USO09725721B2

(12) United States Patent (10) Patent No.: US 9,725,721 B2 MOeller (45) Date of Patent: Aug. 8, 2017

(54) OLIGOMERS WITH IMPROVED (56) References Cited OFF-TARGET PROFILE FOREIGN PATENT DOCUMENTS (71) Applicant: MIRRX THERAPEUTICS, Vejle (DK) WO WO 2005/061710 A1 7/2005 WO WO 2007/031081 A2 3, 2007 (72) Inventor: Thorleif Moeller, Odense SOE (DK) WO WO 2008/061537 A2 5, 2008 WO WO 2008/074328 6, 2008 WO WO 2008,151639 A2 12/2008 (73) Assignee: MIRRX THERAPEUTICS, Vejle WO WO 2009/068033 6, 2009 (DK) WO WO 2009,090.182 A1 T 2009 WO WO 2010, 122538 A1 10, 2010 (*) Notice: Subject to any disclaimer, the term of this WO WO 2011/115818 9, 2011 patent is extended or adjusted under 35 WO WO 2011 115818 A1 9, 2011 U.S.C. 154(b) by 0 days. WO WO 2011, 117353 A1 9, 2011 (21) Appl. No.: 14/431,212 OTHER PUBLICATIONS (22) PCT Filed: Sep. 26, 2013 International Search Report dated Dec. 18, 2013 for corresponding International Patent Application No. PCT/DK2013/050308, filed (86). PCT No.: PCT/DK2O13AOSO3O8 Sep. 26, 2013, 4 pages. International Preliminary Report on Patentability dated Dec. 11, S 371 (c)(1), 2014 for corresponding International Patent Application No. PCT/ (2) Date: Mar. 25, 2015 DK2013/050308, filed Sep. 26, 2013, 6 pages. Takagi-Sato, M. et al., “Fine-Tuning of ENAR. Gapmers as Anti (87) PCT Pub. No.: WO2014/048441 sense Oligonucleotides for Sequence-Specific Inhibition'. Oligo PCT Pub. Date: Apr. 3, 2014 nucleotides, 2007, vol. 17. No. 3, pp. 291-301. Lennox et al., “Chemical modification and design of anti-miRNA (65) Prior Publication Data oligonucleotides.” Gener Therapy (2011)pp. 1111-1120. Stanley T. Crooke: “Principles, Strategies and Applications'. Anti US 2016/O145620 A1 May 26, 2016 sense Drug Technology, 2" Edition, 2007, p. 93. Third Party Observation filed in related European Patent Applica Related U.S. Application Data tion No. 13842025.2, dated Apr. 12, 2017, 15 pages. (60) Provisional application No. 61/705,668, filed on Sep. Jens Kurrecket al.: “Design of antisense oligonucleotides stabilized 26, 2012. by locked nucleic acids.” Nucleic Acids Research, 2002, vol. 30, No. 9, pp. 1911-1918. (30) Foreign Application Priority Data Primary Examiner — Amy Bowman Sep. 26, 2012 (DK) ...... 2012 OO587 (74) Attorney, Agent, or Firm — Merchant & Gould (51) Int. Cl. (57) ABSTRACT C7H 2L/04 (2006.01) The present invention provides oligomers that binds RNA C7H 2L/02 (2006.01) that consists of a lower affinity region and a higher affinity CI2N IS/IT3 (2010.01) region, wherein the monomers in the higher affinity region CI2N IS/II (2006.01) increases the melting temperature (i.e. affinity) of the oli (52) U.S. Cl. gomer base paired to RNA more than the monomers used in CPC ...... CI2N 15/II31 (2013.01); C12N 15/III the lower affinity region, wherein said increase in melting (2013.01); C12N 15/113 (2013.01); C12N temperature is relatively to the alternative use of DNA 23 10/11 (2013.01); C12N 23 10/113 (2013.01); monomers of the same (base) sequence. The oligomers of CI2N 23.10/315 (2013.01); C12N 23.10/321 the invention are useful for binding to target RNA such as (2013.01); C12N 23.10/3231 (2013.01); C12N mRNA and non-coding RNA and have the advantage that 23.10/341 (2013.01); C12N 2320/53 (2013.01) they will be less prone to off-target binding via the lower (58) Field of Classification Search affinity region than via the higher affinity region. CPC ...... C12N 15/113; C12N 2310/11 See application file for complete search history. 7 Claims, 3 Drawing Sheets U.S. Patent Aug. 8, 2017 Sheet 1 of 3 US 9,725,721 B2

On-target binding with various oligomer designs

A

Oligomer A. On-target

Oligomer B

On-targeted r Mn- ESSEE,

C

Oligomer Cre

On-target

Fig. 1 U.S. Patent Aug. 8, 2017 Sheet 2 of 3 US 9,725,721 B2

Off-target binding to competing consensus sequence

A.

Oligomer A Off-target

Oligomer B a 1. Off-target Consensus

COinSensus

Fig. 2 U.S. Patent Aug. 8, 2017 Sheet 3 of 3 US 9,725,721 B2

Off-target binding to non-consensus target

O Cer B sesssssssssssssssssssssssssssssss : 1. Off-target

Off-target-re8:ox:xx H SS

Fig. 3 US 9,725,721 B2 1. 2 OLGOMERS WITH IMPROVED in melting temperature is relatively to the alternative OFF-TARGET PROFILE use of DNA monomers of the same (base) sequence. The oligomers of the invention are useful for binding to This application is a National Stage Application of PCT/ target RNA such as mRNA and non-coding RNA and have DK2013/050308, filed 26 Sep. 2013, which claims benefit of 5 the advantage that they will be less prone to off-target Serial No. PA 2012 00587, filed 26 Sep. 2012 in Denmark, binding via the lower affinity region than via the higher and also claims benefit of Ser. No. 61/705,668, filed 26 Sep. affinity region. 2012 in the United States and which applications are incor porated herein by reference. To the extent appropriate, a BRIEF DESCRIPTION OF THE FIGURES claim of priority is made to each of the above disclosed 10 applications. FIG. 1A show a typical steric block oligomer bound to its intended target (the on-target). The consensus sequence is a BACKGROUND OF THE INVENTION sequence that is shared with other RNAs and is preferably part of a binding site for at cellular molecule, e.g. a micro The present invention relate to oligomers and oligonucle 15 RNA. The sequence adjacent to the consensus sequence is otides that can bind to complementary RNA. Oligonucle denoted unique 1 and is sequence that differs (also herein otides are useful as reagents in Science, research and devel termed 'specificity sequence') among RNAS sharing the opment e.g. for detection and analysis of cellular RNA or for consensus sequence. Base pairs are indicated by lines modulation of the activity of cellular RNA. between the oligomer and the target. The oligomer is uni Oligonucleotides are also being developed as therapeu formly modified with monomer analogues that enhance tics, where they are typically also intended to bind to and affinity toward complementary RNA and the position of modulate the activity of cellular RNA. Oligonucleotides that these modifications is shown by a bold (base pair) line to the bind cellular RNA are herein also termed antisense oligo target. The oligomer may e.g. be a BNA/2"O-methyl RNA nucleotides. oligomer and the affinity enhancing analogues is then BNA Some antisense oligonucleotides act by inducing desta 25 (bicyclic nucleic acids). The illustrated design could just as bilization of their target RNA e.g. by activating RNase H well have been affinity enhancing analogues in every third degradation of the target RNA or by activating the RISC position or even in all positions or any other repeating pathway. Such oligonucleotides are typically described as pattern, which gives a relatively uniform affinity over the gapmers and siRNAS respectively. Another class of oligo length of the oligomer. It is clear that G:C base pairs are nucleotides act as steric blocker and hence prevents the 30 stronger than A: U base pairs. However, for the sake of target RNA from interacting with another macromolecule. simplicity Such sequence specific affinity variations are not One example is splice switching oligonucleotides that considered in the present example. modulate splicing of pre-mRNA. Another example is micro FIG. 1B shows a steric block oligonucleotide of same RNA inhibitors that bind to microRNA and prevent the sequence as oligomer A of FIG. 1A, but with a non-uniform microRNA from binding to its mRNA targets. A thorough 35 distribution of affinity increasing modifications bound to its description of the design, preparation and use of antisense intended target. The affinity of this oligomer toward comple oligonucleotides can be found in “Antisense Drug Technol mentary RNA will not differ dramatically from the oligomer ogy, Principles, Strategies and Applications, 2nd Edition, of 1A. Depending on the monomers chosen, the overall Edited by Stanley Crooke'. affinity may be higher or lower. It is easy to design an antisense oligonucleotide that is 40 FIG. 1C shows a yet another steric block oligonucleotide complementary to a given target RNA and hence is capable of same sequence as oligomer A of FIG. 1A, but with a of binding to the target RNA. However, when attempting to non-uniform distribution of affinity increasing modifications develop antisense oligonucleotides as therapeutics, comple bound to its intended target. The affinity of this oligomer mentarity to the target is not enough. The antisense oligo toward complementary RNA will not differ dramatically nucleotide need to be modified to give it more drug-like 45 from the oligomer of 1A or 1B. Depending on the monomers properties, e.g. better potency, bioavailability and biostabil chosen, the overall affinity may be higher or lower. ity. Moreover, it has turned out that a problem of many In theory, all 3 oligomers have a similar affinity toward antisense oligonucleotides is off-target binding, i.e. binding complementary RNA and would therefore be expected to to non-intended RNAs, which may lead to undesirable have a similar potency, e.g. when used in cells or organisms. effects. To minimize off-target binding, it is typically 50 However, they have a different off-target profile (binds to a attempted to target unique sequences in the RNA transcrip different set of off-targets). tome. Even So, off-target binding of antisense oligonucle FIG. 2 shows the same oligomers as illustrated in FIG. 1, otides is still a problem. One object of the present invention however, bound to an off-target that shares the consensus is to provide antisense oligonucleotides, in particular steric sequence with the intended target. block oligonucleotides that have new and desirable charac 55 The oligomer in 2A will have an intermediate affinity teristics in terms of off-target binding. toward the off-target, whereas the oligomer in 2B will have a lowered affinity toward the off-target because the region SUMMARY OF THE INVENTION complementary to the consensus sequence is a “lower affin ity region’, as seen by the lower density of affinity increas The present invention provides oligomers that can bind 60 ing modifications, such as BNA. I.e. the Boligomer will be RNA consisting of less likely bind to off-targets comprising the consensus a lower affinity region, Sequence. a higher affinity region, On the other hand, the Coligomer in 2C will however be wherein the monomers in the higher affinity region more likely to bind to off-targets comprising the consensus increases the melting temperature (i.e. affinity) of the 65 sequence, because it does so via its "higher affinity region'. oligomer base paired to RNA more than the monomers FIG. 3 shows the same oligomers as illustrated in FIGS. used in the lower affinity region, wherein said increase 1 and 2, however, bound to an off-target that does not share US 9,725,721 B2 3 4 the consensus sequence. It is seen that oligomer B in 3B will Preferably, the monomers in the higher affinity region on do so most potently, because it binds via its higher affinity average (per monomer) increase the melting temperature at region, whereas oligomer C in 3C will do so least potently least 2 degrees Celsius, such as 3, 4, 5, 6, 7 or 8 degrees because it does so via its lower affinity region. I.e. the Celsius more than the monomers in the lower affinity region. increased specificity of oligomer B toward RNA targets that 5 The oligomer is typically an oligonucleotide and prefer share the consensus sequence is achieved at the expense of ably comprise at least two different types of monomers, e.g. decreased specificity toward RNA targets comprising the selected from the group consisting of DNA, RNA, BNA (in specificity region. Oligomer A in FIG. 3A will bind with particular 2',4'-bicyclic nucleic acids (2'4'-BNA) such as intermediate affinity. ENA and LNA (2'-O,4'-C-methylene-beta-D-ribofuranosyl 10 and analogues thereof e.g., amino-LNA, thio-LNA, alfa-L- DISCLOSURE OF THE INVENTION ribo-LNA, beta-D-xylo-LNA, S-cMOE (2',4'-constrained MOE), R-cMOE, cEt (2',4'-constrained ethyl), morpholino, phosphoramidate, FANA, PNA, CENA 2'MOE, 2F, 2'O- The present invention provides oligomers for targeting methyl. RNA, e.g. mRNA and non-coding RNA. 15 It is particular preferred that the higher affinity region A first aspect of the invention is an oligomer of 10-40 comprises 2',4'-bicyclic nucleic acids (2'4'-BNA). monomers that binds RNA comprising (or more preferably In another embodiment, the lower affinity region com consisting of) prises only two types of monomers, preferably selected from a lower affinity region, the group consisting of DNA, RNA, morpholino, phospho a higher affinity region, ramidate, FANA, PNA, UNA, 2MOE, 2F and 2'O-methyl. wherein the monomers in the higher affinity region (alto In another embodiment, the lower affinity region com gether) increases the melting temperature (i.e. affinity) prises only one type of monomer, preferably selected from of the oligomer base paired to complementary RNA the group consisting of morpholino, phosphoramidate, more than the monomers used in the lower affinity FANA, PNA, UNA 2'MOE, 2F, and 2'O-methyl. region (altogether), wherein said increase in melting 25 As is known to the skilled man, different monomers temperature is relatively to the alternative use of DNA generally have different effects on tim. E.g. BNAS have a monomers of the same (base) sequence, strong effect on tim and 2'-modifications have a moderate and wherein the lower affinity region or higher affinity effect. Some modifications like e.g. UNA even decrease tim, region is of 5-12 contiguous monomers, such as 5-10, i.e. have a negative effect. 5-9, 5-8, 6-10, 7-10, 8-10, 6-9 or 6-8 contiguous 30 As used in the present context, 2',4'-bicyclic nucleic acids monomers and wherein the other region is either con are herein classified as monomers that have a strong effect tiguous or consists of two sub regions flanking the on tim. Morpholino, phosphoramidate-, FANA, PNA, region of 5-12 monomers. CENA, 2MOE, 2F, 2"O-methyl monomers are herein clas It should be understood that when referring to “the sified as monomers that have a moderate effect on tim. monomers in the higher affinity region . . . . and “the 35 Monomers that decrease tim are e.g. UNA (unlocked nucleic monomers used in the lower affinity region' in the above, acid). DNA and RNA monomers are herein classified as reference is to the monomers altogether in the region and not neutral monomers with regard to tm (DNA monomers are to the monomers individually. Thus, not every monomer in reference monomers). the higher affinity region does necessarily increase the In a preferred embodiment, the lower affinity region has melting temperature more than individual monomers used in 40 a lower density (number of affinity increasing monomers/ the lower affinity region. total number of monomers in the given region) of monomers Preferably, the monomers in the higher affinity region that have a strong and/or moderate effect on tim than does the (altogether) increases the melting temperature (i.e. affinity) higher affinity region. This feature can usually be used to of the oligomer base paired to RNA at least 5 degrees easily identify oligomers of the invention, e.g. by identifying Celsius more than the monomers used in the lower affinity 45 the lower affinity region within the oligomer. region, Such as at least 5 degrees, 10 degrees, 15 degrees, 20 It follows that oligomers with a uniform distribution of degrees, 25 degrees, 30 degrees, 35 degrees, 40 degrees high affinity/and or moderate monomers are not part of the Celsius more than the monomers used in the lower affinity Scope of this invention, because they do not comprise a region, wherein said increase in melting temperature is lower affinity region. Also oligomers with slight deviations relatively to the alternative use of DNA monomers of the 50 from a uniform distribution are not part of the scope of this Same Sequence. invention, as slight deviations are not enough to create a When referring to the melting temperature (also herein region of lower affinity that differ substantially in affinity (as termed tim ortm value) of the oligomer base paired to RNA, expressed in the melting temperature of the oligonucleotide) the temperature is preferably measured in 100 mM NaCl, 0.1 from the rest of the oligomer. mM EDTA, 10 mM NaH2PO, 5 mM NaHPO, pH 7.0), 55 In many embodiments, the oligomer comprises one or using 1.0 LM concentrations of both oligomer and comple more phosphorothioate linkages. The oligomer may e.g. be mentary RNA. fully modified with phosphorothioate linkages or e.g. one, In a preferred embodiment, the monomers in the higher two or three phosphorothioate linkages at each end. Phos affinity region on average (per monomer) increase the melt phorothioate linkages generally decrease melting tempera ing temperature more than the monomers in the lower 60 ture. However, this may be counteracted e.g. by the use of affinity region. I.e. the increase intm of the lower and higher 2'4' BNA monomers. I.e. the higher affinity region may very affinity region is divided by the number of monomers in the well comprise phosphorothioate linkages. lower and higher affinity region, respectively. It should be Length and Affinity of the Oligomer noted that the increase intm of the lower affinity region may The overall affinity (as expressed by the melting tempera be negative, which is the case when the tim is decreased 65 ture) of the oligomer toward complementary RNA depends relatively to the alternative use of DNA monomers of the on the length of the oligomeras well as the content of affinity Same Sequence. increasing (and sometimes decreasing) monomers. It is an US 9,725,721 B2 5 6 object of the invention to design the oligomer Such that it has In yet another embodiment, neither the 5'end of the lower Sufficient affinity toward its intended target, while displaying affinity region nor the 3'end of the lower affinity region is a improved selectivity and/or potency toward its intended BNA monomer. Even more preferably, neither the two target over other targets sharing an identical sequence monomers at the 5'end of the lower affinity region nor the (which is hence a shared sequence). two monomers at the 5'end of the monomer are BNA The length of the oligomer is preferably between 10 and monomers. This feature further helps define the lower affin 40 monomers. In other embodiments, the length is between ity region within an oligomer of the invention. 10 and 35 monomers, between 10 and 30 monomers, Preferably, the melting temperature of the lower affinity between 10 and 25 monomers. Even more preferably, the region toward complementary RNA is less than 70 degrees length of the oligomer is between 10 and 20 monomers, such 10 Celsius, such as less than 65, 60, 55, 50, 45, 40 and 35 as between 11 and 19 monomers or between 12 and 18 degrees Celsius. The melting temperature in this embodi ment may be determined by measuring the tim against a OOCS. truncated RNA with a length corresponding to the lower When using shorter lengths, it is necessary to use a higher affinity region, i.e. where the truncated RNA cannot base density of monomers that increase the affinity of the oli pair to the higher affinity region of the oligomer. It is even gomer. Particular preferred are 2',4'-bicyclic nucleic acids 15 more preferred that the melting temperature of the lower (24'-BNA) that have a strong effect on affinity. Hence, in a affinity region toward complementary RNA is less than 60 preferred embodiment, at least 30% of the monomers are degrees Celsius, such as less than 55, 50, 45, 40 and 35 2'4'-BNA monomers, e.g. selected from the group consisting degrees Celsius. of LNA (2'-O,4'-C-methylene-beta-D-ribofuranosyl and Preferably, the lower affinity region is contiguous and of analogues thereof e.g., amino-LNA, thio-LNA, alfa-L-ribo 5-12 monomers, such as 5-10, 5-9, 5-8, 6-10, 7-10, 8-10, 6-9 LNA, beta-D-xylo-LNA, S-cMOE (2',4'-constrained MOE), or 6-8 contiguous monomers. Even more preferred is a lower R-cMOE, cEt (2',4'-constrained ethyl). More preferably, at affinity region of 6-10 contiguous monomers. I.e. in a least 35%, such as least 40%, 45%, 50%, 55% or 60% are preferred embodiment, the lower affinity region of the 2'4'-BNA monomers. oligomer can be identified as a region with none or just 1 The melting temperature of the oligomer base paired to 25 BNA monomers over a region of 6-10 monomers, wherein RNA should preferably be at least 60 degrees Celsius. In the density of BNA monomers in the remaining part of the other embodiments, it is preferred that the tim of the oli oligomer has a higher density of BNA monomers, and hence gomer is at least 65 degrees Celsius, at least 70 degrees the remaining part of the oligomer is the higher affinity Celsius, at least 75 degrees Celsius, at least 80 degrees region. Celsius, at least 85 degrees Celsius, at least 90 degrees 30 Uses and Advantages—Selectivity Celsius or at least 95 degrees Celsius. The oligomers of the invention are useful for binding to It is even more preferred that the tim is at least 75 degrees target RNA such as mRNA and non-coding RNA and have Celsius, such as 80, 85, 90 or 95 degrees Celsius. the advantage that they will be less prone to off-target In one embodiment, the lower affinity region is shorter binding via the lower affinity region than via the higher than the higher affinity region. In another region, the lower affinity region. affinity region is longer than the higher affinity region. 35 Consider e.g. two RNA sequences of 10-40 monomers The Lower and Higher Affinity Regions (within RNA targets such as mRNA or non-coding RNA) As mentioned above, it is preferred that the lower affinity that have identical sequences in one region and differ e.g. by region has a lower density of monomers that have a strong 1, 2 or 3 nucleotides over a length of between 5 and 10 and/or moderate effect on tim than does the higher affinity nucleotides in sequence in an adjacent region. The region region. 40 that differs may be termed the “specificity region', whereas When the oligomer comprise 2'4'-BNA monomers, the the identical region may be referred to as the “shared density of these may e.g. 100% in the higher affinity region region'. There could just as well be multiple target RNAs (full modification), 50% (mixmer with 1 BNA per 1 other that share the shared sequence and differ in the adjacent monomer, e.g. 1 BNA monomer in every second position), specificity region. 33% (1 BNA per 2 other monomers, e.g. in every third 45 When the goal is to design an oligomer that preferentially position), 25% etc. The BNAs need not necessarily be bind one RNA sequence (the intended target, herein also evenly dispersed the high affinity region. At any rate, the termed the on-target) over the other target (the off-target), it lower affinity region is easily identified by the lower density is obvious that the oligomer should be complementary to the of 2'4'-BNA monomers. When the higher affinity region intended target. However, before the present invention, it comprises 2'4' BNA monomers, it typically contains at least 50 was not obvious to design and use an oligomer comprising 1 BNA per 4 non-BNA monomers and also typically does a higher affinity region and a lower affinity Such that the not contain 4 or 5 contiguous non-BNA monomers. higher affinity region of the oligomer should be comple The amount of 2'4'-BNA monomers in the higher affinity mentary to the specificity region of the (intended) on-target region is preferably at least 2 fold, or even more preferably and the lower affinity region should be complementary to the 3, 4 or 5 fold higher than in the lower affinity region of the shared region (shared between the off-targets and the on oligomer. 55 target). In this way, off-target binding via the shared region In a preferred embodiment, the lower affinity region does will be minimized because of a lower affinity as expressed not comprise any affinity enhancing monomers selected in tim, while still maintaining high affinity to the intended from the group consisting of 2'4'-BNA, PNA, 2MOE and target. 2F. On the other hand, if a certain degree of off-target binding More preferably, the lower affinity region does not com 60 is acceptable or even desirable, the higher affinity region of prise any 2'4'-BNA monomers. the oligomer should be complementary to the shared region In another embodiment, the lower affinity region com and the lower affinity region should be complementary to the prise at least 5 contiguous monomers, such as 6 or 7 specificity sequence. In this way, off-target binding via the contiguous monomers which are not 2'4'BNA monomers. shared region will be increased. In yet another embodiment, the lower affinity region 65 It is to be understood that oligomers of the invention do comprise at least 5 contiguous monomers, such as 6 or 7 not necessarily overall have less or more binding to off contiguous monomers which are not DNA monomers. targets in the transcriptome of a cell when compared to a US 9,725,721 B2 7 8 uniformly modified oligomer of the same sequence. How comprise a sequence which is shared among other binding ever, they will have decreased binding to off-targets sharing sites of the same cellular macromolecule. Such sequences the same shared sequence (because the lower affinity region are herein referred to as consensus sequences. As described above, it is one objective of the present invention to provide is complementary to the shared sequence). Potentially, this oligomers that have improved (or reduced) selectivity in may be achieved at the expense of more off-target binding 5 terms of targeting an intended target (RNA) over other via the higher affinity region. At any rate, the oligomers of RNAs that share a sequence with the intended target. In a the invention have a different off-target profile as compared preferred embodiment, the shared sequence is a binding site to uniformly modified oligomers of the same sequence. for an RNA binding protein or RNA or and the shared Hence, the oligomers of the invention can be designed to sequence may be represented by a consensus sequence. have less off-target binding among off-targets of interest or 10 One way to avoid or reduce off-target binding to other more off-target binding among off-targets of interest com RNAS sharing the same (consensus) sequence could be to pared to a uniformly modified oligomer of the same avoid the consensus sequence as target, i.e. not included a sequence and similar tim value. sequence complementary to the consensus region in the It should be understood that the oligomer will preferably oligomer. However, when the oligomer is intended to block have perfect complementarity to its intended target. 15 a binding site, this may reduce the activity of the oligomer, The non-obvious characteristics and uses of the oligomer since the binding site is then not completely blocked. Most of the invention will be further described below. often it will be desirable to indeed target the consensus Activity/Mechanism of Action of the Oligomer of the Inven sequence as well as adjacent sequence(s). tion—Steric Block Oligomers with increased selectivity are most preferred. The oligomers of the invention are typically steric block In these embodiments, the lower affinity region is preferably ers, i.e. they bind to target RNA without mediating degra complementary to a consensus sequence. The length of the dation of the target RNA. Steric block oligonucleotides are lower affinity region is preferably the same as the length of well described in the art and are e.g. used as splice Switching the consensus sequence or 1, 2, 3 or 4 monomers longer. The oligonucleotides and microRNA inhibitors. As is known by additional monomers may be added to the 5'end of the lower the skilled man, many monomers can be used in the design affinity region, the 3'end of the lower affinity region or both. of steric block oligonucleotides, since most artificial nucleo 25 Preferred consensus sequences are microRNA binding tide monomers do not allow recruitment of RNase H. See sites in mRNA of a given microRNA, which typically share e.g. Antisense Drug Technology, Principles, Strategies and complementarity to position 2-7 (the so-called seed Applications, 2" Edition, Edited by Stanley Crooke, page sequence) of the microRNA, but differ in sequence outside 93, which mentions 2-O-Me, 2-O-methoxyethyl (MOE), and of this region. I.e. in this instance the consensus sequence of 2-O-aminopropyl, phosphoramidate, and methylphospho 30 the binding site is a sequence that is complementary to the nate derivatives, as well as locked nucleic acid (LNA/2'4'- seed sequence of the microRNA. BNA), peptide nucleic acid (PNA), and morpholino based Another preferred consensus sequence is the seed oligomers. Also mixtures of these monomers do not allow sequence shared among microRNA families that typically RNase H recruitment. RNase H recruitment generally differ outside of the seed sequence. requires at least 5 contiguous DNA monomers and most 35 Hence, in a preferred embodiment, the oligomer of the often 6, 7 or 8 contiguous monomers are used. Therefore, in invention comprise a lower affinity region that can bind to a a preferred embodiment, the oligomer of the invention does RNA sequence that is complementary to position 1-7, 2-7. not comprise contiguous DNA monomers, such as 5, 6, 7 or 1-8 or 2-8 of any of SEQID NOs: 1-2042 or a lower affinity 8 contiguous DNA monomers. region that can bind to a RNA sequence that comprise In one embodiment, the oligomer does not comprise a position 1-7, 2-7, 1-8 or 2-8 of any SEQ ID NOs: 1-2042, Morpholino monomer 40 wherein the positions are counted from the 5' end. The In another embodiment, the oligomer does not comprise region capable of binding to the consensus sequence may be a FANA monomer. referred to as the anti-consensus sequence, which is hence In another embodiment, the oligomer does not comprise comprised within the lower affinity region of the oligomer or a PNA monomer. is identical to the lower affinity region of the oligomer. In In another embodiment, the oligomer does not comprise 45 this embodiment, the oligomer improves selectivity, when a CENA monomer. targeting one microRNA over other microRNA in the same In another embodiment, the oligomer does not comprise family or when targeting one microRNA binding site over a 2"MOE monomer. other binding sites of the same microRNA. In another embodiment, the oligomer does not comprise Likewise, the oligomers may also be used for decreasing a 2F monomer. 50 selectivity. In this embodiment, the oligomer of the inven In another embodiment, the oligomer does not comprise tion comprise a higher affinity region can bind to a RNA a 2"O-methyl monomer. sequence that is complementary to position 1-7, 2-7, 1-8 or In yet another embodiment, the oligomer does not com 2-8 of any of SEQID NOs: 1-2042 or a lower affinity region prise a phosphoramidate monomer. that can bind to a RNA sequence that comprise position 1-7. Preferred Target RNAs and Sequences 2-7, 1-8 or 2-8 of any SEQ ID NOs: 1-2042, wherein the Binding sites for cellular macromolecules such as RNA 55 positions are counted from the 5' end. binding proteins and RNA (e.g. mRNA or microRNA) often Oligomers with increased selectivity are most preferred. TABLE 1.

Human microRNA sequences

NO Sequence (5' to 3') ID and accession

1 AAGAUGUGGAAAAAUUGGAAUC >hsa-miR-576-3p MIMATOOO4796

2 CAGUGGUUUUACCCUAUGGUAG >hsa-miR-140-5p MIMATOOOO431

US 9,725,721 B2 115 116 TABL 1- Continued Human microRNA sequences NO Sequence (5' to 3') ID and accession 2017 UAGGCACACUUAAAGUUAUAGC hsa-mi R-5582-5p MIMATOO22279

2O18 UAGCAGCACGUAAAUAUUGGCG hsa-mi R-16-5p MIMATOOOOO69

2O19 GGGAAAAGGAAGGGGGAGGA hsa-mi R-6124 MIMATOO24597

2O2O GAGCUUGGUCUGUAGCGGUU hsa-mi R-4453. MIMATOO18975

2021. CUGGGAGGUGUGAUAUUGUGGU hsa-mi R-3689b–3p MIMATOO18181.

2O22 UAGGAUUACAAGUGUCGGCCAC hsa-mi R-3159 MIMATOO15 O33

2O23 UUGGGACAUACUUAUGCUAAA hsa-mi R-1302 MIMATOOO5890

2O24 GGCUACAACACAGGACCCGGGC hsa-mi R-187-5p MIMATOOO4561

2O2S UGAGCUGCUGUACCAAAAU hsa-mi R-558 MIMATOOO3222

2O26 AUUGACACCUCUGUGAGUGGA hsa-miR-514b-3p M MATOO15088

2O27 AGGAAUGUUCCUUCUUUGCC hsa-mi R- 613 MIMATOOO3281

2O28 UGGGUGGUCUGGAGAUUUGUGC hsa-miR-1293 MIMA OOO5883

2O29 AGAGUCGGCGACGCCGCCAGC hsa-miR- 4785 MIMA OO 99.49

2O3OUGGGGGAGCCAUGAGAUAAGAGCA hsa-miR-4723-5p M OO19838

2O31 AUCAAAUAAGGACUAGUCUGCA hsa-miR-3671 MIMA 8094

2O32 CGCCCCUCCUGCCCCCACAG hsa-miR-4749-3p M OO19886

2O33 UGAGCCCCUGUGCCGCCCCCAG hsa-miR-1225–3p M OOO5573

2O34 GCUGAUGAUGAUGGUGCUGAAG hsa-miR- 4502 MIMA OO

2O35 GGGAGUCUACAGCAGGG hsa-miR-4294 MIMA OO 6849

2O36 CACUCAGCCUUGAGGGCACUUUC hsa-mi R-512-5p MIMATOOO2822

2O37 GACACAUGACCAUAAAUGCUAA hsa-miR-4 643 MIMA OO

2O38 UAAGUGCUUCCAUGUUUGAGUGU hsa-miR-302d-3p M MA OOOOf 18

2O39 UGCACGGCACUGGGGACACGU hsa-miR-3177-3p M MA OO15054

2O4 O CCUCCUGCCCUCCUUGCUGU hsa-miR-1976 MIMA OOO945.1

2O41 CAACGGAAUCCCAAAAGCAGCUG hsa-mi R-191-5p MIMATOOOO440

2042 CAGCCCUCCUCCCGCACCCAAA hsa-miR-4687–5p M MATOO19774

In one embodiment, the oligomer does not comprise a above) is ensured by a higher density of BNA monomers in lower affinity region that can bind to a RNA sequence that 50 the higher affinity region as compared to the density of BNA is complementary to position 2-7 of SEQ ID NO: 1051. monomers in the lower affinity region. In yet another embodiment, the oligomer is not comple It is preferred that the higher affinity region consist of at mentary to any of SEQID NOs: 1-2041 (over its full length). least 4 monomers, more preferably, at least 5, 6, 7 or 8 Particular preferred oligomers of the invention have a 55 OOCS. length of 10-20 monomers, and consists of Preferably, the density of BNA monomers in the higher a higher affinity region that comprise at least 25% BNA affinity region is at least 2-fold higher than the density of OOCS BNA monomers in the lower affinity region. Even more a lower affinity region of 6-10 contiguous monomers that preferably, the density it at least 3-fold higher such as 4 or comprise less than 25% BNA monomers and wherein 60 the two monomers at the 5' end of the lower affinity 5 fold higher. region and the two monomers at the 3'end of the lower In a preferred embodiment, the lower affinity region does affinity region are non-BNA monomers not comprise any BNA monomers at all. and wherein the oligomer does not comprise a region of The higher affinity region preferably comprise at least 5 contiguous DNA monomers 65 33% (1 BNA monomer/3 monomers in the higher affinity In this embodiment, different affinities of the higher region), 50% (1 BNA/2 monomers), 67% (2 BNA/3 mono affinity region and the lower affinity region (as defined mers), (3 BNA/4 monomers) 75% or 100% BNA monomers. US 9,725,721 B2 117 Preferably, at least one internucleotide linkage is a phos TABLE 2 phorothioate linkage. In another embodiment, all internucle otide linkages are phosphorothioate linkages. Oligomer Tm, 100 mM NaCl Tm, 10 mM NaCl In a related embodiment, the oligomer the lower affinity 123-HCVn >90 86 region consists of 8-10 contiguous monomers and the 124-HCVtm >90 84 remaining monomers of the oligomer are BNA monomers. 125-HCVn >90 8O Typically, both the 5'end of the oligomer and the 3'end of 126-HCVtm 79 65 the oligomer is a BNA monomer. 127-HCVn >90 79 The oligomer may even be of 12-18 monomers or 13-17 128-HCVtm >85 83 OOCS. 10 Preferably, the lower affinity region can base pair to the For the measurements in high salt, only 126HCVtm sticks seed sequence of a microRNA or can base pair to the RNA out with a lower tim value, which is in line with the presence complement of the seed sequence of a microRNA. Human of a UNA monomer that decreases affinity. microRNAs are preferred. For the measurements in low salt, the tim values correlate EXAMPLES 15 well with the content of LNA monomer and again it is seen that one UNA monomer decreases affinity significantly. Example 1 Next, the affinity of another oligomer sequence (Aldo A) toward the same target RNA was measured. This other In the present example, the affinity of various oligomers oligomer is complementary to the same consensus sequence toward an intended target, as well as to an off-target RNA of the target RNA, but differs in the remaining part of the was measured. Finally, the affinity of the oligomers toward Sequence: a target RNA consisting of the consensus sequence was measured. The employed target RNA first was: Target: 5 GAUGGGGGCGACACUCCACCAUGAAU 25 l Aldo Oligomer: GUUGUGAGGUCCGG (135-Hcvtarg) 5 GAUGGGGGCGACACUCCACCAUGAAU 1 indicate a potential G:U base pair. wherein a consensus sequence is underlined. The oligomers used in these measurements were: The sequence of the oligomers complementary to this 30 Lower Affinity Regions are Underlined target RNA: (129-Aldo, uniformly modified) 1GlCimCICmU1GmGlAmGmU1GmUITIG

CUGUGAGGUGGUAC-5 (130-Aldotm, lower affinity region complementary to con 35 sensus sequence) And when bound to each other: 1GlClCICITmGmGmAmGmUmGITITIG (131-Aldotm, lower affinity region complementary to con Target: 5 GAUGGGGGCGACACUCCACCAUGAAU sensus sequence) 1GlClClCmUmGmGmAmGmUmGmUTTIG HCV Oligomer CUGUGAGGUGGUACS" 40 (133-Aldotm, higher affinity region complementary to con First, the following oligomers were employed, wherein sensus sequence) lower affinity regions are underlined: mGmGmCmCITIGIGIAIGITIGITmUmG (123-HCVtm, uniformly modified) ICIAmUmGIGmUIGIGmAIGmUmGITIC TABLE 3 (124-HCVtm, lower affinity region complementary to con 45 sensus sequence) Oligomer Tm, 100 mM NaCl ICIAITIGIGITmGmGmAmGmUmGIUIC 129-Aldotm 77 (125-HCV, tm lower affinity region complementary to con 130-Aldotm 70 sensus sequence) 131-Aldotm 66 ICIAITIGIGmUmGmGmAmGmUmGmUIC 50 133-Aldotm 83 (126-HCVtm, lower affinity region complementary to con sensus sequence) ICIAITIGIGmUmGmGuAmGmUmGmUIC In this set of measurements, the HCV target may be seen (127-HCVtm, lower affinity region complementary to con as an off-target that shares the consensus target with the sensus sequence) 55 intended target of the oligomer. Again the melting tempera ICIAmUmGIGmUmGmGmAmGmUmGITIC ture correlates overall with the content of LNA. Moreover, (128-HCVtm, higher affinity region complementary to con it is seen that affinity increases when the content of LNA sensus sequence) increases in the complementary region. When the LNA mCmAmUmGmGITIGIGIAIGITIGITmC density is higher in the non-complementary region, the m indicates 2"O-methyl RNA melting temperature decreases, see oligomer 131. I indicate LNA (BNA) 60 u indicate unlocked Nucleic Acid (UNA) Next, the affinity of the oligomers toward a complemen The melting temperatures were measured in medium salt tary 8 nucleotide consensus RNA was measured. The affinity concentration, i.e. 100 mM. NaCl, 0.1 mM EDTA, 10 mM of the oligomers toward this 8 nucleotide consensus RNA NaH2PO4, 5 mM Na2HPO4, pH 7.0), using 1.0 M con may be seen as a measure of the affinity of the oligomer centrations of both oligomer and complementary RNA and 65 toward to off-targets comprising the consensus sequence, but in low salt concentration, i.e. in 10 mMNaCl instead of 100 which are not complementary to the oligomer in the regions mM NaCl. adjacent to the consensus sequence. US 9,725,721 B2 119 The consensus RNA was: TABLE 4-continued Oligomer Tm, 100 mM NaCl 5. "ACACUCCA 131-Aldotm 55 132-Aldotm 84 TABLE 4 As seen, less off-target binding (more specific oligomers) Oligomer Tm, 100 mM NaCl can be designed by reducing the LNA content in the comple mentary region. 123-HCVn 77 10 124-HCVtm 59 If the same design principles are used to design antisense 125-HCVn 48 oligomers toward other target sequences comprising a con 126-HCVtm No transition sensus sequence, generally improved specificity can be 127-HCVn 60 128-HCVtm 82 achieved. 129-Aldotm 73 Thus, the teachings of the present invention, including the 130-Aldotm 66 15 findings in the above experiments, should be generally applicable and not limited to certain sequences/targets.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS:

<21 Os SEQ ID NO 1 &211s LENGTH: 22 212s. TYPE RNA <213> ORGANISM: Homo Sapiens <4 OOs SEQUENCE: 1 aagaugugga aaaauluggala luc 22

<21 Os SEQ ID NO 2 &211s LENGTH: 22 212s. TYPE RNA <213> ORGANISM: Homo Sapiens

<4 OOs SEQUENCE: 2

Cagluggluluulu accculaluggu ag 22

<21 Os SEQ ID NO 3 &211s LENGTH: 23 212s. TYPE RNA <213> ORGANISM: Homo Sapiens

<4 OOs SEQUENCE: 3

Caggcaguga culgulucagac gluc 23

<21 Os SEQ ID NO 4 &211s LENGTH: 23 212s. TYPE RNA <213> ORGANISM: Homo Sapiens <4 OOs SEQUENCE: 4 aagaa.gaga C ugaglucaucg aalu 23

<21 Os SEQ ID NO 5 &211s LENGTH: 22 212s. TYPE RNA <213> ORGANISM: Homo Sapiens

<4 OOs SEQUENCE: 5 cucuagaggg aagcgcuuuc lug 22

<21 Os SEQ ID NO 6 &211s LENGTH: 21 212s. TYPE RNA <213> ORGANISM: Homo Sapiens

US 9,725,721 B2 125 126 - Continued <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 22 ulaulacaaggg caa.gcucucul gu. 22

<210s, SEQ ID NO 23 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 23 uluga agagga glugollclugu agc 23

<210s, SEQ ID NO 24 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 24 gaaag.cgcuu cccuulugclug ga 22

<210s, SEQ ID NO 25 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 25 ugaggcuaalu go acuacuuc ac 22

<210s, SEQ ID NO 26 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 26 ulcuaggclugg uaculgcluga 19

<210s, SEQ ID NO 27 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 27 cuuagcaggul lugulaulualuca lulu. 22

<210s, SEQ ID NO 28 &211s LENGTH: 2O 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 28 gaga Caggulu caugclugcula

<210s, SEQ ID NO 29 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 29 caaagugcuul acagugcagg ulag 23

<210s, SEQ ID NO 3 O &211s LENGTH: 21

US 9,725,721 B2 145 146 - Continued <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 101 uluguggclugg ulcallgaggcu aa 22

<210s, SEQ ID NO 102 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 102 alagcaulucull ulcaluugglulug g 21

<210s, SEQ ID NO 103 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 103 ulaulucagaaa ggugccaguc a 21

<210s, SEQ ID NO 104 &211s LENGTH: 24 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 104 aaluulugglululu. Cugaggcacu ulagu. 24

<210s, SEQ ID NO 105 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 105 alugagacuca lugulaaaacau culu. 23

<210s, SEQ ID NO 106 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 106

Cuaulacaauc uaculglucululu, C 21

<210s, SEQ ID NO 107 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 107 lugglulugacca ulagaacaugc gc 22

<210s, SEQ ID NO 108 &211s LENGTH: 18 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 108 aggggg.cggg Cuccgg.cg 18

<210s, SEQ ID NO 109 &211s LENGTH: 23 US 9,725,721 B2 147 148 - Continued

212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 109 agluggalugall gagacucgg llac 23

<210s, SEQ ID NO 110 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 110 aguuluugcag guulugcaucci agc 23

<210s, SEQ ID NO 111 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 111 aguggcaaag ulcuuuc caula u 21

<210s, SEQ ID NO 112 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 112 ulaalucculugc ulac Cugggug aga 23

<210s, SEQ ID NO 113 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 113 lugcggggcula giggculaacag Ca 22

<210s, SEQ ID NO 114 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 114 cCucccacac cca aggculug Ca 22

<210s, SEQ ID NO 115 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 115 agggalaggag gCullugglucull ag 22

<210s, SEQ ID NO 116 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 116

Cculauluculug aluuacuuguu ulc 22

<210s, SEQ ID NO 117 US 9,725,721 B2 149 150 - Continued

&211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 117 cCuccguguu accluguccuC ulag 23

<210s, SEQ ID NO 118 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 118 ulclugu.gagac caaagaacula cul 22

<210s, SEQ ID NO 119 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 119

Caalucago aa guauaculgcc clu. 22

<210s, SEQ ID NO 120 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 120 gull.999acaa gaggaC991C llll 22

<210s, SEQ ID NO 121 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 121 uuugugaccu ggluccaculaa cc 22

<210s, SEQ ID NO 122 &211s LENGTH: 18 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 122

Cagcaguccc ulcCCC clug 18

<210s, SEQ ID NO 123 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 123 lugaggagaluc glucgagglulug g 21

<210s, SEQ ID NO 124 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 124 ulcuCaguaag luggcacucug u. 21

US 9,725,721 B2 153 154 - Continued

<210s, SEQ ID NO 133 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 133

Caggcagaag luggggculgac agg 23

<210s, SEQ ID NO 134 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 134 cagulualucac agugclugalug cul 22

<210s, SEQ ID NO 135 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 135 aggulagaalug aggc.culgaca u 21

<210s, SEQ ID NO 136 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens < 4 OO SEQUENCE: 136 cCuguugaag luguaaluccCC a 21

<210s, SEQ ID NO 137 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 137 agggaaaaaa aaaaggaululu gulc 23

<210s, SEQ ID NO 138 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 138 accgaaga cu glugcgcuaalu cul 22

<210s, SEQ ID NO 139 &211s LENGTH: 27 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 139 accuuculugu aluaagcacug lugcuaaa 27

<210s, SEQ ID NO 140 &211s LENGTH: 2O 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 140 ggggagclugu gigaagcaglla US 9,725,721 B2 155 156 - Continued

<210s, SEQ ID NO 141 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 141 aaluccacgcu gagculuggca ulc 22

<210s, SEQ ID NO 142 &211s LENGTH: 17 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 142 gaga clugggg luggggcc 17

<210s, SEQ ID NO 143 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 143 aaaaguaaluu glugguuluugg cc 22

<210s, SEQ ID NO 144 &211s LENGTH: 18 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 144 lugculuccululu cagagggu 18

<210s, SEQ ID NO 145 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 145 llallagggalull gagc.cgugg C9 22

<210s, SEQ ID NO 146 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 146 cgc.culgcc.ca gcc cucculgc u. 21

<210s, SEQ ID NO 147 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 147 agaclugacgg cluggaggc cc au. 22

<210s, SEQ ID NO 148 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 148 US 9,725,721 B2 157 158 - Continued agcagggclug gigaullgca 19

<210s, SEQ ID NO 149 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 149 cucuagaggg aag.cgcuuuc lug 22

<210s, SEQ ID NO 150 &211s LENGTH: 18 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 150 acguluggclc lugglugglug 18

<210s, SEQ ID NO 151 &211s LENGTH: 2O 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 151 gCucggaclug agc aggluggg

<210s, SEQ ID NO 152 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM; Homo Sapiens <4 OOs, SEQUENCE: 152 llclugca clugu gagluluggclug gCul 23

<210s, SEQ ID NO 153 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 153 lucculucauluc Caccggaguc lug 22

<210s, SEQ ID NO 154 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 154 lugucuculgcll gigglulullcul 19

<210s, SEQ ID NO 155 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OO > SEQUENCE: 155 gagccagulug gaCaggagc 19

<210s, SEQ ID NO 156 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 156

US 9,725,721 B2 161 162 - Continued

<4 OOs, SEQUENCE: 164 ulcuggcugug cluguaalugca g 21

<210s, SEQ ID NO 165 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 165 ggCaggaggg clugu.gcCagg lulug 23

<210s, SEQ ID NO 166 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 166

Cuaululaagga cauuugugaul ulc 22

<210s, SEQ ID NO 167 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 167 ulugullCgull C9gCucgcgll ga. 22

<210s, SEQ ID NO 168 <211 LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 168 caaaacguga ggcgclugcua u 21

<210s, SEQ ID NO 169 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 169 aacaucaculg Caagucullaa Ca 22

<210s, SEQ ID NO 170 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 170 agagauluggu agaaalucagg u. 21

<210s, SEQ ID NO 171 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 171 lugucuacalulu aaugaaaaga gC 22

<210s, SEQ ID NO 172 &211s LENGTH: 24 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

US 9,725,721 B2 165 166 - Continued <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 18O lugcc clugccu guuuucuccu. lulu. 22

<210s, SEQ ID NO 181 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 181 gagglululuggg gaggaululgc ll 21

<210s, SEQ ID NO 182 &211s LENGTH: 18 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 182

C Caguguggc ulcagcgag 18

<210s, SEQ ID NO 183 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 183 aaaggugCuc aaauluagaca u 21

<210s, SEQ ID NO 184 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 184 gCagga Cagg Cagaaglugga ll 21

<210s, SEQ ID NO 185 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 185 uglucagululug ulcalaallac cc Ca 22

<210s, SEQ ID NO 186 &211s LENGTH: 19 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 186 cggcgcgaCC gcc.cgggg 19

<210s, SEQ ID NO 187 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 187

Callaaagulag aaa.gcaculac ul 21

<210s, SEQ ID NO 188 &211s LENGTH: 24 US 9,725,721 B2 167 168 - Continued

212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 188 ugggculaagg gagalugaulug glla 24

<210s, SEQ ID NO 189 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 189 ccucac cauc ccuucugo cu go 22

<210s, SEQ ID NO 190 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 190 ggggggalugu gCalugcluggll ll 21

<210s, SEQ ID NO 191 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 191 cggggcagcu. Caguacagga u 21

<210s, SEQ ID NO 192 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 192 uuluaiacalugg ggguac clugc lug 22

<210s, SEQ ID NO 193 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 193 aaacuaaluau accCaulauluc lug 22

<210s, SEQ ID NO 194 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 194 uuuugo accu ululuggaguga a 21

<210s, SEQ ID NO 195 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 195 luggcaguglla lullgullagclug gll 22

<210s, SEQ ID NO 196 US 9,725,721 B2 169 170 - Continued

&211s LENGTH: 24 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 196 agggcluggac ulcagcggcgg agcul 24

<210s, SEQ ID NO 197 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OO > SEQUENCE: 197 ugaglugaulug aluagcuaugu ulc 22

<210s, SEQ ID NO 198 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 198 llcagcaggca gCugglugca gC 22

<210s, SEQ ID NO 199 &211s LENGTH: 24 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 199 agaugllaugg aallclugulalla llalc 24

<210s, SEQ ID NO 2 OO &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 2OO acuccago'cc cacago cuca gc 22

<210s, SEQ ID NO 2 O1 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 2O1 gaggaaugua gguagaclugu ulu. 22

<210s, SEQ ID NO 202 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 2O2 acaaggugug caugCCugac C 21

<210s, SEQ ID NO 203 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 2O3 gcaaag caca C9gcclugcag aga 23

US 9,725,721 B2 173 174 - Continued

<210s, SEQ ID NO 212 &211s LENGTH: 2O 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 212 agagalugaag C9gggggg.cg

<210s, SEQ ID NO 213 &211s LENGTH: 18 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 213 gggugagggc agglugglull 18

<210s, SEQ ID NO 214 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 214 luggalagggag alaga.gcululla all 22

<210s, SEQ ID NO 215 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens < 4 OO SEQUENCE: 215 caucuu.ac.cg gacaglugclug ga 22

<210s, SEQ ID NO 216 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 216 culgaculgulug ccguccucca g 21

<210s, SEQ ID NO 217 &211s LENGTH: 2O 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 217 aaaggcaulaa alaccalaga.ca

<210s, SEQ ID NO 218 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 218 ulugllalugga laugugugug lall 23

<210s, SEQ ID NO 219 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 219 cuaulacaaccuaculgccuuc cc 22

US 9,725,721 B2 181 182 - Continued

<4 OOs, SEQUENCE: 243 ulaulugcacau luaculaagulug Ca 22

<210s, SEQ ID NO 244 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 244 uucluggallaa caugclugaag cul 22

<210s, SEQ ID NO 245 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 245 aggagcuagc Caggcaulaug Ca 22

<210s, SEQ ID NO 246 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 246 agguguuauc CuaucCaululu go 22

<210s, SEQ ID NO 247 & 211 LENGTH 19 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 247 ggagaluggag gullgcagllg 19

<210s, SEQ ID NO 248 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 248 uucuagaluga gagauauaua lulla 22

<210s, SEQ ID NO 249 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 249 alaulucccuug ulagauaiac cc gg 22

<210s, SEQ ID NO 250 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 250

Cagluggc.cag agcc clugcag lug 22

<210s, SEQ ID NO 251 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens US 9,725,721 B2 183 184 - Continued

<4 OOs, SEQUENCE: 251 aauguggacu ggugugacca aa 22

<210s, SEQ ID NO 252 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 252 aagugc cc cc acagululugag lugc 23

<210s, SEQ ID NO 253 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 253 lugcc cullaaa ggugaaccca gu. 22

<210s, SEQ ID NO 254 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 254 cacgcucaug cacacaccca ca 22

<210 SEQ ID NO 255 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OO > SEQUENCE: 255 ulcacagugaa ccgglucucuu u. 21

<210s, SEQ ID NO 256 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 256 lugugacluggll lugaccagagg gig 22

<210s, SEQ ID NO 257 &211s LENGTH: 24 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OO > SEQUENCE: 257 agaggacccg ulag clugculag aagg 24

<210s, SEQ ID NO 258 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 258 gallalucagcu. Caguaggcac cq 22

<210s, SEQ ID NO 259 &211s LENGTH: 22 212. TYPE : RNA

US 9,725,721 B2 187 188 - Continued

212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 267 uguuccucug ulcucccagac

<210s, SEQ ID NO 268 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 268 aggaggcauc ulugaga aalug ga 22

<210s, SEQ ID NO 269 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 269 lugaggllaglla gulugugugg lull 22

<210s, SEQ ID NO 270 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 270 ulcggggauca lucaugu.cacg aga 23

<210s, SEQ ID NO 271 &211s LENGTH: 2O 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 271 gugllclugggc gga cagcl.gc

<210s, SEQ ID NO 272 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 272 lugu.gcaaaluc caugcaaaac luga 23

<210s, SEQ ID NO 273 &211s LENGTH: 17 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 273

Cagugulucag agalugga 17

<210s, SEQ ID NO 274 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 274 luggllgcggag agggcc caca glug 23

<210s, SEQ ID NO 275 US 9,725,721 B2 189 190 - Continued

&211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OO > SEQUENCE: 275 gcclugcluggg gluggalacclug gll 22

<210s, SEQ ID NO 276 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 276

Cccaguguuc agacluacclug uuc 23

<210s, SEQ ID NO 277 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OO > SEQUENCE: 277 gaga aalugcluggaculaaucu go 22

<210s, SEQ ID NO 278 &211s LENGTH: 17 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OO > SEQUENCE: 278 a 9999.1919 19 lullllll 17

<210s, SEQ ID NO 279 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OO > SEQUENCE: 279 lugulaalacaluc cucgaclugga ag 22

<210s, SEQ ID NO 280 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 280 lugallgallaca gluggagglla g 21

<210s, SEQ ID NO 281 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 281 luggc agggag gCugggaggg g 21

<210s, SEQ ID NO 282 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 282

Cacaggcuula gaaaagacag lu. 21 US 9,725,721 B2 191 192 - Continued

<210s, SEQ ID NO 283 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 283

CCCullugggluc lugalugggglla g 21

<210s, SEQ ID NO 284 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 284 acuuluaiacau ggalagugcuu ulc 22

<210s, SEQ ID NO 285 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 285 lugcluggallca glugglulu.cgag ulc 22

<210s, SEQ ID NO 286 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 286 all.ccculugca giggculgulug gll 23

<210s, SEQ ID NO 287 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OO > SEQUENCE: 287

C cluggaaa.ca Cugagglulugu g 21

<210s, SEQ ID NO 288 &211s LENGTH: 21 212. TYPE : RNA <213> ORGANISM: Homo Sapiens <4 OOs, SEQUENCE: 288 lagugglucag agggcullalug a 21

<210s, SEQ ID NO 289 &211s LENGTH: 23 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 289 ggcaggulucu Cacccucucul agg 23

<210s, SEQ ID NO 290 &211s LENGTH: 22 212. TYPE : RNA <213> ORGANISM: Homo Sapiens

<4 OOs, SEQUENCE: 290 ulugaggagac aluggluggggg CC 22