagaaauauugc
234


 


hsa-miR-195*
MIMAT0004615
CCAAUAUUggcugugcugcucc
235


 


hsa-miR-206
MIMAT0000462
UGGAAUGUaaggaagugugugg
236


 


hsa-miR-320a
MIMAT0000510
AAAAGCUGgguugagagggcga
237


 


hsa-miR-200c*
MIMAT0004657
CGUCUUACccagcaguguuugg
238


 


hsa-miR-200c
MIMAT0000617
UAAUACUGccggguaaugaugga
239


 


hsa-miR-155
MIMAT0000646
UUAAUGCUaaucgugauaggggu
240


 


hsa-miR-155*
MIMAT0004658
CUCCUACAuauuagcauuaaca
241


 


hsa-miR-194*
MIMAT0004671
CCAGUGGGgcugcuguuaucug
242


 


hsa-miR-106b
MIMAT0000680
UAAAGUGCugacagugcagau
243


 


hsa-miR-106b*
MIMAT0004672
CCGCACUGuggguacuugcugc
244


 


hsa-miR-29c*
MIMAT0004673
UGACCGAUuucuccugguguuc
245


 


hsa-miR-29c
MIMAT0000681
UAGCACCAuuugaaaucgguua
246


 


hsa-miR-30c-1*
MIMAT0004674
CUGGGAGAggguuguuuacucc
247


 


hsa-miR-200a*
MIMAT0001620
CAUCUUACcggacagugcugga
248


 


hsa-miR-200a
MIMAT0000682
UAACACUGucugguaacgaugu
249


 


hsa-miR-302a*
MIMAT0000683
ACUUAAACguggauguacuugcu
250


 


hsa-miR-302a
MIMAT0000684
UAAGUGCUuccauguuuugguga
251


 


hsa-miR-219-2-3p
MIMAT0004675
AGAAUUGUggcuggacaucugu
252


 


hsa-miR-34b*
MIMAT0000685
UAGGCAGUgucauuagcugauug
253


 


hsa-miR-34b
MIMAT0004676
CAAUCACUaacuccacugccau
254


 


hsa-miR-34c-5p
MIMAT0000686
AGGCAGUGuaguuagcugauugc
255


 


hsa-miR-34c-3p
MIMAT0004677
AAUCACUAaccacacggccagg
256


 


hsa-miR-299-5p
MIMAT0002890
UGGUUUACcgucccacauacau
257


 


hsa-miR-299-3p
MIMAT0000687
UAUGUGGGaugguaaaccgcuu
258


 


hsa-miR-301a
MIMAT0000688
CAGUGCAAaguauugucaaagc
259


 


hsa-miR-99b
MIMAT0000689
CACCCGUAgaaccgaccuugcg
260


 


hsa-miR-99b*
MIMAT0004678
CAAGCUCGugucuguggguccg
261


 


hsa-miR-296-5p
MIMAT0000690
AGGGCCCCcccucaauccugu
262


 


hsa-miR-296-3p
MIMAT0004679
GAGGGUUGgguggaggcucucc
263


 


hsa-miR-130b*
MIMAT0004680
ACUCUUUCccuguugcacuac
264


 


hsa-miR-130b
MIMAT0000691
CAGUGCAAugaugaaagggcau
265


 


hsa-miR-30e
MIMAT0000692
UGUAAACAuccuugacuggaag
266


 


hsa-miR-30e*
MIMAT0000693
CUUUCAGUcggauguuuacagc
267


 


hsa-miR-26a-2*
MIMAT0004681
CCUAUUCUugauuacuuguuuc
268


 


hsa-miR-361-5p
MIMAT0000703
UUAUCAGAaucuccagggguac
269


 


hsa-miR-361-3p
MIMAT0004682
UCCCCCAGgugugauucugauuu
270


 


hsa-miR-362-5p
MIMAT0000705
AAUCCUUGgaaccuaggugugagu
271


 


hsa-miR-362-3p
MIMAT0004683
AACACACCuauucaaggauuca
272


 


hsa-miR-363*
MIMAT0003385
CGGGUGGAucacgaugcaauuu
273


 


hsa-miR-363
MIMAT0000707
AAUUGCACgguauccaucugua
274


 


hsa-miR-365
MIMAT0000710
UAAUGCCCcuaaaaauccuuau
275


 


hsa-miR-365*
MIMAT0009199
AGGGACUUucaggggcagcugu
276


 


hsa-miR-302b*
MIMAT0000714
ACUUUAACauggaagugcuuuc
277


 


hsa-miR-302b
MIMAT0000715
UAAGUGCUuccauguuuuaguag
278


 


hsa-miR-302c*
MIMAT0000716
UUUAACAUggaguaccugcug
279


 


hsa-miR-302c
MIMAT0000717
UAAGUGCUuccauguuucagugg
280


 


hsa-miR-302d*
MIMAT0004685
ACUUUAACauggaggcacuugc
281


 


hsa-miR-302d
MIMAT0000718
UAAGUGCUuccauguuugagugu
282


 


hsa-miR-367*
MIMAT0004686
ACUGUUGCuaauaugcaacucu
283


 


hsa-miR-367
MIMAT0000719
AAUUGCACuuuagcaaugguga
284


 


hsa-miR-376c
MIMAT0000720
AACAUAGAggaaauuccacgu
285


 


hsa-miR-369-5p
MIMAT0001621
AGAUCGACcguguuauauucgc
286


 


hsa-miR-369-3p
MIMAT0000721
AAUAAUACaugguugaucuuu
287


 


hsa-miR-370
MIMAT0000722
GCCUGCUGggguggaaccuggu
288


 


hsa-miR-371-5p
MIMAT0004687
ACUCAAACugugggggcacu
289


 


hsa-miR-371-3p
MIMAT0000723
AAGUGCCGccaucuuuugagugu
290


 


hsa-miR-372
MIMAT0000724
AAAGUGCUgcgacauuugagcgu
291


 


hsa-miR-373*
MIMAT0000725
ACUCAAAAugggggcgcuuucc
292


 


hsa-miR-373
MIMAT0000726
GAAGUGCUucgauuuuggggugu
293


 


hsa-miR-374a
MIMAT0000727
UUAUAAUAcaaccugauaagug
294


 


hsa-miR-374a*
MIMAT0004688
CUUAUCAGauuguauuguaauu
295


 


hsa-miR-375
MIMAT0000728
UUUGUUCGAuucggcucgcguga
296


 


hsa-miR-376a*
MIMAT0003386
GUAGAUUCuccuucuaugagua
297


 


hsa-miR-376a
MIMAT0000729
AUCAUAGAggaaaauccacgu
298


 


hsa-miR-377*
MIMAT0004689
AGAGGUUGcccuuggugaauuc
299


 


hsa-miR-377
MIMAT0000730
AUCACACAaaggcaacuuuugu
300


 


hsa-miR-378*
MIMAT0000731
CUCCUGACuccagguccugugu
301


 


hsa-miR-378
MIMAT0000732
ACUGGACUuggagucagaagg
302


 


hsa-miR-379
MIMAT0000733
UGGUAGACuauggaacguagg
303


 


hsa-miR-379*
MIMAT0004690
UAUGUAACaugguccacuaacu
304


 


hsa-miR-380*
MIMAT0000734
UGGUUGACcauagaacaugcgc
305


 


hsa-miR-380
MIMAT0000735
UAUGUAAUaugguccacaucuu
306


 


hsa-miR-381
MIMAT0000736
UAUACAAGggcaagcucucugu
307


 


hsa-miR-382
MIMAT0000737
GAAGUUGUucgugguggauucg
308


 


hsa-miR-383
MIMAT0000738
AGAUCAGAaggugauuguggcu
309


 


hsa-miR-340
MIMAT0004692
UUAUAAAGcaaugagacugauu
310


 


hsa-miR-340*
MIMAT0000750
UCCGUCUCaguuacuuuauagc
311


 


hsa-miR-330-5p
MIMAT0004693
UCUCUGGGccugugucuuaggc
312


 


hsa-miR-330-3p
MIMAT0000751
GCAAAGCAcacggccugcagaga
313


 


hsa-miR-328
MIMAT0000752
CUGGCCCUcucugcccuuccgu
314


 


hsa-miR-342-5p
MIMAT0004694
AGGGGUGCuaucugugauuga
315


 


hsa-miR-342-3p
MIMAT0000753
UCUCACACagaaaucgcacccgu
316


 


hsa-miR-337-5p
MIMAT0004695
GAACGGCUucauacaggaguu
317


 


hsa-miR-337-3p
MIMAT0000754
CUCCUAUAugaugccuuucuuc
318


 


hsa-miR-323-5p
MIMAT0004696
AGGUGGUCcguggcgcguucgc
319


 


hsa-miR-323-3p
MIMAT0000755
CACAUUACacggucgaccucu
320


 


hsa-miR-326
MIMAT0000756
CCUCUGGGcccuuccuccag
321


 


hsa-miR-151-5p
MIMAT0004697
UCGAGGAGcucacagucuagu
322


 


hsa-miR-151-3p
MIMAT0000757
CUAGACUGaagcuccuugagg
323


 


hsa-miR-135b
MIMAT0000758
UAUGGCUUuucauuccuauguga
324


 


hsa-miR-135b*
MIMAT0004698
AUGUAGGGcuaaaagccauggg
325


 


hsa-miR-148b*
MIMAT0004699
AAGUUCUGuuauacacucaggc
326


 


hsa-miR-148b
MIMAT0000759
UCAGUGCAucacagaacuuugu
327


 


hsa-miR-331-5p
MIMAT0004700
CUAGGUAUggucccagggaucc
328


 


hsa-miR-331-3p
MIMAT0000760
GCCCCUGGgccuauccuagaa
329


 


hsa-miR-324-5p
MIMAT0000761
CGCAUCCCcuagggcauuggugu
330


 


hsa-miR-324-3p
MIMAT0000762
ACUGCCCCaggugcugcugg
331


 


hsa-miR-338-5p
MIMAT0004701
AACAAUAUccuggugcugagug
332


 


hsa-miR-338-3p
MIMAT0000763
UCCAGCAUcagugauuuuguug
333


 


hsa-miR-339-5p
MIMAT0000764
UCCCUGUCcuccaggagcucacg
334


 


hsa-miR-339-3p
MIMAT0004702
UGAGCGCCucgacgacagagccg
335


 


hsa-miR-335
MIMAT0000765
UCAAGAGCaauaacgaaaaaugu
336


 


hsa-miR-335*
MIMAT0004703
UUUUUCAUuauugcuccugacc
337


 


hsa-miR-133b
MIMAT0000770
UUUGGUCCccuucaaccagcua
338


 


hsa-miR-325
MIMAT0000771
CCUAGUAGguguccaguaagugu
339


 


hsa-miR-345
MIMAT0000772
GCUGACUCcuaguccagggcuc
340


 


hsa-miR-346
MIMAT0000773
UGUCUGCCcgcaugccugccucu
341


 


hsa-miR-384
MIMAT0001075
AUUCCUAGaaauuguucaua
342


 


hsa-miR-196b
MIMAT0001080
UAGGUAGUuuccuguuguuggg
343


 


hsa-miR-196b*
MIMAT0009201
UCGACAGCacgacacugccuuc
344


 


hsa-miR-422a
MIMAT0001339
ACUGGACUuagggucagaaggc
345


 


hsa-miR-423-5p
MIMAT0004748
UGAGGGGCagagagcgagacuuu
346


 


hsa-miR-423-3p
MIMAT0001340
AGCUCGGUcugaggccccucagu
347


 


hsa-miR-424
MIMAT0001341
CAGCAGCAauucauguuuugaa
348


 


hsa-miR-424*
MIMAT0004749
CAAAACGUgaggcgcugcuau
349


 


hsa-miR-425
MIMAT0003393
AAUGACACgaucacucccguuga
350


 


hsa-miR-425*
MIMAT0001343
AUCGGGAAugucguguccgccc
351


 


hsa-miR-18b
MIMAT0001412
UAAGGUGCaucuagugcaguuag
352


 


hsa-miR-18b*
MIMAT0004751
UGCCCUAAaugccccuucuggc
353


 


hsa-miR-20b
MIMAT0001413
CAAAGUGCucauagugcagguag
354


 


hsa-miR-20b*
MIMAT0004752
ACUGUAGUaugggcacuuccag
355


 


hsa-miR-448
MIMAT0001532
UUGCAUAUguaggaugucccau
356


 


hsa-miR-429
MIMAT0001536
UAAUACUGucugguaaaaccgu
357


 


hsa-miR-449a
MIMAT0001541
UGGCAGUGuauuguuagcuggu
358


 


hsa-miR-450a
MIMAT0001545
UUUUGCGAuguguuccuaauau
359


 


hsa-miR-431
MIMAT0001625
UGUCUUGCaggccgucaugca
360


 


hsa-miR-431*
MIMAT0004757
CAGGUCGUcuugcagggcuucu
361


 


hsa-miR-433
MIMAT0001627
AUCAUGAUgggcuccucggugu
362


 


hsa-miR-329
MIMAT0001629
AACACACCugguuaaccucuuu
363


 


hsa-miR-451
MIMAT0001631
AAACCGUUaccauuacugaguu
364


 


hsa-miR-452
MIMAT0001635
AACUGUUUgcagaggaaacuga
365


 


hsa-miR-452*
MIMAT0001636
CUCAUCUGcaaagaaguaagug
366


 


hsa-miR-409-5p
MIMAT0001638
AGGUUACCcgagcaacuuugcau
367


 


hsa-miR-409-3p
MIMAT0001639
GAAUGUUGcucggugaaccccu
368


 


hsa-miR-412
MBIAT0002170
ACUUCACCugguccacuagccgu
369


 


hsa-miR-410
MIMAT0002171
AAUAUAACacagauggccugu
370


 


hsa-miR-376b
MIMAT0002172
AUCAUAGAggaaaauccauguu
371


 


hsa-miR-483-5p
MIMAT0004761
AAGACGGGaggaaagaagggag
372


 


hsa-miR-483-3p
MIMAT0002173
UCACUCCUcuccucccgucuu
373


 


hsa-miR-484
MIMAT0002174
UCAGGCUCaguccccucccgau
374


 


hsa-miR-485-5p
MIMAT0002175
AGAGGCUGgccgugaugaauuc
375


 


hsa-miR-485-3p
MIMAT0002176
GUCAUACAcggcucuccucucu
376


 


hsa-miR-486-5p
MIMAT0002177
UCCUGUACugagcugccccgag
377


 


hsa-miR-486-3p
MIMAT0004762
CGGGGCAGcucaguacaggau
378


 


hsa-miR-487a
MIMAT0002178
AAUCAUACagggacauccaguu
379


 


hsa-miR-488*
MIMAT0002804
CCCAGAUAauggcacucucaa
380


 


hsa-miR-488
MIMAT0004763
UUGAAAGGcuauuucuugguc
381


 


hsa-miR-489
MIMAT0002805
GUGACAUCcauauacggcagc
382


 


hsa-miR-490-5p
MIMAT0004764
CCAUGGAUcuccaggugggu
383


 


hsa-miR-490-3p
MIMAT0002806
CAACCUGGaggacuccaugcug
384


 


hsa-miR-491-5p
MIMAT0002807
AGUGGGGAacccuuccaugagg
385


 


hsa-miR-491-3p
MIMAT0004765
CUUAUGCAagauucccuucuac
386


 


hsa-miR-511
MIMAT0002808
GUGUCUUUugcucugcaguca
387


 


hsa-miR-146b-5p
MIMAT0002809
UGAGAACUgaauuccauaggcu
388


 


hsa-miR-146b-3p
MIMAT0004766
UGCCCUGUggacucaguucugg
389


 


hsa-miR-202*
MIMAT0002810
UUCCUAUGcauauacuucuuug
390


 


hsa-miR-202
MIMAT0002811
AGAGGUAUagggcaugggaa
391


 


hsa-miR-492
MIMAT0002812
AGGACCUGcgggacaagauucuu
392


 


hsa-miR-493*
MIMAT0002813
UUGUACAUgguaggcuuucauu
393


 


hsa-miR-493
MIMAT0003161
UGAAGGUCuacugugugccagg
394


 


hsa-miR-432
MIMAT0002814
UCUUGGAGuaggucauugggugg
395


 


hsa-miR-432*
MIMAT0002815
CUGGAUGGcuccuccaugucu
396


 


hsa-miR-494
MIMAT0002816
UGAAACAUacacgggaaaccuc
397


 


hsa-miR-495
MIMAT0002817
AAACAAACauggugcacuucuu
398


 


hsa-miR-496
MIMAT0002818
UGAGUAUUacauggccaaucuc
399


 


hsa-miR-193b*
MIMAT0004767
CGGGGUUUugagggcgagauga
400


 


hsa-miR-193b
MIMAT0002819
AACUGGCCcucaaagucccgcu
401


 


hsa-miR-497
MIMAT0002820
CAGCAGCAcacugugguuugu
402


 


hsa-miR-497*
MIMAT0004768
CAAACCACacugugguguuaga
403


 


hsa-miR-181d
MIMAT0002821
AACAUUCAuuguugucggugggu
404


 


hsa-miR-512-5p
MIMAT0002822
CACUCAGCcuugagggcacuuuc
405


 


hsa-miR-512-3p
MIMAT0002823
AAGUGCUGucauagcugagguc
406


 


hsa-miR-498
MIMAT0002824
UUUCAAGCcagggggcguuuuuc
407


 


hsa-miR-520e
MIMAT0002S25
AAAGUGCUuccuuuuugaggg
408


 


hsa-miR-515-5p
MIMAT0002826
UUCUCCAAaagaaagcacuuucug
409


 


hsa-miR-515-3p
MIMAT0002827
GAGUGCCUucuuuuggagcguu
410


 


hsa-miR-519e*
MIMAT0002828
UUCUCCAAaagggagcacuuuc
411


 


hsa-miR-519e
MIMAT0002829
AAGUGCCUccuuuuagaguguu
412


 


hsa-miR-520f
MIMAT0002830
AAGUGCUUccuuuuagaggguu
413


 


hsa-miR-519c-5p
MIMAT0002831
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-519c-3p
MIMAT0002832
AAAGUGCAucuuuuuagaggau
415


 


hsa-miR-520a-5p
MIMAT0002833
CUCCAGAGggaaguacuuucu
416


 


hsa-miR-520a-3p
MIMAT0002834
AAAGUGCUucccuuuggacugu
417


 


hsa-miR-526b
MIMAT0002835
CUCUUGAGggaagcacuuucugu
418


 


hsa-miR-526b*
MIMAT0002836
GAAAGUGCuuccuuuuagaggc
419


 


hsa-miR-519b-5p
MIMAT0005454
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-519b-3p
MIMAT0002837
AAAGUGCAuccuuuuagagguu
420


 


hsa-miR-525-5p
MIMAT0002838
CUCCAGAGggaugcacuuucu
421


 


hsa-miR-525-3p
MIMAT0002839
GAAGGCGCuucccuuuagagcg
422


 


hsa-miR-523*
MIMAT0005449
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-523
MIMAT0002840
GAACGCGCuucccuauagagggu
423


 


hsa-miR-518f*
MIMAT0002841
CUCUAGAGggaagcacuuucuc
424


 


hsa-miR-518f
MIMAT0002842
GAAAGCGCuucucuuuagagg
425


 


hsa-miR-520b
MIMAT0002843
AAAGUGCUuccuuuuagaggg
426


 


hsa-miR-518b
MIMAT0002844
CAAAGCGCuccccuuuagaggu
427


 


hsa-miR-526a
MIMAT0002845
CUCUAGAGggaagcacuuucug
428


 


hsa-miR-520c-5p
MIMAT0005455
CUCUAGAGggaagcacuuucug
428


 


hsa-miR-520c-3p
MIMAT0002846
AAAGUGCUuccuuuuagagggu
429


 


hsa-miR-518c*
MIMAT0002847
UCUCUGGAgggaagcacuuucug
430


 


hsa-miR-518c
MIMAT0002848
CAAAGCGCuucucuuuagagugu
431


 


hsa-miR-524-5p
MIMAT0002849
CUACAAAGggaagcacuuucuc
432


 


hsa-miR-524-3p
MIMAT0002850
GAAGGCGCuucccuuuggagu
433


 


hsa-miR-517*
MIMAT0002851
CCUCUAGAuggaagcacugucu
434


 


hsa-miR-517a
MIMAT0002852
AUCGUGCAucccuuuagagugu
435


 


hsa-miR-519d
MIMAT0002853
CAAAGUGCcucccuuuagagug
436


 


hsa-miR-521
MIMAT0002854
AACGCACUucccuuuagagugu
437


 


hsa-miR-520d-5p
MIMAT0002855
CUACAAAGggaagcccuuuc
438


 


hsa-miR-520d-3p
MIMAT0002856
AAAGUGCUucucuuuggugggu
439


 


hsa-miR-517b
MIMAT0002857
UCGUGCAUcccuuuagaguguu
440


 


hsa-miR-520g
MIMAT0002858
ACAAAGUGcuucccuuuagagugu
441


 


hsa-miR-516b
MIMAT0002859
AUCUGGAGguaagaagcacuuu
442


 


hsa-miR-516b*
MIMAT0002860
UGCUUCCUuucagagggu
443


 


hsa-miR-518e*
MIMAT0005450
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-518e
MIMAT0002861
AAAGCGCUucccuucagagug
444


 


hsa-miR-518a-5p
MIMAT0005457
CUGCAAAGggaagcccuuuc
445


 


hsa-miR-518a-3p
MIMAT0002863
GAAAGCGCuucccuuugcugga
446


 


hsa-miR-518d-5p
MIMAT0005456
CUCUAGAGggaagcacuuucug
428


 


hsa-miR-518d-3p
MIMAT0002864
CAAAGCGCuucccuuuggagc
447


 


hsa-miR-517c
MIMAT0002866
AUCGUGCAuccuuuuagagugu
448


 


hsa-miR-520h
MIMAT0002867
ACAAAGUGcuucccuuuagagu
449


 


hsa-miR-522*
MIMAT0005451
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-522
MIMAT0002868
AAAAUGGUucccuuuagagugu
450


 


hsa-miR-519a*
MIMAT0005452
CUCUAGAGggaagcgcuuucug
414


 


hsa-miR-519a
MIMAT0002869
AAAGUGCAuccuuuuagagugu
451


 


hsa-miR-527
MIMAT0002862
CUGCAAAGggaagcccuuuc
445


 


hsa-miR-516a-5p
MIMAT0004770
UUCUCGAGgaaagaagcacuuuc
452


 


hsa-miR-516a-3p
MIMAT0006778
UGCUUCCUuucagagggu
443


 


hsa-miR-499-5p
MIMAT0002870
UUAAGACUugcagugauguuu
453


 


hsa-miR-499-3p
MIMAT0004772
AACAUCACagcaagucugugcu
454


 


hsa-miR-500
MIMAT0004773
UAAUCCUUgcuaccugggugaga
455


 


hsa-miR-500*
MIMAT0002871
AUGCACCUgggcaaggauucug
456


 


hsa-miR-501-5p
MIMAT0002872
AAUCCUUUgucccugggugaga
457


 


hsa-miR-501-3p
MIMAT0004774
AAUGCACCcgggcaaggauucu
458


 


hsa-miR-502-5p
MIMAT0002873
AUCCUUGCuaucugggugcua
459


 


hsa-miR-502-3p
MIMAT0004775
AAUGCACCugggcaaggauuca
460


 


hsa-miR-503
MIMAT0002874
UAGCAGCGggaacaguucugcag
461


 


hsa-miR-504
MIMAT0002875
AGACCCUGgucugcacucuauc
462


 


hsa-miR-505*
MIMAT0004776
GGGAGCCAggaaguauugaugu
463


 


hsa-miR-505
MIMAT0002876
CGUCAACAcuugcugguuuccu
464


 


hsa-miR-513a-5p
MIMAT0002877
UUCACAGGgaggugucau
465


 


hsa-miR-513a-3p
MIMAT0004777
UAAAUUUCaccuuucugagaagg
466


 


hsa-miR-506
MIMAT0002878
UAAGGCACccuucugaguaga
467


 


hsa-miR-507
MIMAT0002879
UUUUGCACcuuuuggagugaa
468


 


hsa-miR-508-5p
MIMAT0004778
UACUCCAGagggcgucacucaug
469


 


hsa-miR-508-3p
MIMAT0002880
UGAUUGUAgccuuuuggaguaga
470


 


hsa-miR-509-5p
MIMAT0004779
UACUGCAGacaguggcaauca
471


 


hsa-miR-509-3p
MIMAT0002881
UGAUUGGUacgucuguggguag
472


 


hsa-miR-510
MIMAT0002882
UACUCAGGagaguggcaaucac
473


 


hsa-miR-514
MIMAT0002883
AUUGACACuucugugaguaga
474


 


hsa-miR-532-5p
MIMAT0002888
CAUGCCUUgaguguaggaccgu
475


 


hsa-miR-532-3p
MIMAT0004780
CCUCCCACacccaaggcuugca
476


 


hsa-miR-455-5p
MIMAT0003150
UAUGUGCCuuuggacuacaucg
477


 


hsa-miR-455-3p
MIMAT0004784
GCAGUCCAugggcauauacac
478


 


hsa-miR-539
MIMAT0003163
GGAGAAAUuauccuuggugugu
479


 


hsa-miR-544
MIMAT0003164
AUUCUGCAuuuuuagcaaguuc
480


 


hsa-miR-545*
MIMAT0004785
UCAGUAAAuguuuauuagauga
481


 


hsa-miR-545
MIMAT0003165
UCAGCAAAcauuuauugugugc
482


 


hsa-miR-487b
MIMAT0003180
AAUCGUACagggucauccacuu
483


 


hsa-miR-551a
MIMAT0003214
GCGACCCAcucuugguuucca
484


 


hsa-miR-552
MIMAT0003215
AACAGGUGacugguuagacaa
485


 


hsa-miR-553
MIMAT0003216
AAAACGGUgagauuuuguuuu
486


 


hsa-miR-554
MIMAT0003217
GCUAGUCCugacucagccagu
487


 


hsa-miR-92b*
MIMAT0004792
AGGGACGGgacgcggugcagug
488


 


hsa-miR-92b
MIMAT0003218
UAUUGCACucgucccggccucc
489


 


hsa-miR-555
MIMAT0003219
AGGGUAAGcugaaccucugau
490


 


hsa-miR-556-5p
MIMAT0003220
GAUGAGCUcauuguaauaugag
491


 


hsa-miR-556-3p
MIMAT0004793
AUAUUACCauuagcucaucuuu
492


 


hsa-miR-557
MIMAT0003221
GUUUGCACgggugggccuugucu
493


 


hsa-miR-558
MIMAT0003222
UGAGCUGCuguaccaaaau
494


 


hsa-miR-559
MIMAT0003223
UAAAGUAAauaugcaccaaaa
495


 


hsa-miR-561
MIMAT0003225
CAAAGUUUaagauccuugaagu
496


 


hsa-miR-562
MIMAT0003226
AAAGUAGCuguaccauuugc
497


 


hsa-miR-563
MIMAT0003227
AGGUUGACauacguuuccc
498


 


hsa-miR-564
MIMAT0003228
AGGCACGGugucagcaggc
499


 


hsa-miR-566
MIMAT0003230
GGGCGCCUgugaucccaac
500


 


hsa-miR-567
MIMAT0003231
AGUAUGUUcuuccaggacagaac
501


 


hsa-miR-568
MIMAT0003232
AUGUAUAAauguauacacac
502


 


hsa-miR-551b*
MIMAT0004794
GAAAUCAAgcgugggugagacc
503


 


hsa-miR-551b
MIMAT0003233
GCGACCCAuacuugguuucag
504


 


hsa-miR-569
MIMAT0003234
AGUUAAUGaauccuggaaagu
505


 


hsa-miR-570
MIMAT0003235
CGAAAACAgcaauuaccuuugc
506


 


hsa-miR-571
MIMAT0003236
UGAGUUGGccaucugagugag
507


 


hsa-miR-572
MIMAT0003237
GUCCGCUCggcgguggccca
508


 


hsa-miR-573
MIMAT0003238
CUGAAGUGauguguaacugaucag
509


 


hsa-miR-574-5p
MIMAT0004795
UGAGUGUGugugugugagugugu
510


 


hsa-miR-574-3p
MIMAT0003239
CACGCUCAugcacacacccaca
511


 


hsa-miR-575
MIMAT0003240
GAGCCAGUuggacaggagc
512


 


hsa-miR-576-5p
MIMAT0003241
AUUCUAAUuucuccacgucuuu
513


 


hsa-miR-576-3p
MIMAT0004796
AAGAUGUGgaaaaauuggaauc
514


 


hsa-miR-577
MIMAT0003242
UAGAUAAAauauugguaccug
515


 


hsa-miR-578
MIMAT0003243
CUUCUUGUgcucuaggauugu
516


 


hsa-miR-579
MIMAT0003244
UUCAUUUGguauaaaccgcgauu
517


 


hsa-miR-580
MIMAT0003245
UUGAGAAUgaugaaucauuagg
518


 


hsa-miR-581
MIMAT0003246
UCUUGUGUucucuagaucagu
519


 


hsa-miR-582-5p
MIMAT0003247
UUACAGUUguucaaccaguuacu
520


 


hsa-miR-582-3p
MIMAT0004797
UAACUGGUugaacaacugaacc
521


 


hsa-miR-583
MIMAT0003248
CAAAGAGGaaggucccauuac
522


 


hsa-miR-584
MIMAT0003249
UUAUGGUUugccugggacugag
523


 


hsa-miR-585
MIMAT0003250
UGGGCGUAucuguaugcua
524


 


hsa-miR-548a-3p
MIMAT0003251
CAAAACUGgcaauuacuuuugc
525


 


hsa-miR-586
MIMAT0003252
UAUGCAUUguauuuuuaggucc
526


 


hsa-miR-587
MIMAT0003253
UUUCCAUAggugaugagucac
527


 


hsa-miR-548b-5p
MIMAT0004798
AAAAGUAAuugugguuuuggcc
528


 


hsa-miR-548b-3p
MIMAT0003254
CAAGAACCucaguugcuuuugu
529


 


hsa-miR-588
MIMAT0003255
UUGGCCACaauggguuagaac
530


 


hsa-miR-589
MIMAT0004799
UGAGAACCacgucugcucugag
531


 


hsa-miR-589*
MIMAT0003256
UCAGAACAaaugccgguucccaga
532


 


hsa-miR-550
MIMAT0004800
AGUGCCUGagggaguaagagccc
533


 


hsa-miR-550*
MIMAT0003257
UGUCUUACucccucaggcacau
534


 


hsa-miR-590-5p
MIMAT0003258
GAGCUUAUucauaaaagugcag
535


 


hsa-miR-590-3p
MIMAT0004801
UAAUUUUAuguauaagcuagu
536


 


hsa-miR-591
MIMAT0003259
AGACCAUGgguucucauugu
537


 


hsa-miR-592
MIMAT0003260
UUGUGUCAauaugcgaugaugu
538


 


hsa-miR-593*
MIMAT0003261
AGGCACCAgccaggcauugcucagc
539


 


hsa-miR-593
MIMAT0004802
UGUCUCUGcugggguuucu
540


 


hsa-miR-595
MIMAT0003263
GAAGUGUGccguggugugucu
541


 


hsa-miR-596
MIMAT0003264
AAGCCUGCccggcuccucggg
542


 


hsa-miR-597
MIMAT0003265
UGUGUCACucgaugaccacugu
543


 


hsa-miR-598
MIMAT0003266
UACGUCAUcguugucaucguca
544


 


hsa-miR-599
MIMAT0003267
GUUGUGUCaguuuaucaaac
545


 


hsa-miR-548a-5p
MIMAT0004803
AAAAGUAAuugcgaguuuuacc
546


 


hsa-miR-600
MIMAT0003268
ACUUACAGacaagagccuugcuc
547


 


hsa-miR-601
MIMAT0003269
UGGUCUAGgauuguuggaggag
548


 


hsa-miR-602
MIMAT0003270
GACACGGGcgacagcugcggccc
549


 


hsa-miR-603
MIMAT0003271
CACACACUgcaauuacuuuugc
550


 


hsa-miR-604
MIMAT0003272
AGGCUGCGgaauucaggac
551


 


hsa-miR-605
MIMAT0003273
UAAAUCCCauggugccuucuccu
552


 


hsa-miR-606
MIMAT0003274
AAACUACUgaaaaucaaagau
553


 


hsa-miR-607
MIMAT0003275
GUUCAAAUccagaucuauaac
554


 


hsa-miR-608
MIMAT0003276
AGGGGUGGuguugggacagcuccgu
555


 


hsa-miR-609
MIMAT0003277
AGGGUGUUucucucaucucu
556


 


hsa-miR-610
MIMAT0003278
UGAGCUAAaugugugcuggga
557


 


hsa-miR-611
MIMAT0003279
GCGAGGACcccucggggucugac
558


 


hsa-miR-612
MIMAT0003280
GCUGGGCAgggcuucugagcuccuu
559


 


hsa-miR-613
MIMAT0003281
AGGAAUGUuccuucuuugcc
560


 


hsa-miR-614
MIMAT0003282
GAACGCCUguucuugccaggugg
561


 


hsa-miR-615-5p
MIMAT0004804
GGGGGUCCccggugcucggauc
562


 


hsa-miR-615-3p
MIMAT0003283
UCCGAGCCugggucucccucu
563


 


hsa-miR-616*
MIMAT0003284
ACUCAAAAcccuucagugacuu
564


 


hsa-miR-616
MIMAT0004805
AGUCAUUGgaggguuugagcag
565


 


hsa-miR-548c-5p
MIMAT0004806
AAAAGUAAuugcggauuuuugcc
566


 


hsa-miR-548c-3p
MIMAT0003285
CAAAAAUCucaauuacuuuugc
567


 


hsa-miR-617
MIMAT0003286
AGACUUCCcauuugaagguggc
568


 


hsa-miR-618
MIMAT0003287
AAACUCUAcuuguccuucugagu
569


 


hsa-miR-619
MIMAT0003288
GACCUGGAcauguuugugcccagu
570


 


hsa-miR-620
MIMAT0003289
AUGGAGAUagauauagaaau
571


 


hsa-miR-621
MIMAT0003290
GGCUAGCAacagcgcuuaccu
572


 


hsa-miR-622
MIMAT0003291
ACAGUCUGcugagguuggagc
573


 


hsa-miR-623
MIMAT0003292
AUCCCUUGcaggggcuguugggu
574


 


hsa-miR-624*
MIMAT0003293
UAGUACCAguaccuuguguuca
575


 


hsa-miR-624
MIMAT0004807
CACAAGGUauugguauuaccu
576


 


hsa-miR-625
MIMAT0003294
AGGGGGAAaguucuaagucc
577


 


hsa-miR-625*
MIMAT0004808
GACUAUAGaacuuucccccuca
578


 


hsa-miR-626
MIMAT0003295
AGCUGUCUgaaaaugucuu
579


 


hsa-miR-627
MIMAT0003296
GUGAGUCUcuaagaaaagagga
580


 


hsa-miR-628-5p
MIMAT0004809
AUGCUGACauauuuacuagagg
581


 


hsa-miR-628-3p
MIMAT0003297
UCUAGUAAgaguggcagucga
582


 


hsa-miR-629
MIMAT0004810
UGGGUUUAcguugggagaacu
583


 


hsa-miR-629*
MIMAT0003298
GUUCUCCCaacguaagcccagc
584


 


hsa-miR-630
MIMAT0003299
AGUAUUCUguaccagggaaggu
585


 


hsa-miR-631
MIMAT0003300
AGACCUGGcccagaccucagc
586


 


hsa-miR-33b
MIMAT0003301
GUGCAUUGcuguugcauugc
587


 


hsa-miR-33b*
MIMAT0004811
CAGUGCCUcggcagugcagccc
588


 


hsa-miR-632
MIMAT0003302
GUGUCUGCuuccuguggga
589


 


hsa-miR-633
MIMAT0003303
CUAAUAGUaucuaccacaauaaa
590


 


hsa-miR-634
MIMAT0003304
AACCAGCAccccaacuuuggac
591


 


hsa-miR-635
MIMAT0003305
ACUUGGGCacugaaacaaugucc
592


 


hsa-miR-636
MIMAT0003306
UGUGCUUGcucgucccgcccgca
593


 


hsa-miR-637
MIMAT0003307
ACUGGGGGcuuucgggcucugcgu
594


 


hsa-miR-638
MIMAT0003308
AGGGAUCGcgggcggguggcggccu
595


 


hsa-miR-639
MIMAT0003309
AUCGCUGCgguugcgagcgcugu
596


 


hsa-miR-640
MIMAT0003310
AUGAUCCAggaaccugccucu
597


 


hsa-miR-641
MIMAT0003311
AAAGACAUaggauagagucaccuc
598


 


hsa-miR-642
MIMAT0003312
GUCCCUCUccaaaugugucuug
599


 


hsa-miR-643
MIMAT0003313
ACUUGUAUgcuagcucagguag
600


 


hsa-miR-644
MIMAT0003314
AGUGUGGCuuucuuagagc
601


 


hsa-miR-645
MIMAT0003315
UCUAGGCUgguacugcuga
602


 


hsa-miR-646
MIMAT0003316
AAGCAGCUgccucugaggc
603


 


hsa-miR-647
MIMAT0003317
GUGGCUGCacucacuuccuuc
604


 


hsa-miR-648
MIMAT0003318
AAGUGUGCagggcacuggu
605


 


hsa-miR-649
MIMAT0003319
AAACCUGUguuguucaagaguc
606


 


hsa-miR-650
MIMAT0003320
AGGAGGCAgcgcucucaggac
607


 


hsa-miR-651
MIMAT0003321
UUUAGGAUaagcuugacuuuug
608


 


hsa-miR-652
MIMAT0003322
AAUGGCGCcacuaggguugug
609


 


hsa-miR-548d-5p
MIMAT0004812
AAAAGUAAuugugguuuuugcc
610


 


hsa-miR-548d-3p
MIMAT0003323
CAAAAACCacaguuucuuuugc
611


 


hsa-miR-661
MIMAT0003324
UGCCUGGGucucuggccugcgcgu
612


 


hsa-miR-662
MIMAT0003325
UCCCACGUuguggcccagcag
613


 


hsa-miR-663
MIMAT0003326
AGGCGGGGcgccgcgggaccgc
614


 


hsa-miR-449b
MIMAT0003327
AGGCAGUGuauuguuagcuggc
615


 


hsa-miR-449b*
MIMAT0009203
CAGCCACAacuacccugccacu
616


 


hsa-miR-653
MIMAT0003328
GUGUUGAAacaaucucuacug
617


 


hsa-miR-411
MIMAT0003329
UAGUAGACcguauagcguacg
618


 


hsa-miR-411*
MIMAT0004813
UAUGUAACacgguccacuaacc
619


 


hsa-miR-654-5p
MIMAT0003330
UGGUGGGCcgcagaacaugugc
620


 


hsa-miR-654-3p
MIMAT0004814
UAUGUCUGcugaccaucaccuu
621


 


hsa-miR-655
MIMAT0003331
AUAAUACAugguuaaccucuuu
622


 


hsa-miR-656
MIMAT0003332
AAUAUUAUacagucaaccucu
623


 


hsa-miR-549
MIMAT0003333
UGACAACUauggaugagcucu
624


 


hsa-miR-657
MIMAT0003335
GGCAGGUUcucacccucucuagg
625


 


hsa-miR-658
MIMAT0003336
GGCGGAGGgaaguagguccguuggu
626


 


hsa-miR-659
MIMAT0003337
CUUGGUUCaggagggucccca
627


 


hsa-miR-660
MIMAT0003338
UACCCAUUgcauaucggaguug
628


 


hsa-miR-421
MIMAT0003339
AUCAACAGacauuaauugggcgc
629


 


hsa-miR-542-5p
MIMAT0003340
UCGGGGAUcaucaugucacgaga
630


 


hsa-miR-542-3p
MIMAT0003389
UGUGACAGauugauaacugaaa
631


 


hsa-miR-758
MIMAT0003879
UUUGUGACcugguccacuaacc
632


 


hsa-miR-1264
MIMAT0005791
CAAGUCUUauuugagcaccuguu
633


 


hsa-miR-671-5p
MIMAT0003880
AGGAAGCCcuggaggggcuggag
634


 


hsa-miR-671-3p
MIMAT0004819
UCCGGUUCucagggcuccacc
635


 


hsa-miR-668
MIMAT0003881
UGUCACUCggcucggcccacuac
636


 


hsa-miR-767-5p
MIMAT0003882
UGCACCAUgguugucugagcaug
637


 


hsa-miR-767-3p
MIMAT0003883
UCUGCUCAuaccccaugguuucu
638


 


hsa-miR-1224-5p
MIMAT0005458
GUGAGGACucgggaggugg
639


 


hsa-miR-1224-3p
MIMAT0005459
CCCCACCUccucucuccucag
640


 


hsa-miR-320b
MIMAT0005792
AAAAGCUGgguugagagggcaa
641


 


hsa-miR-320c
MIMAT0005793
AAAAGCUGgguugagagggu
642


 


hsa-miR-1296
MIMAT0005794
UUAGGGCCcuggcuccaucucc
643


 


hsa-miR-1468
MIMAT0006789
CUCCGUUUgccuguuucgcug
644


 


hsa-miR-1323
MIMAT0005795
UCAAAACUgaggggcauuuucu
645


 


hsa-miR-1271
MIMAT0005796
CUUGGCACcuagcaagcacuca
646


 


hsa-miR-1301
MIMAT0005797
UUGCAGCUgccugggagugacuuc
647


 


hsa-miR-454*
MIMAT0003884
ACCCUAUCaauauugucucugc
648


 


hsa-miR-454
MIMAT0003885
UAGUGCAAuauugcuuauagggu
649


 


hsa-miR-1185
MIMAT0005798
AGAGGAUAcccuuuguauguu
650


 


hsa-miR-449c
MIMAT0010251
UAGGCAGUguauugcuagcggcugu
651


 


hsa-miR-449c*
MIMAT0013771
UUGCUAGUugcacuccucucugu
652


 


hsa-miR-1283
MIMAT0005799
UCUACAAAggaaagcgcuuucu
653


 


hsa-miR-769-5p
MIMAT0003886
UGAGACCUcuggguucugagcu
654


 


hsa-miR-769-3p
MIMAT0003887
CUGGGAUCuccggggucuugguu
655


 


hsa-miR-766
MIMAT0003888
ACUCCAGCcccacagccucagc
656


 


hsa-miR-762
MIMAT0010313
GGGGCUGGggccggggccgagc
657


 


hsa-miR-802
MIMAT0004185
CAGUAACAaagauucauccuugu
658


 


hsa-miR-670
MIMAT0010357
GUCCCUGAguguauguggug
659


 


hsa-miR-1298
MIMAT0005800
UUCAUUCGgcuguccagaugua
660


 


hsa-miR-2113
MIMAT0009206
AUUUGUGCuuggcucugucac
661


 


hsa-miR-761
MIMAT0010364
GCAGCAGGgugaaacugacaca
662


 


hsa-miR-764
MIMAT0010367
GCAGGUGCucacuuguccuccu
663


 


hsa-miR-759
MIMAT0010497
GCAGAGUGcaaacaauuuugac
664


 


hsa-miR-765
MIMAT0003945
UGGAGGAGaaggaaggugaug
665


 


hsa-miR-770-5p
MIMAT0003948
UCCAGUACcacgugucagggcca
666


 


hsa-miR-675
MIMAT0004284
UGGUGCGGagagggcccacagug
667


 


hsa-miR-675*
MIMAT0006790
CUGUAUGCccucaccgcuca
668


 


hsa-miR-298
MIMAT0004901
AGCAGAAGcagggagguucuccca
669


 


hsa-miR-891a
MIMAT0004902
UGCAACGAaccuaccacuga
670


 


hsa-miR-300
MIMAT0004903
UAUACAAGggcagacucucucu
671


 


hsa-miR-886-5p
MIMAT0004905
CGGGUCGGaguuagcucaagcgg
672


 


hsa-miR-886-3p
MIMAT0004906
CGCGGGUGcuuacugacccuu
673


 


hsa-miR-892a
MIMAT0004907
CACUGUGUccuuucugcguag
674


 


hsa-miR-220b
MIMAT0004908
CCACCACCgugucugacacuu
675


 


hsa-miR-450b-5p
MIMAT0004909
UUUUGCAAuauguuccugaaua
676


 


hsa-miR-450b-3p
MIMAT0004910
UUGGGAUCauuuugcauccaua
677


 


hsa-miR-874
MIMAT0004911
CUGCCCUGgcccgagggaccga
678


 


hsa-miR-890
MIMAT0004912
UACUUGGAaaggcaucaguug
679


 


hsa-miR-891b
MIMAT0004913
UGCAACUUaccugagucauuga
680


 


hsa-miR-220c
MIMAT0004915
ACACAGGGcuguugugaagacu
681


 


hsa-miR-888
MIMAT0004916
UACUCAAAaagcugucaguca
682


 


hsa-miR-888*
MIMAT0004917
GACUGACAccucuuugggugaa
683


 


hsa-miR-892b
MIMAT0004918
CACUGGCUccuuucuggguaga
684


 


hsa-miR-541*
MIMAT0004919
AAAGGAUUcugcugucggucccacu
685


 


hsa-miR-541
MIMAT0004920
UGGUGGGCacagaaucuggacu
686


 


hsa-miR-889
MIMAT0004921
UUAAUAUCggacaaccauugu
687


 


hsa-miR-875-5p
MIMAT0004922
UAUACCUCaguuuuaucaggug
688


 


hsa-miR-875-3p
MIMAT0004923
CCUGGAAAcacugagguugug
689


 


hsa-miR-876-5p
MIMAT0004924
UGGAUUUCuuugugaaucacca
690


 


hsa-miR-876-3p
MIMAT0004925
UGGUGGUUuacaaaguaauuca
691


 


hsa-miR-708
MIMAT0004926
AAGGAGCUuacaaucuagcuggg
692


 


hsa-miR-708*
MIMAT0004927
CAACUAGAcugugagcuucuag
693


 


hsa-miR-147b
MIMAT0004928
GUGUGCGGaaaugcuucugcua
694


 


hsa-miR-190b
MIMAT0004929
UGAUAUGUuugauauuggguu
695


 


hsa-miR-744
MIMAT0004945
UGCGGGGCuagggcuaacagca
696


 


hsa-miR-744*
MIMAT0004946
CUGUUGCCacuaaccucaaccu
697


 


hsa-miR-885-5p
MIMAT0004947
UCCAUUACacuacccugccucu
698


 


hsa-miR-885-3p
MIMAT0004948
AGGCAGCGggguguaguggaua
699


 


hsa-miR-877
MIMAT0004949
GUAGAGGAgauggcgcaggg
700


 


hsa-miR-877*
MIMAT0004950
UCCUCUUCucccuccucccag
701


 


hsa-miR-887
MIMAT0004951
GUGAACGGagcgccaucccgagg
702


 


hsa-miR-665
MIMAT0004952
ACCAGGAGgcugaggccccu
703


 


hsa-miR-873
MIMAT0004953
GCAGGAACuugugagucuccu
704


 


hsa-miR-543
MIMAT0004954
AAACAUUCgcggugcacuucuu
705


 


hsa-miR-374b
MIMAT0004955
AUAUAAUAcaaccugcuaagug
706


 


hsa-miR-374b*
MIMAT0004956
CUUAGCAGguuguauuaucauu
707


 


hsa-miR-760
MIMAT0004957
CGGCUCUGggucugugggga
708


 


hsa-miR-301b
MIMAT0004958
CAGUGCAAugauauugucaaagc
709


 


hsa-miR-216b
MIMAT0004959
AAAUCUCUgcaggcaaauguga
710


 


hsa-miR-208b
MIMAT0004960
AUAAGACGaacaaaagguuugu
711


 


hsa-miR-920
MIMAT0004970
GGGGAGCUguggaagcagua
712


 


hsa-miR-921
MIMAT0004971
CUAGUGAGggacagaaccaggauuc
713


 


hsa-miR-922
MIMAT0004972
GCAGCAGAgaauaggacuacguc
714


 


hsa-miR-924
MIMAT0004974
AGAGUCUUgugaugucuugc
715


 


hsa-miR-509-3-5p
MIMAT0004975
UACUGCAGacguggcaaucaug
716


 


hsa-miR-933
MIMAT0004976
UGUGCGCAgggagaccucuccc
717


 


hsa-miR-934
MIMAT0004977
UGUCUACUacuggagacacugg
718


 


hsa-miR-935
MIMAT0004978
CCAGUUACcgcuuccgcuaccgc
719


 


hsa-miR-936
MIMAT0004979
ACAGUAGAgggaggaaucgcag
720


 


hsa-miR-937
MIMAT0004980
AUCCGCGCucugacucucugcc
721


 


hsa-miR-938
MIMAT0004981
UGCCCUUAaaggugaacccagu
722


 


hsa-miR-939
MIMAT0004982
UGGGGAGCugaggcucugggggug
723


 


hsa-miR-940
MIMAT0004983
AAGGCAGGgcccccgcucccc
724


 


hsa-miR-941
MIMAT0004984
CACCCGGCugugugcacaugugc
725


 


hsa-miR-942
MIMAT0004985
UCUUCUCUguuuuggccaugug
726


 


hsa-miR-943
MIMAT0004986
CUGACUGUugccguccuccag
727


 


hsa-miR-944
MIMAT0004987
AAAUUAUUguacaucggaugag
728


 


hsa-miR-297
MIMAT0004450
AUGUAUGUgugcaugugcaug
729


 


hsa-miR-1178
MIMAT0005823
UUGCUCACuguucuucccuag
730


 


hsa-miR-1179
MIMAT0005824
AAGCAUUCuuucauugguugg
731


 


hsa-miR-1180
MIMAT0005825
UUUCCGGCucgcgugggugugu
732


 


hsa-miR-1181
MIMAT0005826
CCGUCGCCgccacccgagccg
733


 


hsa-miR-1182
MLMAT0005827
GAGGGUCUugggagggaugugac
734


 


hsa-miR-1183
MIMAT0005828
CACUGUAGgugauggugagagugggca
735


 


hsa-miR-1184
MIMAT0005829
CCUGCAGCgacuugauggcuucc
736


 


hsa-miR-1225-5p
MIMAT0005572
GUGGGUACggcccagugggggg
737


 


hsa-miR-1225-3p
MIMAT0005573
UGAGCCCCugugccgcccccag
738


 


hsa-miR-1226*
MIMAT0005576
GUGAGGGCaugcaggccuggaugggg
739


 


hsa-miR-1226
MIMAT0005577
UCACCAGCccuguguucccuag
740


 


hsa-miR-1227
MIMAT0005580
CGUGCCACccuuuuccccag
741


 


hsa-miR-1228*
MIMAT0005582
GUGGGCGGgggcaggugugug
742


 


hsa-miR-1228
MIMAT0005583
UCACACCUgccucgcccccc
743


 


hsa-miR-1229
MIMAT0005584
CUCUCACCacugcccucccacag
744


 


hsa-miR-1231
MIMAT0005586
GUGUCUGGgcggacagcugc
745


 


hsa-miR-1233
MLMAT0005588
UGAGCCCUguccucccgcag
746


 


hsa-miR-1234
MIMAT0005589
UCGGCCUGaccacccaccccac
747


 


hsa-miR-1236
MIMAT0005591
CCUCUUCCccuugucucuccag
748


 


hsa-miR-1237
MIMAT0005592
UCCUUCUGcuccgucccccag
749


 


hsa-miR-1238
MIMAT0005593
CUUCCUCGucugucugcccc
750


 


hsa-miR-1200
MIMAT0005863
CUCCUGAGccauucugagccuc
751


 


hsa-miR-1201
MIMAT0005864
AGCCUGAUuaaacacaugcucuga
752


 


hsa-miR-1202
MIMAT0005865
GUGCCAGCugcagugggggag
753


 


hsa-miR-1203
MIMAT0005866
CCCGGAGCcaggaugcagcuc
754


 


hsa-miR-663b
MIMAT0005867
GGUGGCCCggccgugccugagg
755


 


hsa-miR-1204
MIMAT0005868
UCGUGGCCuggucuccauuau
756


 


hsa-miR-1205
MIMAT0005869
UCUGCAGGguuugcuuugag
757


 


hsa-miR-1206
MIMAT0005870
UGUUCAUGuagauguuuaagc
758


 


hsa-miR-1207-5p
MIMAT0005871
UGGCAGGGaggcugggagggg
759


 


hsa-miR-1207-3p
MIMAT0005872
UCAGCUGGcccucauuuc
760


 


hsa-miR-1208
MIMAT0005873
UCACUGUUcagacaggcgga
761


 


hsa-miR-548e
MIMAT0005874
AAAAACUGagacuacuuuugca
762


 


hsa-miR-548j
MIMAT0005875
AAAAGUAAuugcggucuuuggu
763


 


hsa-miR-1285
MIMAT0005876
UCUGGGCAacaaagugagaccu
764


 


hsa-miR-1286
MIMAT0005877
UGCAGGACcaagaugagcccu
765


 


hsa-miR-1287
MIMAT0005878
UGCUGGAUcagugguucgaguc
766


 


hsa-miR-1289
MIMAT0005879
UGGAGUCCaggaaucugcauuuu
767


 


hsa-miR-1290
MIMAT0005880
UGGAUUUUuggaucaggga
768


 


hsa-miR-1291
MIMAT0005881
UGGCCCUGacugaagaccagcagu
769


 


hsa-miR-548k
MIMAT0005882
AAAAGUACuugcggauuuugcu
770


 


hsa-miR-1293
MIMAT0005883
UGGGUGGUcuggagauuugugc
771


 


hsa-miR-1294
MIMAT0005884
UGUGAGGUuggcauuguugucu
772


 


hsa-miR-1295
MIMAT0005885
UUAGGCCGcagaucuggguga
773


 


hsa-miR-1297
MIMAT0005886
UUCAAGUAauucaggug
774


 


hsa-miR-1299
MIMAT0005887
UUCUGGAAuucugugugaggga
775


 


hsa-miR-5481
MIMAT0005889
AAAAGUAUuugcggguuuuguc
776


 


hsa-miR-1302
MIMAT0005890
UUGGGACAuacuuaugcuaaa
777


 


hsa-miR-1303
MIMAT0005891
UUUAGAGAcggggucuugcucu
778


 


hsa-miR-1304
MIMAT0005892
UUUGAGGCuacagugagaugug
779


 


hsa-miR-1305
MIMAT0005893
UUUUCAACucuaaugggagaga
780


 


hsa-miR-1243
MIMAT0005894
AACUGGAUcaauuauaggagug
781


 


hsa-miR-548f
MIMAT0005895
AAAAACUGuaauuacuuuu
782


 


hsa-miR-1244
MIMAT0005896
AAGUAGUUgguuuguaugagaugguu
783


 


hsa-miR-1245
MIMAT0005897
AAGUGAUCuaaaggccuacau
784


 


hsa-miR-1246
MIMAT0005898
AAUGGAUUuuuggagcagg
785


 


hsa-miR-1247
MIMAT0005899
ACCCGUCCcguucguccccgga
786


 


hsa-miR-1248
MIMAT0005900
ACCUUCUUguauaagcacugugcuaaa
787


 


hsa-miR-1249
MIMAT0005901
ACGCCCUUcccccccuucuuca
788


 


hsa-miR-1250
MIMAT0005902
ACGGUGCUggauguggccuuu
789


 


hsa-miR-1251
MIMAT0005903
ACUCUAGCugccaaaggcgcu
790


 


hsa-miR-1253
MIMAT0005904
AGAGAAGAagaucagccugca
791


 


hsa-miR-1254
MIMAT0005905
AGCCUGGAagcuggagccugcagu
792


 


hsa-miR-1255a
MIMAT0005906
AGGAUGAGcaaagaaaguagauu
793


 


hsa-miR-1256
MIMAT0005907
AGGCAUUGacuucucacuagcu
794


 


hsa-miR-1257
MIMAT0005908
AGUGAAUGauggguucugacc
795


 


hsa-miR-1258
MIMAT0005909
AGUUAGGAuuaggucguggaa
796


 


hsa-miR-1259
MIMAT0005910
AUAUAUGAugacuuagcuuuu
797


 


hsa-miR-1260
MIMAT0005911
AUCCCACCucugccacca
798


 


hsa-miR-548g
MIMAT0005912
AAAACUGUaauuacuuuuguac
799


 


hsa-miR-1261
MIMAT0005913
AUGGAUAAggcuuuggcuu
800


 


hsa-miR-1262
MIMAT0005914
AUGGGUGAauuuguagaaggau
801


 


hsa-miR-1263
MIMAT0005915
AUGGUACCcuggcauacugagu
802


 


hsa-miR-548n
MIMAT0005916
CAAAAGUAauuguggauuuugu
803


 


hsa-miR-548m
MIMAT0005917
CAAAGGUAuuugugguuuuug
804


 


hsa-miR-1265
MIMAT0005918
CAGGAUGUggucaaguguuguu
805


 


hsa-miR-548o
MIMAT0005919
CCAAAACUgcaguuacuuuugc
806


 


hsa-miR-1266
MIMAT0005920
CCUCAGGGcuguagaacagggcu
807


 


hsa-miR-1267
MIMAT0005921
CCUGUUGAaguguaaucccca
808


 


hsa-miR-1268
MIMAT0005922
CGGGCGUGgugguggggg
809


 


hsa-miR-1269
MIMAT0005923
CUGGACUGagccgugcuacugg
810


 


hsa-miR-1270
MIMAT0005924
CUGGAGAUauggaagagcugugu
811


 


hsa-miR-1272
MIMAT0005925
GAUGAUGAuggcagcaaauucugaaa
812


 


hsa-miR-1273
MIMAT0005926
GGGCGACAaagcaagacucuuucuu
813


 


hsa-miR-1274a
MIMAT0005927
GUCCCUGUucaggcgcca
814


 


hsa-miR-548h
MIMAT0005928
AAAAGUAAucgcgguuuuuguc
815


 


hsa-miR-1275
MIMAT0005929
GUGGGGGAgaggcuguc
816


 


hsa-miR-1276
MIMAT0005930
UAAAGAGCccuguggagaca
817


 


hsa-miR-302e
MIMAT0005931
UAAGUGCUuccaugcuu
818


 


hsa-miR-302f
MIMAT0005932
UAAUUGCUuccauguuu
819


 


hsa-miR-1277
MIMAT0005933
UACGUAGAuauauauguauuuu
820


 


hsa-miR-548p
MIMAT0005934
UAGCAAAAacugcaguuacuuu
821


 


hsa-miR-548i
MIMAT0005935
AAAAGUAAuugcggauuuugcc
822


 


hsa-miR-1278
MIMAT0005936
UAGUACUGugcauaucaucuau
823


 


hsa-miR-1279
MIMAT0005937
UCAUAUUGcuucuuucu
824


 


hsa-miR-1274b
MIMAT0005938
UCCCUGUUcgggcgcca
825


 


hsa-miR-1281
MIMAT0005939
UCGCCUCCuccucuccc
826


 


hsa-miR-1282
MIMAT0005940
UCGUUUGCcuuuuucugcuu
827


 


hsa-miR-1284
MIMAT0005941
UCUAUACAgacccuggcuuuuc
828


 


hsa-miR-1288
MIMAT0005942
UGGACUGCccugaucuggaga
829


 


hsa-miR-1292
MIMAT0005943
UGGGAACGgguuccggcagacgcug
830


 


hsa-miR-1252
MIMAT0005944
AGAAGGAAauugaauucauuua
831


 


hsa-miR-1255b
MIMAT0005945
CGGAUGAGcaaagaaagugguu
832


 


hsa-miR-1280
MIMAT0005946
UCCCACCGcugccaccc
833


 


hsa-miR-1308
MIMAT0005947
GCAUGGGUgguucagugg
834


 


hsa-miR-664*
MIMAT0005948
ACUGGCUAgggauaaugauuggau
835


 


hsa-miR-664
MIMAT0005949
UAUUCAUUuauccccagccuaca
836


 


hsa-miR-1306
MIMAT0005950
ACGUUGGCucugguggug
837


 


hsa-miR-1307
MIMAT0005951
ACUCGGCGuggcgucggucgug
838


 


hsa-miR-513b
MIMAT0005788
UUCACAAGgaggugucauuuau
839


 


hsa-miR-513c
MIMAT0005789
UUCUCAAGgaggugucguuuau
840


 


hsa-miR-1321
MIMAT0005952
CAGGGAGGugaaugugau
841


 


hsa-miR-1322
MIMAT0005953
GAUGAUGCugcugaugcug
842


 


hsa-miR-720
MIMAT0005954
UCUCCCUGgggccucca
843


 


hsa-miR-1197
MIMAT0005955
UAGGACACauggucuacuucu
844


 


hsa-miR-1324
MIMAT0005956
CCAGACAGaauucuaugcacuuuc
845


 


hsa-miR-1469
MIMAT0007347
CUCGGCGCggggcgcgggcucc
846


 


hsa-miR-1470
MIMAT0007348
GCCCUCCGcccgugcaccccg
847


 


hsa-miR-1471
MIMAT0007349
GCCCGCGUguggagccaggugu
848


 


hsa-miR-1537
MIMAT0007399
AAAACCGUcuaguuacaguugu
849


 


hsa-miR-1538
MIMAT0007400
CGGCCCGGgcugcugcuguuccu
850


 


hsa-miR-1539
MIMAT0007401
UCCUGCGCgucccagaugccc
851


 


hsa-miR-103-as
MIMAT0007402
UCAUAGCCcuguacaaugcugcu
852


 


hsa-miR-320d
MIMAT0006764
AAAAGCUGgguugagagga
853


 


hsa-miR-1825
MIMAT0006765
UCCAGUGCccuccucucc
854


 


hsa-miR-1826
MIMAT0006766
AUUGAUCAucgacacuucgaacgcaau
855


 


hsa-miR-1827
MIMAT0006767
UGAGGCAGuagauugaau
856


 


hsa-miR-1908
MIMAT0007881
CGGCGGGGacggcgauugguc
857


 


hsa-miR-1909*
MIMAT0007882
UGAGUGCCggugccugcccug
858


 


hsa-miR-1909
MIMAT0007883
CGCAGGGGccgggugcucaccg
859


 


hsa-miR-1910
MIMAT0007884
CCAGUCCUgugccugccgccu
860


 


hsa-miR-1911
MIMAT0007885
UGAGUACCgccaugucuguuggg
861


 


hsa-miR-1911*
MIMAT0007886
CACCAGGCauuguggucucc
862


 


hsa-miR-1912
MIMAT0007887
UACCCAGAgcaugcagugugaa
863


 


hsa-miR-1913
MIMAT0007888
UCUGCCCCcuccgcugcugcca
864


 


hsa-miR-1914
MIMAT0007889
CCCUGUGCccggcccacuucug
865


 


hsa-miR-1914*
MIMAT0007890
GGAGGGGUcccgcacugggagg
866


 


hsa-miR-1915*
MIMAT0007891
ACCUUGCCuugcugcccgggcc
867


 


hsa-miR-1915
MIMAT0007892
CCCCAGGGcgacgcggcggg
868


 


hsa-miR-1972
MIMAT0009447
UCAGGCCAggcacaguggcuca
869


 


hsa-miR-1973
MIMAT0009448
ACCGUGCAaagguagcaua
870


 


hsa-miR-1975
MIMAT0009450
CCCCCACAaccgcgcuugacuagcu
871


 


hsa-miR-1976
MIMAT0009451
CCUCCUGCccuccuugcugu
872


 


hsa-miR-1979
MIMAT0009454
CUCCCACUgcuucacuugacua
873


 


hsa-miR-2052
MIMAT0009977
UGUUUUGAuaacaguaaugu
874


 


hsa-miR-2053
MIMAT0009978
GUGUUAAUuaaaccucuauuuac
875


 


hsa-miR-2054
MIMAT0009979
CUGUAAUAuaaauuuaauuuauu
876


 


hsa-miR-2110
MIMAT0010133
UUGGGGAAacggccgcugagug
877


 


hsa-miR-2114
MIMAT0011156
UAGUCCCUuccuugaagcgguc
878


 


hsa-miR-2114*
MIMAT0011157
CGAGCCUCaagcaagggacuu
879


 


hsa-miR-2115
MIMAT0011158
AGCUUCCAugacuccugaugga
880


 


hsa-miR-2115*
MIMAT0411159
CAUCAGAAuucauggaggcuag
881


 


hsa-miR-2116
MIMAT0011160
GGUUCUUAgcauaggaggucu
882


 


hsa-miR-2116*
MIMAT0011161
CCUCCCAUgccaagaacuccc
883


 


hsa-miR-2117
MIMAT0011162
UGUUCUCUuugccaaggacag
884


 


hsa-miR-548q
MIMAT0011163
GCUGGUGCaaaaguaauggcgg
885


 


hsa-miR-2276
MIMAT0011775
UCUGCAAGugucagaggcgagg
886


 


hsa-miR-2277
MIMAT0011777
UGACAGCGcccugccuggcuc
887


 


hsa-miR-2278
MIMAT0011778
GAGAGCAGuguguguugccugg
888


 


hsa-miR-711
MIMAT0012734
GGGACCCAgggagagacguaag
889


 


hsa-miR-718
MIMAT0012735
CUUCCGCCccgccgggcgucg
890


 


hsa-miR-2861
MIMAT0013802
GGGGCCUGgcggugggcgg
891


 


hsa-miR-2909
MIMAT0013863
GUUAGGGCcaacaucucuugg
892


 


hsa-miR-3115
MIMAT0014977
AUAUGGGUuuacuaguuggu
893


 


hsa-miR-3116
MIMAT0014978
UGCCUGGAacauaguagggacu
894


 


hsa-miR-3117
MIMAT0014979
AUAGGACUcauauagugccag
895


 


hsa-miR-3118
MIMAT0014980
UGUGACUGcauuauaaaauucu
896


 


hsa-miR-3119
MIMAT0014981
UGGCUUUUaacuuugauggc
897


 


hsa-miR-3120
MIMAT0014982
CACAGCAAguguagacaggca
898


 


hsa-miR-3121
MIMAT0014983
UAAAUAGAguaggcaaaggaca
899


 


hsa-miR-3122
MIMAT0014984
GUUGGGACaagaggacggucuu
900


 


hsa-miR-3123
MIMAT0014985
CAGAGAAUuguuuaauc
901


 


hsa-miR-3124
MIMAT0014986
UUCGCGGGcgaaggcaaaguc
902


 


hsa-miR-548s
MIMAT0014987
AUGGCCAAaacugcaguuauuuu
903


 


hsa-miR-3125
MIMAT0014988
UAGAGGAAgcuguggagaga
904


 


hsa-miR-3126-5p
MIMAT0014989
UGAGGGACagaugccagaagca
905


 


hsa-miR-3126-3p
MIMAT0015377
CAUCUGGCauccgucacacaga
906


 


hsa-miR-3127
MIMAT0014990
AUCAGGGCuuguggaaugggaag
907


 


hsa-miR-3128
MIMAT0014991
UCUGGCAAguaaaaaacucucau
908


 


hsa-miR-3129
MIMAT0014992
GCAGUAGUguagagauugguuu
909


 


hsa-miR-3130-5p
MIMAT0014995
UACCCAGUcuccggugcagcc
910


 


hsa-miR-3130-3p
MIMAT0014994
GCUGCACCggagacuggguaa
911


 


hsa-miR-3131
MIMAT0014996
UCGAGGACugguggaagggccuu
912


 


hsa-miR-3132
MIMAT0014997
UGGGUAGAgaaggagcucagagga
913


 


hsa-miR-3133
MIMAT0014998
UAAAGAACucuuaaaacccaau
914


 


hsa-miR-378b
MIMAT0014999
ACUGGACUuggaggcagaa
915


 


hsa-miR-3134
MIMAT0015000
UGAUGGAUaaaagacuacauauu
916


 


hsa-miR-3135
MIMAT0015001
UGCCUAGGcugagacugcagug
917


 


hsa-miR-466
MIMAT0015002
AUACACAUacacgcaacacacau
918


 


hsa-miR-3136
MIMAT0015003
CUGACUGAauagguagggucauu
919


 


hsa-miR-544b
MIMAT0015004
ACCUGAGGuugugcauuucuaa
920


 


hsa-miR-3137
MIMAT0015005
UCUGUAGCcugggagcaauggggu
921


 


hsa-miR-3138
MIMAT0015006
UGUGGACAgugagguagagggagu
922


 


hsa-miR-3139
MIMAT0015007
UAGGAGCUcaacagaugccuguu
923


 


hsa-miR-3140
MIMAT0015008
AGCUUUUGggaauucagguagu
924


 


hsa-miR-548t
MIMAT0015009
CAAAAGUGaucgugguuuuug
925


 


hsa-miR-3141
MIMAT0015010
GAGGGCGGguggaggagga
926


 


hsa-miR-3142
MIMAT0015011
AAGGCCUUucugaaccuucaga
927


 


hsa-miR-3143
MIMAT0015012
AUAACAUUguaaagcgcuucuuucg
928


 


hsa-miR-548u
MIMAT0015013
CAAAGACUgcaauuacuuuugcg
929


 


hsa-miR-3144-5p
MIMAT0015014
AGGGGACCaaagagauauauag
930


 


hsa-miR-3144-3p
MIMAT0015015
AUAUACCUguucggucucuuua
931


 


hsa-miR-3145
MIMAT0015016
AGAUAUUUugaguguuuggaauug
932


 


hsa-miR-1273c
MIMAT0015017
GGCGACAAaacgagacccuguc
933


 


hsa-miR-3146
MIMAT0015018
CAUGCUAGgauagaaagaaugg
934


 


Hsa-miR-3147
MIMAT0015019
GGUUGGGCagugaggaggguguga
935


 


hsa-miR-548v
MIMAT0015020
AGCUACAGuuacuuuugcacca
936


 


hsa-miR-3148
MIMAT0015021
UGGAAAAAacuggugugugcuu
937


 


hsa-miR-3149
MIMAT0015022
UUUGUAUGgauauguguguguau
938


 


hsa-miR-3150
MIMAT0015023
CUGGGGAGauccucgagguugg
939


 


hsa-miR-3151
MIMAT0015024
GGUGGGGCaaugggaucaggu
940


 


hsa-miR-3152
MIMAT0015025
UGUGUUAGaauaggggcaauaa
941


 


hsa-miR-3153
MIMAT0015026
GGGGAAAGcgaguagggacauuu
942


 


hsa-miR-3074
MIMAT0015027
GAUAUCAGcucaguaggcaccg
943


 


hsa-miR-3154
MIMAT0015028
CAGAAGGGgaguugggagcaga
944


 


hsa-miR-3155
MIMAT0015029
CCAGGCUCugcagugggaacu
945


 


hsa-miR-3156
MIMAT0015030
AAAGAUCUggaaaugggagaca
946


 


hsa-miR-3157
MIMAT0015031
UUCAGCCAggcuagugcagucu
947


 


hsa-miR-3158
MIMAT0015032
AAGGGCUUccucucugcaggac
948


 


hsa-miR-3159
MIMAT0015033
UAGGAUUAcaagugucggccac
949


 


hsa-miR-3160
MIMAT0015034
AGAGCUGAgacuagaaagccca
950


 


hsa-miR-3161
MIMAT0015035
CUGAUAAGaacagaggcccagau
951


 


hsa-miR-3162
MIMAT0015036
UUAGGGAGuagaaggguggggag
952


 


hsa-miR-3163
MIMAT0015037
UAUAAAAUgagggcaguaagac
953


 


hsa-miR-3164
MIMAT0015038
UGUGACUUuaagggaaauggcg
954


 


hsa-miR-3165
MIMAT0015039
AGGUGGAUgcaaugugaccuca
955


 


hsa-miR-3166
MIMAT0015040
CGCAGACAaugccuacuggccua
956


 


hsa-miR-1260b
MIMAT0015041
AUCCCACCacugccaccau
957


 


hsa-miR-3167
MIMAT0015042
AGGAUUUCagaaauacuggugu
958


 


hsa-miR-3168
MIMAT0015043
GAGUUCUAcagucagac
959


 


hsa-miR-3169
MIMAT0015044
UAGGACUGugcuuggcacauag
960


 


hsa-miR-3170
MIMAT0015045
CUGGGGUUcugagacagacagu
961


 


hsa-miR-3171
MIMAT0015046
AGAUGUAUggaaucuguauauac
962


 


hsa-miR-3172
MIMAT0015047
UGGGGUUUugcaguccuua
963


 


hsa-miR-3173
MIMAT0015048
AAAGGAGGaaauaggcaggcca
964


 


hsa-miR-1193
MIMAT0015049
GGGAUGGUagaccggugacgugc
965


 


hsa-miR-323b-5p
MIMAT0001630
AGGUUGUCcguggugaguucgca
966


 


hsa-miR-323b-3p
MIMAT0015050
CCCAAUACacggucgaccucuu
967


 


hsa-miR-3174
MIMAT0015051
UAGUGAGUuagagaugcagagcc
968


 


hsa-miR-3175
MIMAT0015052
CGGGGAGAgaacgcagugacgu
969


 


hsa-miR-3176
MIMAT0015053
ACUGGCCUgggacuaccgg
970


 


hsa-miR-3177
MIMAT0015054
UGCACGGCacuggggacacgu
971


 


hsa-miR-3178
MIMAT0015055
GGGGCGCGgccggaucg
972


 


hsa-miR-3179
MIMAT0015056
AGAAGGGGugaaauuuaaacgu
973


 


hsa-miR-3180-5p
MIMAT0015057
CUUCCAGAcgcuccgccccacgucg
974


 


hsa-miR-3180-3p
MIMAT0015058
UGGGGCGGagcuuccggaggcc
975


 


hsa-miR-548w
MIMAT0015060
AAAAGUAAcugcgguuuuugccu
976


 


hsa-miR-3181
MIMAT0015061
AUCGGGCCcucggcgccgg
977


 


hsa-miR-3182
MIMAT0015062
GCUUCUGUaguguaguc
978


 


hsa-miR-3183
MIMAT0015063
GCCUCUCUcggagucgcucgga
979


 


hsa-miR-3184
MIMAT0015064
UGAGGGGCcucagaccgagcuuuu
980


 


hsa-miR-3185
MIMAT0015065
AGAAGAAGgcggucggucugcgg
981


 


hsa-miR-3065-5p
MIMAT0015066
UCAACAAAaucacugaugcugga
982


 


hsa-miR-3065-3p
MIMAT0015378
UCAGCACCaggauauuguuggag
983


 


hsa-miR-3186-5p
MIMAT0015067
CAGGCGUCugucuacguggcuu
984


 


hsa-miR-3186-3p
MIMAT0015068
UCACGCGGagagauggcuuug
985


 


hsa-miR-3187
MIMAT0015069
UUGGCCAUggggcugcgcgg
986


 


hsa-miR-3188
MIMAT0015070
AGAGGCUUugugcggauacgggg
987


 


hsa-miR-3189
MIMAT0015071
CCCUUGGGucugaugggguag
988


 


hsa-miR-320e
MIMAT0015072
AAAGCUGGguugagaagg
989


 


hsa-miR-3190-5p
MIMAT0015073
UGUGGAAGguagacggccagaga
990


 


hsa-miR-3190-3p
MIMAT0015074
UGGAAGGUagacggccagagag
991


 


hsa-miR-3191
MIMAT0015075
UGGGGACGuagcuggccagacag
992


 


hsa-miR-3192
MIMAT0015076
UCUGGGAGguuguagcaguggaa
993


 


hsa-miR-3193
MIMAT0015077
UCCUGCGUaggaucugaggagu
994


 


hsa-miR-3194
MIMAT0015078
GGCCAGCCaccaggagggcug
995


 


hsa-miR-3195
MIMAT0015079
CGCGCCGGgcccggguu
996


 


hsa-miR-3196
MIMAT0015080
CGGGGCGGcaggggccuc
997


 


hsa-miR-548x
MIMAT0015081
UAAAAACUgcaauuacuuuca
998


 


hsa-miR-3197
MIMAT0015082
GGAGGCGCaggcucggaaaggcg
999


 


hsa-miR-3198
MIMAT0015083
GUGGAGUCcuggggaauggaga
1000


 


hsa-miR-3199
MIMAT0015084
AGGGACUGccuuaggagaaaguu
1001


 


hsa-miR-3200
MIMAT0015085
CACCUUGCgcuacucaggucug
1002


 


hsa-miR-3201
MIMAT0015086
GGGAUAUGaagaaaaau
1003


 


hsa-miR-514b-5p
MIMAT0015087
UUCUCAAGagggaggcaaucau
1004


 


hsa-miR-514b-3p
MIMAT0015088
AUUGACACcucugugagugga
1005


 


hsa-miR-3202
MIMAT0015089
UGGAAGGGagaagagcuuuaau
1006


 


hsa-miR-1273d
MIMAT0015090
GAACCCAUgagguugaggcugcagu
1007


 


hsa-miR-4295
MIMAT0016844
CAGUGCAAuguuuuccuu
1008


 


hsa-miR-4296
MIMAT0016845
AUGUGGGCucaggcuca
1009


 


hsa-miR-4297
MIMAT0016846
UGCCUUCCugucugug
1010


 


hsa-miR-378c
MIMAT0016847
ACUGGACUuggagucagaagagugg
1011


 


hsa-miR-4293
MIMAT0016848
CAGCCUGAcaggaacag
1012


 


hsa-miR-4294
MIMAT0016849
GGGAGUCUacagcaggg
1013


 


hsa-miR-4301
MIMAT0016850
UCCCACUAcuucacuuguga
1014


 


hsa-miR-4299
MIMAT0016851
GCUGGUGAcaugagaggc
1015


 


hsa-miR-4298
MIMAT0016852
CUGGGACAggaggaggaggcag
1016


 


hsa-miR-4300
MIMAT0016853
UGGGAGCUggacuacuuc
1017


 


hsa-miR-4304
MIMAT0016854
CCGGCAUGuccagggca
1018


 


hsa-miR-4302
MIMAT0016855
CCAGUGUGgcucagcgag
1019


 


hsa-miR-4303
MIMAT0016856
UUCUGAGCugaggacag
1020


 


hsa-miR-4305
MIMAT0016857
CCUAGACAccuccaguuc
1021


 


hsa-miR-4306
MIMAT0016858
UGGAGAGAaaggcagua
1022


 


hsa-miR-4309
MIMAT0016859
CUGGAGUCuaggauucca
1023


 


hsa-miR-4307
MIMAT0016860
AAUGUUUUuuccuguuucc
1024


 


hsa-miR-4308
MIMAT0016861
UCCCUGGAguuucuucuu
1025


 


hsa-miR-4310
MIMAT0016862
GCAGCAUUcauguccc
1026


 


hsa-miR-4311
MIMAT0016863
GAAAGAGAgcugagugug
1027


 


hsa-miR-4312
MIMAT0016864
GGCCUUGUuccugucccca
1028


 


hsa-miR-4313
MIMAT0016865
AGCCCCCUggccccaaaccc
1029


 


hsa-miR-4315
MIMAT0016866
CCGCUUUCugagcuggac
1030


 


hsa-miR-4316
MIMAT0016867
GGUGAGGCuagcuggug
1031


 


hsa-miR-4314
MIMAT0016868
CUCUGGGAaaugggacag
1032


 


hsa-miR-4318
MIMAT0016869
CACUGUGGguacaugcu
1033


 


hsa-miR-4319
MIMAT0016870
UCCCUGAGcaaagccac
1034


 


hsa-miR-4320
MIMAT0016871
GGGAUUCUguagcuuccu
1035


 


hsa-miR-4317
MIMAT0016872
ACAUUGCCagggaguuu
1036


 


hsa-miR-4322
MIMAT0016873
CUGUGGGCucagcgcgugggg
1037


 


hsa-miR-4321
MIMAT0016874
UUAGCGGUggaccgcccugcg
1038


 


hsa-miR-4323
MIMAT0016875
CAGCCCCAcagccucaga
1039


 


hsa-miR-4324
MIMAT0016876
CCCUGAGAcccuaaccuuaa
1040


 


hsa-miR-4256
MIMAT0016877
AUCUGACCugaugaaggu
1041


 


hsa-miR-4257
MIMAT0016878
CCAGAGGUggggacugag
1042


 


hsa-miR-4258
MIMAT0016879
CCCCGCCAccgccuugg
1043


 


hsa-miR-4259
MIMAT0016880
CAGUUGGGucuaggggucagga
1044


 


hsa-miR-4260
MIMAT0016881
CUUGGGGCauggaguccca
1045


 


hsa-miR-4253
MIMAT0016882
AGGGCAUGuccagggggu
1046


 


hsa-miR-4251
MIMAT0016883
CCUGAGAAaagggccaa
1047


 


hsa-miR-4254
MIMAT0016884
GCCUGGAGcuacuccaccaucuc
1048


 


hsa-miR-4255
MIMAT0016885
CAGUGUUCagagaugga
1049


 


hsa-miR-4252
MIMAT0016886
GGCCACUGagucagcacca
1050


 


hsa-miR-4325
MIMAT0016887
UUGCACUUgucucaguga
1051


 


hsa-miR-4326
MIMAT0016888
UGUUCCUCugucucccagac
1052


 


hsa-miR-4327
MIMAT0016889
GGCUUGCAugggggacugg
1053


 


hsa-miR-4261
MIMAT0016890
AGGAAACAgggaccca
1054


 


hsa-miR-4265
MIMAT0016891
CUGUGGGCucagcucuggg
1055


 


hsa-miR-4266
MIMAT0016892
CUAGGAGGccuuggcc
1056


 


hsa-miR-4267
MIMAT0016893
UCCAGCUCgguggcac
1057


 


hsa-miR-4262
MIMAT0016894
GACAUUCAgacuaccug
1058


 


hsa-miR-2355
MIMAT0016895
AUCCCCAGauacaauggacaa
1059


 


hsa-miR-4268
MIMAT0016896
GGCUCCUCcucucaggaugug
1060


 


hsa-miR-4269
MIMAT0016897
GCAGGCACagacagcccuggc
1061


 


hsa-miR-4263
MIMAT0016898
AUUCUAAGugccuuggcc
1062


 


hsa-miR-4264
MIMAT0016899
ACUCAGUCauggucauu
1063


 


hsa-miR-4270
MIMAT0016900
UCAGGGAGucaggggagggc
1064


 


hsa-miR-4271
MIMAT0016901
GGGGGAAGaaaaggugggg
1065


 


hsa-miR-4272
MIMAT0016902
CAUUCAACuagugauugu
1066


 


hsa-miR-4273
MIMAT0416903
GUGUUCUCugauggacag
1067


 


hsa-miR-4276
MIMAT0016904
CUCAGUGAcucaugugc
1068


 


hsa-miR-4275
MIMAT0016905
CCAAUUACcacuucuuu
1069


 


hsa-miR-4274
MIMAT0016906
CAGCAGUCccucccccug
1070


 


hsa-miR-4281
MIMAT0016907
GGGUCCCGgggagggggg
1071


 


hsa-miR-4277
MIMAT0016908
GCAGUUCUgagcacaguacac
1072


 


hsa-miR-4279
MIMAT0016909
CUCUCCUCccggcuuc
1073


 


hsa-miR-4278
MIMAT0016910
CUAGGGGGuuugcccuug
1074


 


hsa-miR-4280
MIMAT0016911
GAGUGUAGuucugagcagagc
1075


 


hsa-miR-4282
MIMAT0016912
UAAAAUUUgcauccagga
1076


 


hsa-miR-4285
MIMAT0016913
GCGGCGAGuccgacucau
1077


 


hsa-miR-4283
MIMAT0016914
UGGGGCUCagcgaguuu
1078


 


hsa-miR-4284
MIMAT0016915
GGGCUCACaucaccccau
1079


 


hsa-miR-4286
MIMAT0016916
ACCCCACUccugguacc
1080


 


hsa-miR-4287
MIMAT0016917
UCUCCCUUgagggcacuuu
1081


 


hsa-miR-4288
MIMAT0016918
UUGUCUGCugaguuucc
1082


 


hsa-miR-4292
MIMAT0016919
CCCCUGGGccggccuugg
1083


 


hsa-miR-4289
MIMAT0016920
GCAUUGUGcagggcuauca
1084


 


hsa-miR-4290
MIMAT0016921
UGCCCUCCuuucuucccuc
1085


 


hsa-miR-4291
MIMAT0016922
UUCAGCAGgaacagcu
1086


 


hsa-miR-4329
MIMAT0016923
CCUGAGACccuaguuccac
1087


 


hsa-miR-4330
MIMAT0016924
CCUCAGAUcagagccuugc
1088


 


hsa-miR-500b
MIMAT0016925
AAUCCUUGcuaccugggu
1089


 


hsa-miR-4328
MIMAT0016926
CCAGUUUUcccaggauu
1090









EXAMPLE 1
Inhibition of VAMP3 Expression by Single Stranded miR-124 Analogs
RT-qPCR Assays—
HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 6000 cells/well 24 hours prior to transfection.
Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine RNAiMax (Invitrogen) with a final miRNA concentration of 10 nM for the data in FIGS. 1 and 2, and ranging from 30 nM down to 0.01 nM along a 12-point titration curve for the data in FIG. 3.
24 hours after transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan® Gene Expression Cells-to-CTTM Kit (Applied Biosystems/Ambion) to extract RNA, synthesize cDNA, and perform RT-qPCR using a VAMP3-specific probe (Applied Biosystems) on an ABI Prism 7900HT Sequence Detector.
Reverse transcription conditions were as follows: 60 minutes at 37° C., followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GUSB mRNA levels were used for data normalization. Knockdown of VAMP3 was calculated as the two-fold change in VAMP3 cDNA measured in experimentally-treated cells relative to the VAMP3 eDNA measured in non-targeting control-treated cells.
Reporter Assays—
HCT-116 cells were cultured in McCoy's 5A Medium (Mediatech Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These cells were plated in 96-well culture plates at a density of 25,000 cells/well 24 hours prior to transfection.
Transfection was carried out using Opti-MEM I Reduced Serum Media (Gibco) and Lipofectamine 2000 (Invitrogen) with a final miRNA concentration of 10 nM for the data in FIGS. 4 and 5, and ranging from 30 nM down to 0.01 nM along a 6-point titration curve for the data in FIG. 6. miRNAs were co-transfected with siCHECK2 vectors (Genscript) containing cloned target inserts consisting of a tandem repeat of a seed match to miR-124 (2×; FIG. 6A) or a full-length match to miR-124 (2×FL; FIG. 6B).
Twenty-four hours after transfection, transfection medium was replaced with fresh growth medium. Fourty-eight hours after transfection, cells were lysed and both Firefly- and Renilla-Luciferase activity were measured using the Dual-GloTM Luciferase Assay System (Promega) on a Wallac EnVision 2103 Multilabel Reader (PerkinElmer). Firefly-Luciferase activity was used to normalize Renilla-Luciferase activity, and the final data was calculated as two-fold change of the Renilla-Luciferase signal in experimentally-treated cells relative to non-targeting control-treated cells.
Oligonucleotide Synthesis—
Oligonucleotides were synthesized using protocols well known in the art (solid phase synthesis) using commercially available phosphoramidites, then purified by reversed phase solid phase extraction (SPE). The C3 (C33H43N2O5P) and C6 (C36H49N2O5P) phosphoramidites were purchased from ChemGenes.
Briefly, the single strand oligonucleotides were synthesized using phosphoramidite chemistry on an automated solid-phase synthesizer, using procedures as are generally known in the art (see, for example, U.S. application Ser. No. 12/064,014, published as US 20090176725). A synthesis column was packed with solid support derivatized with the first nucleoside residue (natural or chemically modified). Synthesis was initiated by detritylation of the acid labile 5′-O-dimethoxytrityl group to release the 5′-hydroxyl. A suitably protected phosphoramidite and a suitable activator in acetonitrile were delivered simultaneously to the synthesis column resulting in coupling of the amidite to the 5′-hydroxyl. The column was then washed with a solvent, such as acetonitrile. An oxidizing solution, such as an iodine solution was pumped through the column to oxidize the phosphite triester linkage P(III) to its phosphotriester P(V) analog. Unreacted 5′-hydroxyl groups were capped using reagents such as acetic anhydride in the presence of 2,6-lutidine and N-methylimidazole. The elongation cycle was resumed with the detritylation step for the next phosphoramidite incorporation. This process was repeated until the desired sequence was synthesized. The synthesis concluded with the final 5′-terminus protecting group (trityl or 5′-O-dimethoxytrityl).
Upon completion of the synthesis, the solid-support and associated oligonucleotide were dried under argon pressure or vacuum. Aqueous base was added and the mixture was heated to effect cleavage of the succinyl linkage, removal of the cyanoethyl phosphate protecting group, and deprotection of the exocyclic amine protection.
The following process was performed on single strands that do not contain ribonucleotides. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with DMSO, which is combined with the filtrate. The resultant basic solution allows for retention of the 5′-O-dimethoxytrityl group to remain on the 5′ terminal position (trityl-on).
For single strands that contain ribonucleotides, the following process was performed. After treating the solid support with the aqueous base, the mixture was filtered to separate the solid support from the deprotected crude synthesis material. The solid support was then rinsed with dimethylsulfoxide (DMSO), which was combined with the filtrate. Fluoride reagent, such as triethylamine trihydrofluoride, was added to the mixture, and the solution was heated. The reaction was quenched with suitable buffer to provide a solution of crude single strand with the 5′-O-dimethoxytrityl group on the final 5′ terminal position.
The trityl—on solution of each crude single strand was purified using chromatographic purification, such as SPE RPC purification. The hydrophobic nature of the trityl group permits stronger retention of the desired full-length oligo than the non-tritylated truncated failure sequences. The failure sequences were selectively washed from the resin with a suitable solvent, such as low percent acetonitrile. Retained oligonucleotides were then detritylated on-column with trifluoroacetic acid to remove the acid-labile trityl group. Residual acid was washed from the column, a salt exchange was performed, and a final desalting of the material commenced. The full-length oligo was recovered in a purified form with an aqueous-organic solvent. The final product was then analyzed for purity (HPLC), identity (Maldi-TOF MS), and yield (UV A260). The oligos were dried via lyophilization or vacuum condensation.
Results—
The ability of single-stranded miR-124 analogs to inhibit expression of a known target, VAMP3, was tested, wherein the miR-124 analogs comprise either a C3 spacer substituted for one nucleotide, or a C6 spacer substituted for two nucleotides, at various positions along the strand.
The passenger strand sequence of the miR-124 used in this study is 5′-GCAUUCACCGCGUGCCUUAAAU-3′ (SEQ ID NO: 1091), and the guide strand sequence is 5′UUAAGGCACGCGGUGAAUGCCA-3′ (SEQ ID NO: 1092). The miR-124 analogs tested, as well as control molecules, are described in Table 2 and below.








TABLE 2





Name
Sequence (5′ → 3′)*







G/P
(guide) UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1092)



(passenger) GCAUUCACCGCGUGCCUUAAAU (SEQ ID NO: 1091)


 


21-8p-c3spacer-20
(guide) UAAGGCACGCGGUGAAUGC(C3-spacer)A (SEQ ID NO: 1093)


 


21-8p-c3spacer-19
(guide) UAAGGCACGCGGUGAAUG(C3-spacer)CA (SEQ ID NO: 1094)


(FIGS. 1, 3)



 


c3pos19spacer (FIG. 2)



 


c3pos19 (FIG. 4)



 


21-8p-c3spacer-18
(guide) UAAGGCACGCGGUGAAU(C3-spacer)CCA (SEQ ID NO: 1095)


 


21-8p-c3spacer-17
(guide) UAAGGCACGCGGUGAA(C3-spacer)GCCA (SEQ ID NO: 1096)


 


21-8p-c3spacer-16
(guide) UAAGGCACGCGGUGA(C3-spacer)UGCCA (SEQ ID NO: 1097)


 


21-8p-c3spacer-15
(guide) UAAGGCACGCGGUG(C3-spacer)AUGCCA (SEQ ID NO: 1098)


(FIGS. 1, 3)



 


c3pos15spacer (FIG. 2)



 


21-8p-c3spacer-14
(guide) UAAGGCACGCGGU(C3-spacer)AAUGCCA (SEQ ID NO: 1099)


(FIGS. 1, 3)



 


c3posl4 (FIG. 4)



 


21-8p-c3spacer-3
(guide) UAAGGCACGCGG(C3-spacer)GAAUGCCA (SEQ ID NO: 1100)


 


21-8p-c3spacer-12
(guide) UAAGGCACGCG(C3-spacer)UGAAUGCCA (SEQ ID NO: 1101)


 


21-8p-c3spacer-11
(guide) UAAGGCACGC(C3-spacer)GUGAAUGCCA (SEQ ID NO: 1102)


 


21-8p-c3spacer-10
(guide) UAAGGCACG(C3-spacer)GGUGAAUGCCA (SEQ ID NO: 1103)


 


21-8p-c3spacer-9
(guide) UAAGGCAC(C3-spacer)CGGUGAAUGCCA (SEQ ID NO: 1104)


 


21-8p-c3spacer-8
(guide) UAAGGCA(C3-spacer)GCGGUGAAUGCCA (SEQ ID NO: 1105)


 


21-8p-c3space-7
(guide) UAAGGC(C3-spacer)CGCGGUGAAUGCCA (SEQ ID NO: 1106)


 


21-8p-c3spacer-6
(guide) UAAGG(C3-spacer)ACGCGGUGAAUGCCA (SEQ ID NO: 1107)


 


21-8p-c3spacer-5
(guide) UAAG(C3-spacer)CACGCGGUGAAUGCCA (SEQ ID NO: 1108)


 


21-8p-c3spacer-4
(guide) UAA(C3-spacer)GCACGCGGUGAAUGCCA (SEQ ID NO: 1109)


 


21-8p-c3spacer-3
(guide) UA(C3-spacer)GGCACGCGGUGAAUGCCA (SEQ ID NO: 1110)


 


21-8p-c3spacer-2
(guide) U(C3-spacer)AGGCACGCGGUGAAUGCCA (SEQ ID NO: 1111)


 


21-8p-c6spacerdel2-19
(guide) UAAGGCACGCGGUGAAUG(C6-spacer)A (SEQ ID NO: 1112)


 


21-8p-c6spacerdel2-18
(guide) UAAGGCACGCGGUGAAU(C6-spacer)CA (SEQ ID NO: 1113)


 


21-8p-c6spacerdel2-17
(guide) UAAGGCACGCGGUGAA(C6-spacer)CCA (SEQ ID NO: 1114)


 


21-8p-c6spacerdel2-16
(guide) UAAGGCACGCGGUGA(C6-spacer)GCCA (SEQ ID NO: 1115)


 


21-8p-c6spacerdel2-15
(guide) UAAGGCACGCGGUG(C6-spacer)UGCCA (SEQ ID NO: 1116)


(FIGS. 1, 3)



 


c6del2pos15spacer



(FIG. 2)



 


21-8p-c6spacerdel2-14
(guide) UAAGGCACGCGGU(C6-spacer)AUGCCA (SEQ ID NO: 1117)


 


21-8p-c6spacerdel2-13
(guide) UAAGGCACGCGG(C6-spacer)AAUGCCA (SEQ ID NO: 1118)


 


21-8p-c6spacerdel2-12
(guide) UAAGGCACGCG(C6-spacer)GAAUGCCA (SEQ ID NO: 1119)


 


21-8p-c6spacerdel2-11
(guide) UAAGGCACGC(C6-spacer)UGAAUGCCA (SEQ ID NO: 1120)


 


21-8p-c6spacerdel2-10
(guide) UAAGGCACG(C6-spacer)GUGAAUGCCA (SEQ ID NO: 1121)


 


21-8p-c6spacerdel2-9
(guide) UAAGGCAC(C6-spacer)GGUGAAUGCCA (SEQ ID NO: 1122)


 


21-8p-c6spacerdel2-8
(guide) UAAGGCA(C6-spacer)CGGUGAAUGCCA (SEQ ID NO: 1123)


 


21-8p-c6spacerdel2-7
(guide) UAAGGC(C6-spacer)GCGGUGAAUGCCA (SEQ ID NO: 1124)


 


UC3
(passenger) B gUaUgaCCgaCUaCgCgUatt B (SEQ ID NO: 1125)



(guide) UACGCGUAGUCGGUCAUACUU SEQ ID NO: 1126)


 


miR-124 (FIG. 1)
(guide) UUAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1127)


 


pG (FIG. 2)



 


124(21)-8p-16rrr
(guide) UAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1128)


 


124(21)-8p (FIG. 1)



 


G-all-Fluoro (FIG. 2)
(guide) UAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1129)


 


pG-Fluoro (FIG. 4)



 


GAPDH
(guide) AAGUUGUCAUGGAUGACCUUU (SEQ ID NO: 1131)



(passenger) B aggUCaUCCaUgaCaaCUUtt B (SEQ ID NO: 1130)


 


Renilla
(guide) UAGUUGCGGACAAUCUGGAtt (SEQ ID NO: 1133)



(passenger) UCCAGAUUGUCCGCAACUAtt (SEQ ID NO: 1132)


 


C6delpos15spacer/P
(guide) UAAGGCACGCGGUG(C6-spacer)UGCCA (SEQ ID NO: 1116)



(passenger) GCAUUCACCGCGUGCCUUAAAU (SEQ ID NO: 1091)


 


pG-Fluoro/Pshort
(guide) UAAGGCACGCGGUGAAUGCCA (SEQ ID NO: 1129)



(passenger) GCAUUCACCGCGUGCCUUAAU (SEQ ID NO: 1134)





*A, U, C, and G = 2′-deoxy-2′-fluoro A, U, C, and G


A, U, C, and G = 2′-O-methyl (2′-OMe) A, U, C, and G


a, g, c and u = deoxy A, U, C, and G


t = thymidine


A, C, G, and U = ribose A, C, G or U


B = inverted abasic






All of the single-stranded molecules in Table 2 contain a 5′ phosphate cap.
“G/P” represents double-stranded miR-124, wherein the duplex has two nucleotide overhangs on the 3′ ends of the passenger and guide strands. The guide strand of the G/P duplex is 22 nucleotides in length.
SEQ ID NOs: 1093-1124 represent analogs of the single-stranded miR-124 guide strand. Each of these molecules are a 21-nucleotide version of the miR-124 guide strand that is present in the G/P duplex, missing the 5′-uracil nucleotide that is present in the 22-nucleotide G/P miR-124 guide strand. All of the nucleotides in these 21-mer analogs, with the exception of the 3′ adenosine, and the adjacent cytosine (if present), are chemically modified on the ribose moiety with 2′-fluoro (depicted as italicized nucleotides in Table 2). The 3′ adenosine, and the adjacent cytosine (if present), are chemically modified on the ribose moiety with 2′-O-methyl (depicted as underlined nucleotides in Table 2). Finally, the 21-mer analogs of the miR-124 guide strand contain either a C3-spacer substituted for one nucleotide (the “c3spacer” analogs) or a C6-spacer substituted for two nucleotides (the “c6spacerdel2” analogs) at the various denoted positions along the strand. For example “21-8p-c3spacer-20” represents a 21-mer miR-124 guide strand analog containing an ethylene glycol spacer in the place of the nucleotide at position 20 within the 21-nucleotide miR-124 guide strand, linking the nucleotides at position 19 and position 21. As another example, the analog labeled “21-Sp-c6spacerdel2-19” represents a 21-met miR-124 guide strand analog containing a hexane spacer in the place of the nucleotides at positions 19 and 20 within the 21-nucleotide miR-124 guide strand, linking the nucleotides at positions 18 and 21. Some of the 21-nucleotide miR-124 guide strand analogs have different names in accompanying Figures, as noted in Table 2. For example, the 21-mer miR-124 guide strand analog represented by SEQ ID NO: 1116 is called “21-8p-c6spacerdel2-15” in FIGS. 1 and 3 and “c6del2pos15spacer” in FIG. 2.
“UC3” represents a non-targeting, chemically-modified duplex.
“124(21)-8p-16rrr” represents an analog of the 21-nucleotide version of the miR-124 guide strand. This molecule does not contain an internal spacer. All of the nucleotides are modified with 2′-fluoro, with the exception of nucleotides 16-18, which are RNA, and nucleotides 21 and 22, which are modified with 2′-O-methyl.
“124(21)-8p” represents a 21-nucleotide version of the miR-124 guide, wherein nucleotides 1-20 are modified with 2′-fluoro and nucleotides 20 and 21 are modified with 2′-O-methyl.
“124(21)-8p” is the name of this analog in FIG. 1; “G-all Fluoro” is the name of this analog in FIG. 2; and, “pG-Fluoro” is the name of this analog in FIG. 4.
“miR-124” is the single-stranded guide strand of the G/P duplex. It is 22 nucleotides in length and unmodified.
“C6delpos15spacer/P” represents a double-stranded miR-124 duplex, wherein the guide strand has the structure of “21-Sp-c6spacerdel2-15” (SEQ ID NO: 1116), and the passenger strand is the 22-nucleotide miR-124 passenger strand (SEQ ID NO: 1091).
FIGS. 1 and 2 show the degree of inhibition of VAMP3 target expression by the single-stranded miR-124 analogs described in Table 2 using the RT-qPCR assay described above. FIG. 1 shows the degree of inhibition by the single-stranded miR-124 analogs containing a C3 spacer. FIG. 2 shows the degree of inhibition by the single-stranded miR-124 analogs containing a C6 spacer. The longer bars in each figure indicate greater knockdown of VAMP3. The duplicate bars indicate biological replicates, each representing data from a separate well of cells (on two separate plates) transfected with the indicated nucleic acid molecules. The spacer appears to be most well-tolerated at position 19, and in the vicinity of position 15, of the miR-124 analogs.
The graph in FIG. 3 depicts the dose-dependent response of VAMP3 expression to a subset of the analogs tested in FIGS. 1 and 2 (see Table 2 for sequences). VAMP3 expression is depicted along the y-axis, thus data points with lower values along this axis indicate greater VAMP3 expression knockdown. The dose of the miR-124 analog tested is depicted along the x-axis, ranging from the lowest doses on the left to the highest doses on the right. Although the double stranded versions (G/P and c6del2pos15spacer/P) are more potent than the single-stranded analogs, it is worth noting that the single-stranded “G-all Fluoro” analog (no internal spacer, nucleotides 1-20 are 2′-fluoro, nucleotides 20 and 21 are 2′O-methyl) behaves almost identically to comparable single-stranded analogs with a C3 spacer replacing position 15 (c3pos15spacer) or position 19 (c3pos19spacer).
FIGS. 4 and 5 show data from a screen of the same single-stranded miR-124 analogs tested in FIGS. 1 and 2, measuring knockdown of a co-transfected luciferase reporter that carries two target sites matching the seed region of miR-124. Thus, the data from this assay is a representation of the miRNA activity of the tested analogs. FIG. 4 shows the degree of inhibition by the single-stranded miR-124 analogs that contain a C3 spacer. FIG. 5 shows the degree of inhibition by the single-stranded miR-124 analogs that contain a C6 spacer. The duplicate bars indicate biological replicates, each representing data from a separate well of cells (on two separate plates) transfected with the indicated molecules. Again, the longer bars indicate greater inhibition, showing that the analogs that contain a spacer at position 19, or in the vicinity of position 15, have the greatest knockdown activity.
The graphs in FIG. 6 depict the dose-dependent response of target expression inhibition of two different luciferase reporters to a subset of the analogs tested in FIG. 3. In FIG. 6A, the inhibition activity shown is against a luciferase reporter with two matches to the miR-124 seed region. Thus, this is a representation of the miRNA activity of the tested analogs. In FIG. 6B, the inhibition activity shown is against a luciferase reporter with two full-length matches to miR-124 and, thus, represents the siRNA activity of the tested analogs. In both A and B, the G/P curves represent activity by a miR-124 duplex made up entirely of RNA. The pG-Fluoro/Pshort curves represent activity by a guide strand that is predominantly modified with 2′-fluoro nucleotides duplexed to an all-RNA passenger strand. The pG-Fluoro curves represent the activity of a single-stranded miR-124 guide strand analog that is predominantly modified with 2′-fluoro nucleotides. The c3pos14 and c3pos19 curves represent the activity of analogs of pG-Fluoro, only differing from pG-Fluoro by containing a 3-carbon spacer (C3-spacer) substituted for position 14 (“c3pos14”) or position 19 (“c3pos19”). All of the single-stranded analogs show less potency than either duplexes against the reporter with only seed matches, but they are effectively equivalent to each other across all concentrations and show similar activity to the duplexes at the highest concentration (FIG. 6A). Against the reporter with full-length matches, the all-RNA duplex (“G/P”) was still the strongest performer, but the spacer-containing single-stranded analogs had activity as strong as or stronger than the duplex with the 2′-fluoro guide strand (“pG-Fluoro/Pshort”) and the single-strand (“pG-Fluoro”).
EXAMPLE 2
Single-Strand RNAi Knockdown of ApoB mRNA
RT-qPCR Assays (Primary Screens and Dose-Response Curves)—
Hepa1-6 cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin-steptomycin, and 1% sodium bicarbonate. These cells were plated in a 96-well culture plates at a density of 3000 cells/well 24 hours prior to transfection.
Transfections were performed using Opti-MEM I Reduced Serum Media and Lipofectamine RNAiMAX per the manufacturer's directions. Final single-stranded siRNA concentrations were 100 nM and 10 nM.
Twenty-four hours post-transfection, cells were washed with phosphate-buffered saline and processed using the TaqMan Gene Expression Cells-to-CTTM Kit, per manufacturer's instructions, to extract RNA, synthesize cDNA, and perform RT-qPCR using an ApoB specific Taqman primer/probe set on an ABI Prism 7900HT Sequence Detector.
Reverse transcription conditions were as follows: 60 minutes at 37° C. followed by 5 minutes at 95° C. RT-qPCR conditions were as follows: 2 minutes at 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. GADPH mRNA levels were used for data normalization.
Knockdown of ApoB was calculated as the two-fold change in ApoB eDNA measured in experimentally-treated cells relative to the ApoB cDNA measured in non-targeting, control-treated cells.
Results—
The knockdown of ApoB mRNA was measured using single strand (guide strand) oligonucleotides with a C3 spacer incorporated at either position 15, 16, 17, 18, or 19 (relative to the 5′ of the oligo) at two different concentrations (100 nM and 10 nM). The results are shown in FIG. 7. All of the single strand molecules tested are composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotides and a 5′ phosphate. The two 3′ terminal nucleotides of each molecule are 2′-O-methyl nucleotides. Single strand molecule “485” (see FIG. 7; 5′-UUAAGAGAAGCCUUACUGGUU-3′ (SEQ ID NO: 1135)) is a 21-nucleotide molecule that does not contain a C3 spacer and targets ApoB mRNA at nucleotide position 485. A C3 spacer is incorporated into molecule 485 at positions 15 (“485 c3@pos15”; SEQ ID NO: 1136), 16 (“485 c3@pos16”; SEQ ID NO: 1137), 17 (“485 c3@pos17”; SEQ ID NO: 1138), 18 (“485 c3@pos18”; SEQ ID NO: 1139) or 19 (“485 c3@pos19”; SEQ ID NO: 1140) (i.e., the spacer takes the place of the indicated nucleotide of the 485 molecule). For example, signal strand molecule “485 c3@pos15” is represented by: 5′-UUAAGAGAAGCCUU(C3-spacer)CUGGUU-3′; SEQ ID NO: 1136). As shown in FIG. 7, inclusion of the C3 spacer at positions 18 or 19 is both well tolerated and improves mRNA knockdown at two different concentrations (100 nM and 10 nM). This data indicates that incorporation of a non-nucleotide C3 carbon spacer at the 3′ end of a single strand RNA interference oligonucleotide improves the potency of mRNA knockdown (FIG. 7).
To evaluate whether the incorporation of a C3 spacer in single strands was more broadly applicable, 30 different single strand sequences targeting ApoB, each with a C3 spacer at either position 18 (FIG. 8) or position 19 (FIG. 9), were evaluated at two concentrations (100 nM and 10 nM). In FIGS. 8 and 9, the single-stranded molecules are notated by the position within the ApoB mRNA which they target. All of the single-stranded molecules tested are composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotides and a 5′ phosphate. The two 3′ terminal nucleotides of each molecule are 2′-O-methyl nucleotides. The sequence identifiers (SEQ ID NOs:) of the single stranded RNAi molecules evaluated in FIGS. 8 and 9 are listed in Table 3.










TABLE 3





ApoB





Target
C3-position 18
C3-position 19
No spacer


Region
(FIGS. 8 and 10)
(FIGS. 9 and 10)
(FIG. 10)


















19
SEQ ID NO: 1141
SEQ ID NO: 1170
SEQ ID NO: 1199


248
SEQ ID NO: 1142
SEQ ID NO: 1171
SEQ ID NO: 1200


397
SEQ ID NO: 1143
SEQ ID NO: 1172
SEQ ID NO: 1201


485
SEQ ID NO: 1139
SEQ ID NO: 1140
SEQ ID NO: 1135


601
SEQ ID NO: 1144
SEQ ID NO: 1173
SEQ ID NO: 1202


719
SEQ ID NO: 1145
SEQ ID NO: 1174
SEQ ID NO: 1203


780
SEQ ID NO: 1146
SEQ ID NO: 1175
SEQ ID NO: 1204


1124
SEQ ID NO: 1147
SEQ ID NO: 1176
SEQ ID NO: 1205


1445
SEQ ID NO: 1148
SEQ ID NO: 1177
SEQ ID NO: 1206


1446
SEQ ID NO: 1149
SEQ ID NO: 1178
SEQ ID NO: 1207


1611
SEQ ID NO: 1150
SEQ ID NO: 1179
SEQ ID NO: 1208


1983
SEQ ID NO: 1151
SEQ ID NO: 1180
SEQ ID NO: 1209


3214
SEQ ID NO: 1152
SEQ ID NO: 1181
SEQ ID NO: 1210


3614
SEQ ID NO: 1153
SEQ ID NO: 1182
SEQ ID NO: 1211


4542
SEQ ID NO: 1154
SEQ ID NO: 1183
SEQ ID NO: 1212


6548
SEQ ID NO: 1155
SEQ ID NO: 1184
SEQ ID NO: 1213


6930
SEQ ID NO: 1156
SEQ ID NO: 1185
SEQ ID NO: 1214


6981
SEQ ID NO: 1157
SEQ ID NO: 1186
SEQ ID NO: 1215


7044
SEQ ID NO: 1158
SEQ ID NO: 1187
SEQ ID NO: 1216


9414
SEQ ID NO: 1159
SEQ ID NO: 1188
SEQ ID NO: 1217


9514
SEQ ID NO: 1160
SEQ ID NO: 1189
SEQ ID NO: 1218


9621
SEQ ID NO: 1161
SEQ ID NO: 1190
SEQ ID NO: 1219


10162
SEQ ID NO: 1162
SEQ ID NO: 1191
SEQ ID NO: 1220


10167
SEQ ID NO: 1163
SEQ ID NO: 1192
SEQ ID NO: 1221


10168
SEQ ID NO: 1164
SEQ ID NO: 1193
SEQ ID NO: 1222


10219
SEQ ID NO: 1165
SEQ ID NO: 1194
SEQ ID NO: 1223


10455
SEQ ID NO: 1166
SEQ ID NO: 1195
SEQ ID NO: 1224


10517
SEQ ID NO: 1167
SEQ ID NO: 1196
SEQ ID NO: 1225


12673
SEQ ID NO: 1168
SEQ ID NO: 1197
SEQ ID NO: 1226


13666
SEQ ID NO: 1169
SEQ ID NO: 1198
SEQ ID NO: 1227









In FIGS. 8 and 9, the data was normalized to the corresponding single-stranded molecule without the C3 spacer. Knockdown amounts that are equivalent to the single-stranded controls (without C3 spacer) would be centered at 0, while positive values indicate that incorporation of the C3 spacer confers an improvement in mRNA knockdown. Negative values indicate a deleterious effect of C3 spacer inclusion. Note that due to experimental variation with in vitro assays, only values greater than +0.5 or less than −0.5 are considered significant. For example, inclusion of the C3 spacer in ApoB molecule 485 at position 18 does not have a significant improvement over the single strand control since the difference in knockdown shown in the FIG. 8 is less than 0.5. However, inclusion of the C3 spacer in the same single strand guide molecule at position 19 has a significant improvement in knockdown (see FIG. 9). Overall, inclusion of the C3 spacer at position 19 is preferred as incorporation at this position seems to improve the potency of mRNA knockdown for the majority of the 30 different sequences tested (73%).
In FIG. 10, ApoB mRNA knockdown at 100 nM concentration using single stranded molecules targeting each of the 30 different ApoB target sites tested in FIGS. 8 and 9 were compared—single strands without C3 spacer (“all-flu-p”), with C3 spacer at position 18 (“all-flu-c3-18-p”), and with C3 spacer at position 19 (“all-flu-c3-19-p”). All of the single-stranded molecules tested are composed of 2′-deoxy-2′-fluoro nucleotides at both pyrimidine and purine nucleotides and a 5′ phosphate. The two 3′ terminal nucleotides of each molecule are 2′-O-methyl nucleotides. The sequence identifiers (SEQ ID NOs:) of the single stranded RNAi molecules evaluated in FIG. 10 are listed in Table 3. FIG. 10 demonstrates the range in overall efficacy of mRNA knockdown for different single-stranded sequences. For example, single strand molecules targeting ApoB target site 485 is maximally effective, while others like those targeting ApoB target site 780 or 10219 have limited mRNA knockdown. For each of the 30 different sequences, the mRNA knockdown shown in FIG. 10 was normalized to the corresponding strands which do not contain the C3 spacer (“all-flu-p”).

## Claims

1. A single-stranded RNA molecule that mediates RNA interference against a target RNA, wherein said single-stranded RNA molecule comprises:
(a) a nucleic acid portion comprising a first nucleotide portion (N1) and a second nucleotide portion (N2) that are not self complementary, wherein said nucleic acid portion comprises at least 8 nucleotides that can base pair with a target site of the target RNA, and wherein the total number of nucleotides within the nucleic acid portion is from 8 to 26 nucleotides;
(b) an internal spacer portion, wherein said spacer portion comprises at least a first non-nucleotide spacer portion (S1) that covalently links the first and second nucleotide portions wherein said single-stranded RNA molecule comprises the following structure:


5′N1-S1-N2 3′

wherein:
(i) N1 contains either one nucleotide or a contiguous stretch of nucleotides;
(ii) S1 contains one or more non-nucleotide spacers covalently linking N1 and N2;
(iii) N2 contains either one nucleotide or a contiguous stretch of nucleotides;
(iv) S1 is at the 3′ end of N1 and at the 5′ end of N2; and
(v) wherein said single-stranded RNA molecule has one 3′ end; and

(c) a ligand.

2. The molecule of claim 1, wherein S1 is an aliphatic or aromatic organic group.

3. The molecule of claim 2, wherein S1 is a C1-C12 alkyl chain that is optionally substituted.

4. The molecule of claim 3, wherein said alkyl chain is optionally substituted with cholesterol.

5. The molecule of claim 2, wherein S1 is selected from a group consisting of a C3 alkyl, a C6 alkyl, and polyethylene glycol.

6. The molecule of claim 1, wherein N1 is 13 to 20 nucleotides long.

7. The molecule of claim 1, wherein the total number of nucleotides within the nucleic acid portion is about 19 to about 21 nucleotides.

8. The molecule of claim 1, wherein the target site is within a untranslated region of the target RNA.

9. The molecule of claim 8, wherein the at least 8 nucleotides that can base pair with the target site is the whole or a part of a seed sequence of a naturally-occurring, endogenous miRNA nucleotide sequence.

10. The molecule of claim 9, wherein S1 takes the place of from one to 4 internal nucleotides of the naturally-occurring, endogenous miRNA nucleotide sequence.

11. The molecule of claim 10, wherein the nucleic acid portion of the molecule is at least 50% homologous to the naturally-occurring, endogenous miRNA nucleotide sequence.

12. The molecule of claim 1, wherein the target site is within a gene coding region of the target RNA.

13. The molecule of claim 1, wherein the nucleic acid portion of the molecule is at least 90% complementary to the target site.

14. The molecule of claim 1, wherein the nucleic acid portion comprises at least 20 nucleotides that can base pair with the target site.

15. The molecule of claim 1, wherein the nucleic acid portion further comprises a third nucleotide portion (N3) and the internal spacer portion further comprises a second non-nucleotide spacer portion (S2).

16. The molecule of claim 1, wherein at least one nucleotide has a modified sugar.

17. The molecule of claim 1, wherein at least one nucleotide has a modified internucleoside linkage.

18. The molecule of claim 1, having a terminal cap at the 5′-end, the 3′-end, or both the 5′- and 3′-ends.

19. The molecule of claim 1, wherein the ligand is a terminal group.

20. The molecule of claim 1, wherein the ligand is a ligand for a cellular receptor.

21. The molecule of claim 20, wherein the ligand is a carbohydrate.

22. The molecule of claim 21, wherein the carbohydrate is an N-acetyl-D-galactosamine.

23. The molecule of claim 1, wherein the ligand is attached by a linker.

24. A composition comprising the single-stranded RNA molecule of claim 1 and a pharmaceutically acceptable carrier.

25. The composition of claim 24, further comprising a liposome, a hydrogel, a cyclodextrin, a biodegradable nanocapsule, a bioadhesive microsphere, or a proteinaceous vector.

26. A method of reducing the expression of an endogenous RNA target gene in a cell comprising administering a composition of claim 24.